NL9301525A - Novel Lactobacillus strains, proteins and sequences thereof, as well as methods for the use of these strains, proteins and sequences. - Google Patents

Abstract

PURPOSE:To obtain Lactobacillus helveticus SBT2171 or its mutants which is useful for aging cheese because it has strong protease activity to hydrolyze peptides causing the bitterness and bad smells. CONSTITUTION:The Lactobacillus helveticus SBT2171 (Deposit no.-CBS-40493) or its mutants which has activity of removing bitterness caused by casein trypsin hydrolysates. It is preferred that said microorganism is used to effect the hydrolysis of physiologically active peptides.

Description

Title: Novel Lactobacillus strains, proteins and sequences thereof, as well as methods for the use of these strains, proteins and sequences

The invention relates to a new Lacto-bacillus strain, enzymes and other proteins obtained from that strain, nucleotide sequences encoding such proteins, enzymes or other parts of the Lactobacillus organism and uses of the organisms, enzymes and / or other proteins, as well as the nucleotide sequences.

Lactic acid bacteria are used in the fermentation of a large number of products, especially in the fermentation of foodstuffs. In the past, the most important property of these bacteria was that they could convert carbohydrates (sugars) present into lactic acid, so that the fermented product was better preserved.

In the current processes in the dairy industry, other properties, such as the development of a taste and a constant quality of the product, have become at least as important.

To obtain dairy products with all the desired properties by fermentation, specific starter cultures comprising a mixture of different microorganisms have been developed. Micro-organisms with special properties are therefore of great importance for the dairy industry.

For example in the preparation of cheese, where maturation can take from several weeks to two years, depending on the type of cheese.

This entails high costs due to the long-term refrigerated storage, capital immobilization and production losses due to atypical fermentation, resulting in, for example, a bitter taste.

Accelerating the ripening process could yield significant cost savings.

Lactococci are widely used in the dairy industry as an organism as part of a starter culture. In particular, the conversion of fresh cheese curd to mature cheese is determined by the proteolytic breakdown of casein using these Lactococci starter cultures.

The proteolytic system of this Lactococci consists of a number of proteinases and a number of peptidases and has been the subject of a number of studies (Tan, PST et al, J. Dairy Res. 60 p. 269-286, 1993 and Visser, S., J .Dairy Sci.

76, pp. 329-350, 1993).

In contrast to the above mentioned Lactococcus strains, Lactobacillus species are usually not used as starter cultures in cheese ripening.

However, cultures of Lactobacillus species develop in situ as a number of cheese ripens. Apparently these varieties do play a role in the ripening of those cheeses (Marth, EH, J. Dairy Sci. 46, p. 869-890, 1963 and Peterson et al, J. Dairy Sci. 73, p. 1395-1410, 1990).

Furthermore, it is believed that a number of Lactobacillus species have high proteolytic activity (Arora et al, J. Dairy Sci.73, p.264-273,1990a) and that the ability of these Lactobacillus species to hydrolyze peptides is necessary to accumulate of bitter peptides in cheese. A number of Lactobacillus proteolytic enzymes have been studied (3, 4, 5, 6, 16, 17, 21, 22, 23, 24 and 30)

The invention provides a new microorganism containing a proteolytic system with a higher activity than any other Lactobacilli known to date and a higher activity than most other lactic acid bacteria, including some Lactococci.

The new microorganism is characterized by a unique combination of properties that makes it very suitable for use in fermentation processes, in particular for cheese ripening.

Characteristics of the new microorganism include in comparison with other somewhat similar microorganisms of the same genus or of the genus Lactococcus, shown in Figures 1a-c, Figures 2 and 3, Table 2 and Table 6.

The new microorganism has been deposited with the Central Bureau for Fungal Cultures in Baarn under Art 22B of the ROW and the Budapest Treaty under number CBS 404.93 (Article 31F paragraph 3 of the patent regulations applies).

Briefly, the properties of the new microorganism come down to the following.

Lactobacillus Helveticus (internal code SBT 2171) forms regular gram-positive rods, can grow anaerobically, is obligatory saccharoclastic, can use glucose, mannose, galactose and lactose as a substrate and has an average very high proteolytic activity.

This proteolytic activity is manifested in particular in the debittering of a tryptic degradation product of casein (the so-called bitter peptides in cheese).

