Starter strains expressing a protease of Lactobacillus bulgaricus

The present invention relates to the proteolytic system of Lactobacillus bulgaricus, and especially to the use of the novel recombinant protease PrtP to develop efficient starter strains with accelerated fermentation properties.

Background of the invention

The proteolytic system is essential to ensure a rapid growth in milk and supplies auxotrophic lactic acid bacteria with amino acids from caseins, the major milk proteins. This system is complex and extensively studied in lactococci (Kok et al., FEMS Microbiol. Rev, 87, 15-42, 1990; Smid et al., Appl. Env. Microb., 57, 2447-2452, 1991; Pritchard et al., FEMS Microbiol. Rev., 12, 179-206, 1993).

The first step of milk casein degradation is performed by a cell surface proteinase, PrtP. According to their substrate specificity, two types of PrtPs were distinguished. PI type preferentially cleaves β-casein, and the PIII type degrades α, β and κ caseins. Lactococcal PrtPs are serine protases and show extensive homology with subtilisins secreted by Bacillus genus. Lactococcal proteinases are synthetized as inactive preproproteins. A N-terminal signal peptide of 33 residues is removed during crossing through the cytoplasmic membrane and the C- terminus remains anchored in the cell-envelope. Then a maturation process leads to the removal of the pro region (154 residues) and involves a membrane located lipoprotein, PrtM. This 33 κDa protein is encoded by a gene (prtM) located immediately upstream and in opposite direction of the prtP gene. Selfdigestion of the carboxy terminus of PrtP results in the release of the enzyme into the culture medium. Incubation of lactococci cells in a calcium free buffer stimulates the PrtP selfdigestion and release. Lactobacilli have been investigated in a lesser extent, but display a high cell surface proteinase activity with substrate specificity differing from that of lactococci (Bosman et al., FEMS Microbial. Rev., 12, p. 72, 1993).

Cell surface proteinases from several lactobacilli were purified: Lb. paracasei subsp. paracasei NCDO 151, Lb. Helveticus CNRZ 303 and L89, Lb. bulgaricus CNRZ 397 (Zevaco et al., Le Lait, 68, 393-408, 1988; Laloi et al., Appl. Microbiol. Biotech., 36, 196-204, 1991; Naes et al., J. Gen. Microbiol., 138, 313-318, 1992; Martin Hernandez et al., Appl. Microbiol. Biotech., 40, 828-834, 1994). The sequence of the gene coding for the proteinase from Lb. paracasei was cloned and the deduced amino acid sequence shows 1902 residues and a high homology (96,6%) with lactococcal PrtPs. The presence of a prtM gene suggests a maturation process similar to the lactococcal PrtP maturation. In contrast, thermophilic lactobacilli proteinases appear different. Lb. helveticus CNRZ 303 proteinase is characterized by an original proteolytic specificity towards α_s1 casein. The proteinases of Lb. Bulgaricus CNRZ397 and Lb. helveticus L89 cannot be released from the cell-wall by the procedure using calcium free buffer. Moreover, the Lb. bulgaricus enzyme is sensible to serine-and cysteine protease inhibitors.

The cell-wall proteinase of Lb. bulgaricus CNRZ397 displays however quite different biochemical properties. This enzyme has been purified from cell-wall extracts obtained after lysozyme treatment and osmotic shock of whole cells. Inhibitors of serine proteases have no effect on this enzyme characterised as a cysteine-protease with a molecular mass of 170 κDa and able to hydrolyse alpha- and beta-caseins. Optimal pH of enzyme activity is 5.5 instead of 7.5 to 8.0 for Lb. helveticus proteinase. The level of the cell-wall proteinase synthesis depends on the nature of culture medium: cells grown in a milk medium exhibit a higher activity than cells developed in MRS (rich peptide medium).

The present invention has the objective to use an effective gene of a protease to develop dairy starter strains with accelerated fermentation properties. This protease should be active without a further maturation step involving another protein. Moreover, the level of activity of this protease should be sufficient, but not too high, to permit an accelerated fermentation of dairy products, without accelerating the degradation of these products at chilled temperature during storage. Finally, this protease should produce amino acid and peptides precursors involved in the formation of typical dairy flavours.

Summary of the invention

Accordingly, the invention provides the recombinant protease PrtP of Lactobacillus bulgaricus having the amino-acid sequence SEQ ID NO:2, and functional derivatives of this protease(see the sequence listing below).