In addition to the microorganism itself which becomes available through the invention, the invention also provides for the use of said organism or parts thereof in, for example, fermentation processes or proteolytic steps in processes.

For example, in fermentation processes, the organisms according to the invention are used as part of a starter culture for cheese ripening, but the organisms according to the invention can also be added at a later stage than as a starter culture. In addition, the micro-organisms are used, for example, for the proteolysis of biologically active peptides, such as, for example, neuropeptides, endorphins, LHRH, ACTH, etc.

They can also be used for other hydrolyses of proteins, for example for food preparations.

In addition to the application of the micro-organisms per se, as stated previously, parts of the micro-organisms can also be used. This applies in particular to the new proteins according to the invention which originate from the new organism. Those new proteins also include active fragments of these proteins or derivatives, fusions and / or mutants of these proteins that

Have similar activity. The new proteins according to the invention can be used alone or in combination with other proteins (whether or not according to the invention) in various cleavages of proteins and / or peptides. It is particularly preferred, for example, in cheese ripening, to use a combination of peptidases and proteinases according to the invention.

In addition, the proteins of the invention can also be added to conventional starter cultures, for example such as those based on Lactococcus species.

Also, the new proteins according to the invention, like the micro-organisms, can also be used separately for other proteolytic purposes, such as, for example, in the proteolysis of biologically active peptides such as LHRH, ACTH, endorphins, etc.

Antibodies can be raised against the proteins and other parts of the micro-organisms. These antibodies also belong to the invention. For example, they can be used to suppress certain proteolytic activities of the new microorganism, which may be less desirable in some circumstances.

In addition, they can be used in the purification of the proteins of the invention, by affinity chromatography.

They can also be used in the detection (detection) of micro-organisms according to the invention. This can be useful in the search for further suitable Lactobacillus strains and, for example, in the quality control of the final product.

Methods to raise antibodies may be characterized as standard techniques. In general, a mammal, usually a rodent, is injected with a preparation against which antibodies are to be raised, generally in combination with an adjuvant.

After a number of repeated injections (boosters), a sufficiently high antibody titer is generally achieved. The antibodies can then be purified from the ascites. Today it is often chosen to produce monoclonal antibodies. The same immunization schedule applies to these antibodies. Only now the spleen cells of the immunized animal are removed and fused with a myeloma, or immortalized using a virus transformation (e.g. EBV). This provides a continuous source of antibodies with identical specificity and affinity.

Where antibodies are mentioned in this application, they include both polyclonal and monoclonal antibodies, as well as antigen-binding fragments thereof. Many techniques are now known to obtain antigen binding fragments.

Fab fragments and Fab'2 fragments can be easily obtained by chemical cleavage. Smaller binding fragments up to MRUs (Molecular Recognition Units: only consist of a CDR (complementarity determining region)) are usually prepared by recombinant DNA techniques.

Antibodies with two specificities or linked to other proteins can also be prepared by these recombinant DNA techniques. All of these and other techniques are useful for obtaining antibodies of the invention.

Recombinant DNA techniques are also very suitable for applying sequences of the new micro-organisms according to the invention. For example, sequences encoding proteolytic enzymes in cells other than Lactobacillus can be expressed to yield a purer preparation with a higher yield. Expressing such a protein in a host cell (e.g., E. Coli or other prokaryotes, Saccharomyces or other yeasts, fungi, insect cells, plant cells and / or mammalian cells) is usually done by the DNA sequence, which can be isolated from the organism, or can be obtained by isolating mRNA from the organism and preparing cDNA from it, introducing it into a suitable vector for transfection or transformation of the host cell under the control of some suitable regulatory elements, such as promoters, enhancers, etc.

Again, for these sequences it will not always be necessary to use the entire sequence encoding a protein. Often a part will also be able to have the desired activity.

Other sequences can also be linked to the sequences according to the invention, for example to produce fusion proteins.

In addition, the sequences can be used in amplification techniques or hybridization techniques which are now also widely known to the average skilled person.

The invention is further illustrated by the following examples.

MATERIAL AND METHODS Organisms

In the examples, the following Lactobacillus and Lactococcus species were used.