The invention provides also two usefull portions of the protease PrtP, which are the prepro portion having the amino-acid sequence from amino-acid 1 - 192 of SEQ ID NO:2, and the anchorage portion having the amino-acid sequence from amino-acid 1883 to at least amino-acid 1915 of SEQ ID NO:2.

The present invention relates to a DNA encoding the protease PrtP, its anchorage portion or its prepro portion. In particular this DNA may be the prtP gene having the nucleotide sequence from nucleotide 794 to 6631 of SEQ ID NO:1 (see the sequence listing below).

The invention also relates to the L. bulgaricus promoter of the prtP gene having the nucleotide sequence from nucleotide 588 to 793 of SEQ ID NO:1.

The invention also relates to a recombinant cell comprising a DNA molecule according to the invention.

The invention further relates to a method for producing the recombinant protease PrtP comprising, cultivating recombinant cells according to the invention in a suitable growth medium under conditions that the cells express the said recombinant protease, and optionally isolating the said recombinant protease in the form of a concentrate.

In a further aspect of the invention, the use of these recombinant cells is provided for the manufacture of fermented dairy products.

Short description of the drawings

• Figure 1: fermentation of a milk by Lactococcus lactis recombinant strains expressing the protease PrtP (see example 2: transformants with plasmid pLL82).
• Figure 2: fermentation of a milk by Lactococcus lactis recombinant strains containing but not expressing the protease PrtP (see example 2: plasmid pLL81).
• Figure 3: restriction map of the prtP region.

Detailed description of the invention

For purpose of this disclosure and claims, the term "prtP" refers to the gene encoding the related protease "PrtP"of Lactobacillus bulgaricus.

Within the context of the present invention the expression "functional derivative" may comprise all amino acid sequences which differ by substitution, deletion, addition of some amino acids but which keep their original activities or functions. A protein may be generally considered as a derivative to another protein, if its sequence is at least 50% identical to the protein, preferably at least 70%, in particular 90%. In the present disclosure, the identity is determined by the ratio between the number of amino acids of a derivative sequence which are identical to those of PrtP and the total number of or amino acids of the said derivative sequence.

Likewise, the expression "a DNA encoding the protease PrtP" may comprise any DNA encoding the same or a derivative protein due to the degeneracy of the genetic code or to the cross species variation. A DNA molecule may be generally considered as a derivative to another DNA molecule, if its sequence is at least 40% identical to the another DNA molecule, preferably at least 60%, in particular 80%. In the present disclosure, the identity is determined by the ratio between the number of bases of a derivative sequence which are identical to those of nucleotides 794 to 6631 from SEQ ID NO:1 and the total number of bases of the said derivative sequence.

The DNA molecule according to the present invention may be obtained in substantially purified form, by using the method described in the following examples, from any strain of Lactobacillus bulgaricus, more particularly from the strain L. bulgaricus NCDO1489 (NCDO comes from the former name "National Collection of Dairy Organisms" of the NCFB - National Collection of Food Bacteria - which in an International Depository Authority under the Budapest Treaty; see NCFB catalogue of strains, 1986, p 85, No 1489).

Alternatively, the DNA molecule may be recovered also from other genera or species of bacteria by use of DNA probes derived from a DNA sequence of the invention in a stringent hybridisation assay.

In a further aspect, the DNA molecule may also be synthesised from sequences given in the sequence listing below, multiplied in vitro for instance by using the polymerase chain reaction or multiplied in vivo for instance in bacteria of the species Escherichia coli, Lactococcus lactis, or Streptococcus thermophilus.

The DNA molecule according to the invention comprises at least an encoding part of at least 20 base pairs, or even 50, 100 or 200, of a DNA sequence selected from the group comprising the DNA sequence SEQ ID NO:1 in particular from nucleotide 794 to 6631 of SEQ ID NO:1 encoding the prtP gene.

The DNA molecule according to the invention may also comprise a sequence which is a derivative (see definition) to the above sequences.

The DNA molecule according to the invention can be present in a vector, such as a replicative plasmid or an integrative circular or linearized non replicative plasmid, for example.

The DNA molecule can also comprise, operably linked to the said DNA, regulatory sequences native to the organism from which derives the nucleotide sequence. The said native regulatory sequences can be a promoter, a terminator, a Shine-Dalgarno sequence, an enhancer, or a sequence encoding a leader peptide, for example, that regulate expression of prtP.

In another embodiment, regulatory sequences can be native sequences that regulate a different gene in the said organism of origin or that regulate a different gene in a foreign organism, for example. A regulatory sequence other than the native regulatory sequence will generally be selected for its high efficiency. It is also possible to select as regulatory sequence a promoter on the basis of other desirable characteristics, for example thermo inducibility, or a sequence encoding a peptide signal which will permit excretion of the protein.

If heterologous expression is preferred, meaning that the genes of the invention are expressed in another organism than the original host (strain, variety, species, genus, family, order, class or division) the regulatory sequence is preferably derived from an organism similar or equal to the expression host. For example, if the host is a yeast cell, then the regulatory sequence will be derived from a yeast cell. The promoter suitable for constitutive expression, preferably in a fungal host, may be a promoter from the following genes: glycerolaldhehyde-3-phosphate dehydrogenase, phospho-glycerate kinase, triose phosphate isomerase and acetamidase, for example. Promoter suitable for inducible expression, preferably in a fungal host, may be a promoter from the following genes: endoxylanase IIA, glucoamylase A, cellobiosehydrolase, amylase, invertase, alcohol dehydrogenase and amyloglucosidase. The selection of a desirable regulatory sequence operably linked to a sequence of the invention and capable of directing the expression of the said nucleotide sequence is considered to be obvious to one skilled in the art.

The DNA molecule according to the invention may also comprise a selection marker to discriminate host cells into which the recombinant DNA material has been introduced from cells that do not comprise the said recombinant material. It may also comprise at least one suitable replication origin. Suitable transformation methods and suitable expression vectors provided with a suitable transcription promoter, suitable transcription termination signals and suitable marker genes for selecting transformed cells are already known in the literature for many organisms including different bacteria, fungal and plant species.

Over expression of proteins of the invention may be achieved by incorporation of the DNA molecule of the present invention in an expression host, the said DNA molecule comprising one or more regulatory sequences which serve to increase expression levels of the protein(s) of the invention. The over expression can be further achieved by introducing a multicopy of the DNA molecule of the invention, for example.

The invention also encompasses a recombinant cell comprising the DNA molecule of the invention. These cells may be derived from the group of fungal cells in particular of the genus Aspergillus, yeast cells in particular of the genera Saccharomyces, Kluyveromyces, Hansenula and Pichia, bacterial cells in particular Gram-positive bacteria of the genera Bacillus, Lactobacillus, Streptococcus in particular Streptococcus thermophilus, Bifidobacteria, Staphylococcus and Lactococcus in particular Lactococcus lactis, and plant cells in particular of the tree and vegetable groups, for example.

Recombinant cells may comprise the DNA molecule of the invention stably integrated into the chromosome or on a replicative plasmid. Preferably, the DNA molecule of the invention is integrated into the chromosome by using the process described in EP564966, i.e.,

• (1) transforming a host strain microorganism with a donor plasmid which does not replicate in the host strain, wherein the donor plasmid comprises a vector backbone and a sequence comprising a promoterless foreign gene (prtP or similar genes) operably integrated into at least a part of an operon of the host strain, maintaining the frame and the function of the genomic operon of the host strain;
• (2) identifying cointegrate transformants in which the complete donor plasmid is integrated into the genomic operon of the host strain;
• and (3) selecting an integrant transformant from the cointegrate transformants, wherein the genome of the selected integrant transformant does not include the vector backbone of the donor plasmid but does include the foreign gene, which is operably integrated into the conserved genomic operon and which is stably maintained and expressed due to selective pressure on the correct functioning of the essential cistron upon growth in a standard medium.

Progeny of an expression host comprising a DNA molecule according to the invention is also included in the present invention. Accordingly, a preferred embodiment of the invention is directed to a cell comprising a recombinant DNA molecule of the invention in any of the embodiments described above, wherein the said cell is able to integrate the PrtP protease or functional derivatives into the cell wall or the cell membrane or secrete the enzymes into the periplasmic space or the culture medium. The secreting route to be followed by recombinant proteins according to the invention will depend on the selected host cell and the composition of the recombinant DNA according to the invention. Most preferably, however, the protein will be bound to the outer cell-wall. To this end, the cell according to the invention can comprise recombinant DNA further comprising operably linked DNA encoding foreign leader sequences (pre or prepro), for example.