Lb. acidophilus, Lb.brevis, Lb.buchneri, Lb.casei ssp.casei, Lb.delbrueckii ssp.bulgaricus, Lb.delbrueckii ssp.delbrueckii, Lb.delbrueckii ssp.lactis, Lb.fermentum,

Lb.helveticus, Lb.paracasei ssp.paracasei, Lb.plantarum, Lb.rhamnosus, Lactococcus lactis ssp.cremoris, Lactococcus lactis ssp.lactis, Lactococcus lactis ssp.diacetylactis. Strains with an SBT number have been obtained from the Snow Brand Type Culture Collection, Tokyo, Japan. All strains were obtained from the National Collection of Food Bacteria (NCFB); Agricultural and Food Research Council; Institute of Food Research, Reading, Great Britain. Lactococcus lactis ssp. Cremoris AMI, E *, HP, SK112 and Wg2 and Lactococcus lactis ssp.lactis ML3 were obtained from the Netherlands Institute for Dairy Research (NIZO), Ede.

The organisms were routinely subcultured (3X) in MRS broth (Merck-Darmstadt, Germany) at 30 or 37 ° C and stored as "stock cultures" in MRS broth with 10% v / v glycerol at -55 ° C.

Growth curves

Growth curves of each strain were obtained by inoculating 1% seed culture from the stock culture in 10 ml MRS or MRX, followed by a second inoculation: the pre-culture, from which 1% was inoculated into the growth tubes (13 x 150 mm) containing 10 ml MRS. Growth was monitored by measuring the optical density (OD) at 650 nm for several hours.

Preparation of cell-free extract

The cells were grown in MRS broth (400 ml), harvested in the late log phase by centrifugation at 7500 xg for 10 min at 40 C, washed in HEPES (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid ) with 10 mM CaCl 2 at pH = 7 and finally suspended in 10 ml HEPES.

For the second screening, the cells were grown in MRX. MRX is MRS supplemented with 15 mM CaCl2 and modified according to Hugenholtz et al. (15).

Cell extracts were obtained by sonicating the cells over a period of 10 min. (Vibra cell; Sonics & Materials Inc. Danbury, Connecticut, USA; 50% output, 20% pulse) at 10 min rest at 0 ° C. Cell-free extracts were obtained by centrifuging the cell mass for 10 min at 30,000 x g at 40 ° C.

Enzyme phagings

Proteolytic activity was measured with a wide variety of synthetic and chromogenic substrates as described for Lactococcus species (see Table 1).

The hydrolysis of the p-nitroanilide substrates was determined by the method of Exterkate (12). The enzyme mixtures contain 20 μΐ cell extract and 200 μΐ HEPES (pH 7).

The reactions were initiated by adding 200 μΐ of a 2 mM p-nitroanilide solution. Incubation was made at 30 ° C for several minutes.

Reactions were stopped by adding 100 μΐ 52% (v / v) acetic acid. The reaction mixtures were centrifuged at 1400 rpm for 5 min. The amount of p-nitroanilide in the supernatant was measured at 410 nm (U-2000 spectrophotometer, Hitachi). The concentration of the p-nitroanilide was calculated with the molar absorption coefficient (ε 8800 M ~ 1cm ~ 1).

In some cases, the release of p-nitroanilide was continuously measured.

The hydrolysis of the β-naphthylamide substrates was determined by a modified method according to Elleman (11). The enzyme mixture contained 20 μΐ cell extract and 200 μΐ HEPES 100 mM pH = 7.

The reactions were started by adding 200 μΐ of a 2 mM β-naphthylamide substrate solution in water and incubated at 30 ° C for several minutes. The reactions were stopped by adding 200 μΐ 1M acetate, pH = 4, which 1mg / ml Fast Garnet GBC ?? and 5% (w / v) Brij 35. The reaction mixture was allowed to reach optimal color development for 10 min and then centrifuged at 14000 rpm for 5 min. The amount of β-naphthylamide in the supernatant was calculated using the molar absorption coefficient (ε 20000 M ~ 1cm ~ 1).

The hydrolysis of non-chromogenic synthetic peptides was determined by measuring the amount of amino acids released by the modified cadmium-ninhydrin method, as described by Doi et al (8). The enzyme mixtures contained 100 μl of diluted cell extract in 100 mM HEPES pH 7. Reactions were initiated by adding 100 μl of a 2 mM peptide solution and incubated at 30 ° C for several minutes. Reactions were stopped using 1.5 ml Cd-ninhydrin reagent and subsequently incubated for several minutes at 84 ° C. After cooling, the mixtures were centrifuged at 14000 rpm and the absorbance was measured at 515 nm with a Vitalab 10 spectrophotometer (Vital scientific, Dieren).