The invention is also directed to a process for producing recombinant protein(s) according to the invention comprising, providing recombinant cells according to the invention in a suitable growth medium under conditions that the cells express the said recombinant protein(s), and optionally isolating the said recombinant protein(s) in the form of a concentrate. The selection of the appropriate medium may be based on the choice of expression host and/or based on the regulatory requirements of the DNA recombinant material. Such media are well-known to those skilled in the art.

After fermentation, the cells can be removed from the fermentation broth by centrifugation or filtration. Depending on whether the host cells have secreted the recombinant protein(s) of the invention into the medium or whether the said protein(s) are still connected to the host cells in some way either in the cytoplasm, in the periplasmic space or attached to or in the membrane or cell wall, the cells can undergo further treatment to obtain the recombinant protein(s). In the latter case, where the recombinant protein(s) are still connected to the cells, recovery may be accomplished by rupturing the cells for example by high pressure, sonication, enzymatic digestion or simply by cell autolysis followed by subsequent isolation of the desired product. In this context, the method described by Laloi et al. is incorporated by reference (Appl. Microb. Biotech., 36 , 196-204, 1991). The protein(s) can be separated from the cell mass by various methods, such as ultrafiltration, and then subsequently precipitated with an organic solvent. The isolated protein(s) may be further purified by conventional methods such as precipitation and/or chromatography.

The invention is also directed to the use of recombinant cells according to the invention, for the manufacture of fermented dairy products, in particular yoghurt, acidified milk and cheese.

Indeed, of all processes involved in the ripening from cheese, for example Cheddar and Gouda, the degradation of milk proteins by proteinases from starter cultures is the most important process. Ideally, the objective in accelerating ripening should be to accelerate all the desirable reactions involved in ripening in a balanced way while controlling the undesirable ones. However, since the key reactions responsible for the unique flavour are not known in most cases, a more or less empirical approach has to be adopted. Since the glycolytic reactions occur very quickly and since the modification of lactose in most cheeses is not the fermentation rate limiting step, there is apparently no need to accelerate glycolysis and subsequent reactions. Likewise, lipolysis is not important, is in fact undesirable, in most varieties. Therefore, attempts to accelerate ripening have concentrated on proteolysis. Essentially, there are two approaches to accelerate cheese ripening. Although elevated temperatures may give satisfactory results with a 50% reduction in ripening time, industry appears to be reticent to apply this technique. Development of engineered "super" starters, which may accelerate ripening are therefore awaited.

On the other hand, although yoghurt starter cultures are considered to be only weakly proteolytic, S. thermophilus and L. bulgaricus may, during the fermentation, cause a significant degree of proteolysis, and this activity may be important for the following reasons:

• (a) The enzymatic hydrolysis of milk proteins results in the liberation of peptides of varying sizes and free amino acids, and these possible changes can affect the physical structure of yoghurt.
• (b) The liberation of amino acids into the milk is essential for the growth of S. thermophilus. Indeed S. thermophilus does not possess substantial extracellular proteolytic activity and the amino acid and free peptide content of milk is not high enough to promote its full growth.
• (c) Although amino acids and peptides may not contribute directly to the flavour of yoghurt, they do act as precursors for the multitude of reactions which produce flavour compounds.

Therefore, there is a need to develop dairy starter strains with accelerated fermentation properties, or capable to full grow in a milk (S. thermophilus for instance).

However, these starters should not exhibit a too high protease activity, otherwise the advantage of the accelerated ripening or fermentation is counter-balanced by an accelerated degradation of the product. Therefore, the protease activity should be carefully chosen, so that at chilled temperature it does not degrade the product.

It has been surprisingly observed, that the recombinant protease PrtP according to the invention fulfils these needs.

In order to improve the shelf stability of dairy products fermented by cells according to the invention, it is possible to make dependent the gene encoding of the said PrtP to a temperature or pH sensitive promotor selected to be inhibited at chilled temperatures, that is to say 9°C to 12°C, or at acid pH, for example lower than 5.5. Such promotors are already known to the skilled person.

Alternatively, the gene itself may be mutated and rendered sensitive to temperature or pH. Therefore, it is possible to use a recombinant cell according to the invention, said cell expressing a recombinant derivative of the protease PrtP according to the invention, said derivative exhibiting decreased activity at a rate of at least 20% less than the protease having the amino-acid sequence SEQ ID NO:2, under the storage conditions of the fermented dairy product, but where the said derivative retains at least 90% of its activity at the production conditions of the fermented dairy product when compared to the protease having the amino-acid sequence SEQ ID NO:2.

Preferably, the derivative protease according to the invention exhibits decreased activity at a temperature below 20°C, or at a pH below 5.5.

Such variants and recombinant cells are readily obtainable by use of a process comprising:

• a) inserting the prtP gene in a vector;
• b) performing mutations on a plurality of vectors of step a);
• c) selecting those vectors of step b) in which said gene is mutated so that when a selected vector is used to transform an organism, expression of the selected gene produces a variant enzyme which exhibits decreased activity at a rate of at least 20% less than the PrtP enzyme having the amino acid sequence SEQ ID NO:2., under the storage conditions of the fermented dairy product, but where the said derivative retains at least 90% of its activity at the production conditions of the fermented dairy product when compared with the PrtP enzyme having the amino acid sequence SEQ ID NO:2.
• d) inserting and expressing a selected gene coding for the variant enzyme of step c) into an organism suitable for the production of the fermented food product.

This method has already been used for the development of β-galactosidase variants, as described in EP 402 450, this method being thus incorporated by reference.

The present invention is further illustrated hereafter, and not limited, by a supplemental description which refers examples of characterisation of DNA molecules, cells, proteins and use according to the invention, in which all parts, ratios, and percentages are expressed on a weight basis unless otherwise stated, with reference to the accompanying drawings.

Methods involving DNA techniques were essentially performed as described in the Book of Sambrook et al. (Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, U.S.A., 1989).

Example 1:

DNA sequencing of the prtP gene

From the lacZ gene of the L. bulgaricus NCDO 1489 strain, reverse PCR allowed us to pick up the DNA region downstream to the lacZ gene. (See figure 3). The PCR fragment was cloned in pUC19 and sequenced (Yanisch et al., Gene, 33, 103-119, 1991). Search for protein homology in protein libraries revealed an identity of 27.8% between the deduced amino acid sequence with the PrtP from Lb. paracasei NCDO151. Then, the region downstream lacZ and including the prtP-like gene was determined in a cascade PCR strategy using synthetic primers as the known sequence progressed. A restriction map of the prtP gene location from L. bulgaricus is presented in Fig. 3.

The nucleotide sequence of the prtP gene is presented in the sequence listing below (see nucleotides 794 to 6631 of SEQ ID NO:1).

The prtP gene is under the control of a promoter located from nucleotide around 588 to 793 of SEQ ID NO:1.

The open reading frame (ORF: nucleotide 794-6631) displays homology with lactococal prtP genes. The first encoding DNA codon (ATG) is preceded by a Shine-Dalgarno sequence matching well the 165 rRNA from lactobacilli species (see nucleotide 784 to 790 of SEQ ID NO:1). Moreover, the N-terminus amino acid sequence translated looks like a signal sequence of PrtPs. The upstream contains three characteristic boxes and might be the promoter region (see nucleotide 632 to 659 of SEQ ID NO:1). Moreover, 5 nucleotides upstream -35 box, there is a third recognition signal which is called UP-element and characterised by oligo A and T stretches (see nucleotide 608-627 of SEQ ID NO:1). The UP element has already been found in lactococcal prtP and Lb. delbrueckii subsp. lactis promoters and is known to enhance transcription (Kok et al., Appl. Environ. Microbiol., 54, 231-238, 1988).

The coding sequence of prtP gene is 5,841 nucleotides long which is in the range of length of ptrP genes. The GC content of prtP gene is estimated at 47.6% which is very similar to the ratio described for the prtP genes of Lb. paracasei and Lc. lactis NCDO 763 (47.5 and 47.2%, respectively). It is interesting to stress that Lb. bulgaricus genome exhibits a GC content of 54% and Lc. lactis of 38% (Kilpper-Balz et al., Cur. Microbiol., 7, 245-250, 1982). So, prtP gene significantly differs from other L. bulgaricus genes (lacZ, lacS and pepIP) which show a CG content of 52.5, 53.1 and 56.9%, respectively (Leong et al., J. Bacteriol., 173, 1951-1957, 1991, Atlan et al., Microbiol., 140, 527-535, 1994).

The expected proteinase PrtP consists of 1,946 amino acids and is characterised by a predicted Mr of 212,271.

The amino acid sequence of this PrtP protease is presented in the listing sequence below (see SEQ ID NO:2). This length fits in well with other described PrtPs: 1,902 residues for Lb. paracasei, Lc. cremoris Wg2 and Lc. lactis NCDO 763, and 1,962 residues for Lc. cremoris SK11 associated with a duplication of 60 amino acids at the COOH extremity.

L. bulgaricus PrtP shows 27% of identity with Lb. paracasei PrtP over the first 1,806 residues and up to 39.5% when only the first 820 residues were compared. The C-terminus part of the cell surface proteinase did not share any homology with a PrtP proteinase.

The major amino acid of PrtP is lysine (12%) instead of leucine for other Lb.bulgaricus enzymes (LacS, Beta-galactosidase and PepIP) or threonine for other lactococcal and Lb. paracasei PrtPs. Codon usage was investigated and is similar to other L. bulgaricus enzymes (LacS, β-galactosidase and PepIP) for lysine and glycine; but serine, aspartic acid, and valine are preferentially coded as lactococci. Finally, alanine codon preference is specific for PrtP.

PrtP and cell envelope location

At the amino acid level, the homology of sequences between N-terminus regions of PrtP exhibits a very positively charged N-extremity followed by an α-helix with a high content in leucine and alanine. This domain closely resembles signal peptides of exported proteins of Gram positive bacteria. The prepro portion of the prtP enzyme is thus located from amino-acid 1 to 192 of SEQ ID NO:2.

The absence of a prtM-like gene located immediately upstream or downstream the prtP gene and expressing a functional chaperone gives rise to two hypotheses. The maturation and export of PrtP could be processed by a PrtM-like chaperone encoded by a gene located anywhere on the chromosome; however, it cannot be ruled out that a general chaperone acts on different extracytoplasmic proteins, PrtP included. On the other hand, the PrtP may preferably not be processed by a PrtM-like chaperone, since the recombinant PrtP is functional in different host system (see examples 2 and 3).

In contrast to the proteinases PrtPs, PrtP from L. bulgaricus is never recovered in the culture medium and, consequently, is devoid of the ability of removal of the C-extremity by selfdigestion. A portion of the prtP enzyme is thus believed to be an anchorage sequence which permits to the enzyme to be stably maintained in the outer membrane of the cell. This portion is thus located from amino-acid 1883 to at least amino-acid 1915 of SEQ ID NO:2.

Once the first step of casein catabolism, the peptides released by the PrtP action are different from those resulting from the hydrolysis by PrtPs. Other steps of the proteolytic system of Lb. bulgaricus are suspected of being different. For instance, Lb. bulgaricus exhibits high activities towards substrates containing proline (Sasaki et al., FEMS Microbiol. Rev. 12, p 75D16, 1993).

Example 2

L. bulgaricus NCDO 1489 DNA was purified by the spooling method (Delley et al., Appl. Environ. Microbiol., 56, 1967-1970, 1990). Cell wall associated protease gene (prtP) was amplified by PCR using the polymerases Biotag (bioprobe, FR) and a 1/20 dilution of Pfu (cloned Pfu DNA polymerase Stratagene, USA) and the conditions 92°C for 1mn, 55°C for 2 mn and 72°C for 6mn (Saiki et al., Science, 239, 487-491, 1988). To this end, the prtP gene was amplified without its promotor (primers having respectively for sequence the nucleotides 763-787 and 6831-6858 of SEQ ID NO:1) and with its promotor (primers having respectively for sequence the nucleotides 557-583 and 6831-6858 of SEQ ID NO:1). PCR products were cleaved with BamHI and XbaI and ligated in XbaI/BglII digested pNZ124 (Platteeuw et al., Applied and Env. Microbio., 60, 587-593,1994).

Recombinant plasmids pLL81 (promoter free prtP) and pLL82 (prtP with promoter) were introduced in Lactococcus lactis MG1363 (plasmid free) by electroporation (Holo and Nes, Applied and Env. Microb., 55, 3119-3123, 1989).

Transformants were grown in M17 with 1% glucose (Difco laboratory) in the presence of 5 µg/ml chloramphenicol. As an example, a transformant comprising the plasmid pLL82a (the prtP gene with its promoter) has been deposited at the Pasteur Institute, 28 rue du Dr. Roux, F-75724 Paris 15, France, where it received, on May 24, 1996, the deposit number CNCM I-1713.

Transformants were precultured in 10% reconstituted skim-milk containing 0.1% yeast extract, 1% glucose and 5 µg/ml chloramphenicol. Growth was followed in pasteurised (80°C, 30mn) 10% reconstituted skim-milk containing 1% glucose and 5 µg/ml chloramphenicol by impedence measurement (RABIT: rapid automated bacterial impedence technique, Don Whitley scientific limited, England).

Figure 1 presents the fermentation results: plot 1 designates the Lactococcus lactis MG1363 strain without plasmid, plots 2 to 4 designate 3 independent Lactococcus lactis transformants comprising the plasmid pLL82 (prtP with promotor). As a control Figure 2 shows fermentation results with Lactococcus lactis transformants containing plasmid pLL81 (prtP without promoter): plot 1 designates the Lactococcus lactis MG1363 strain without plasmid, plots 2 to 4 designate 3 independent Lactococcus lactis transformants comprising the plasmid pLL81.

Example 3

Plasmids pLL81 and pLL82 of Example 2 were used to transform a Streptococcus thermophilus. The transformants were precultured and cultured in the same manner as described in Example 2. The results are similar to those presented in figures 1 and 2.

Example 4

A frozen mixed starter comprising a mixture of a strain Streptococcus thermophilus containing an expressed Lb. bulgaricus prtP gene (see example 3) and the strain Lactobacillus delbruckii subsp. bulgaricus CNCM I-1420 is prepared as follows. To this end, a skimmed milk is reconstituted using 10% of skimmed milk powder, 1% of yeast extract is added, and the mixture is sterilized to 115°C for 30 minutes and then cooled to a temperature of approximately 42°C. To this milk there is added 1% of a fresh preculture of the strain S. thermophilus and 2% of a fresh preculture of the strain L. delbruckii subsp. bulgaricus. The milk is subsequently incubated at a temperature of approximately 42°C until a pH of 4.7 is reached. The milk is cooled to a temperature of 4°C. Then 5% of sterile glycerol is added and the mixture is frozen at -75°C.

Stirred yoghurts are then prepared by direct inoculation with this frozen starter. To this end, the milk is prepared from full-cream milk comprising 3.7% of fat and 2.5% of skimmed milk powder. 40 1 of this milk is sterilised at 105°C for 2 minutes, subsequently homogenised at 75°C and 300 bar (first stage) and finally cooled to a temperature of approximately 43°C. This milk is then fermented with 10ml of the frozen starter at 43°C until a pH of approximately 4.65 is reached and then it is cooled to 4°C. The results show that we gain time during the fermentation, in comparison with traditional starters. Moreover, the yoghurt is stable during several weeks at 4°C.

Example 5

An acidified milk is prepared traditionally with the same S. thermophilus strain expressing the Lb. bulgaricus prtP gene (see example 3). The results show that the milk can be acidified up to a pH below 4.9. Moreover, the acidified milk is stable during several weeks at 4°C.

Example 6

Lactococcus lactis CNCM I-1713 is used in a conventional process for making Cheddar cheese. To a milk is added 0.8% of a fully ripened bulk starter containing a high population of a commercial Leuconostoc mesenteroides subsp. cremoris and the strain Lactococcus lactis CNCM I-1713, and 5.10⁶ CFU/ml of a commercial dried Lactobacillus plantarum. The mixture is allowed to ripen for 1 hour after which single-strength calf rennet is added. The proper coagulum is formed within 30 minutes at which point cutting occured. Standard cheesemaking procedures are followed to provide a cheddared curd which are milled when a pH of 5.4 is reached. The milled curd is salted to provide a finished product containing 1.8% salt. The finished hooped curd has a final moisture content of 37% and fat content of 33%. The blocks are then shrink film wrapped and aged at 10°C for 3 months which results in the production of a typical aged (6 months) cheddar cheese flavour. Longer aging at 10°C results in strongly aged cheddar cheese flavors which simulate 12 month flavors with only 6 month storage. The flavor development rate can be substantially reduced by lowering the aging temperature from 10°C to 7°C or below.