The specific peptidase activity was defined as the amount of enzyme required to produce 1 μηιοί of L-Leucine equivalent amino acid from the substrate per minute under the experimental conditions (26).

The hydrolysis of resorufine-labeled casein (Boehringer Mannheim GmbH, Germany) was determined as described in the protocol given by the manufacturer, in 50 mM HEPES at 37 ° C.

All mixtures were brought to 30 ° C before the reactions were started.

The APIZYM determination

The APIZYM kits (LRA ZYM AP 1 through 6) were obtained from API SYSTEM (La Balme Les Grottes, France). Cell (free) extract 70 μΐ (65 or 125 mg protein / ml) was added to each of the cups containing dehydrated chromogenic enzyme substrate and the mixtures incubated at 37 ° C for two or four hours. The reactions were initiated by adding the enzyme mixtures which rehydrated the chromogenic substrates. Hydrolytic activity of the respective enzymes in the cell extracts on the β-naphtylamide substrate resulted in the release of the β-naphtylamide group. The reaction was stopped by adding 50 μΐ ZYM A reagent and the color was developed by adding 50 μΐ ZYM B. Activities were determined by comparing the color that developed in five minutes with the color chart provided by the manufacturer.

SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and preparative slab gel electrophoresis was performed with a Mini-Protean II and a Protean II xi (Bio-Rad Laboratories, Richmond, California, USA), respectively, and performed as described in the instructions provided by the manufacturer. The molecular sizes of the enzymes were estimated using pre-stained SDS-PAGE standards in the interval from 14,400 to 97,400 Da (Bio-Rad Laboratories, Richmond, California, USA).

Inrouno blotting

Proteins separated on a gel were transferred to sheets of polyvinylidene difluoride (PVDF) with a Mini Trans-Blott cell (Bio-Rad Laboratories, Richmond, California, USA).

Far. States) according to the manufacturer's instructions. The PVDF membranes were saturated with 1% skim milk and incubated with antibodies to various proteolytic enzymes: polyclonal and monoclonal antibodies at dilutions of 1/4000 to 1/8000 and 1/10, respectively. After washing with 10 mM Tris-HCl (pH 8.0) containing 150 mM NaCl and 0.05% Tween-20 (TBST), the membrane was incubated with alkaline phosphatase conjugated to goat anti-rabbit immunoglobulin serum or goat anti mouse (Bio-Rad Laboratories, Richmond, California, USA) diluted 1/3000. The PVDF membrane was washed with TBST and the enzyme-antibody complex was visualized by incubating the membrane in carbonate buffer (0.1 M NaHCO3, 1.0 mM MgCl2 (pH 9.8) with 1% (v / v) nitro blue tetrazolium (30 mg / ml) in 70% dimethylformamide and 1% (v / v) 5-bromo-4-chloro-3-indolylphosphate disodium salt (15 mg / ml) in 100% dimethylformamide. Department of Microbiology, of the University of Groningen (14, 18, 27).

Detection of peptidase activity on polvaclavlamide gel

Proteins in the cell-free extract were separated on preparative gel electrophoresis (7.5%). The gel was cut vertically into 7 mm strips and subsequently washed (2x) with 0.1 mM HEPES (pH 7) for 10 minutes. The strips were incubated in a 10 ml solution containing 1 mM β-naphthylamide substrate and diluted in 100 mM HEPES pH 7 for 2 to 60 minutes (depending on activity) at 37 °. The peptidase activity was visualized within 5 minutes after addition of a 10 ml Fast Garnet GBC solution (2 mg / ml water) (Sarath et al.). In the alternative, peptidase activity was monitored in the gel strips by the detection of leucine in a coupled enzyme reaction in which orthodianisidine is oxidized to a reddish brown color. The gel strips were incubated for 60 min at 37 ° C in an 8.4 ml solution containing 9 ml 100 mM HEPES (pH 7.5), 160 μu of a 50 mM Leu-Leu or Leu-Leu-Leu solution, 80 μΐ 23.3 mM ortho-dianisidine in 0.1 N hydrochloric acid, 80 μΐ 5000 units / ml peroxidase in 3.2 M ammonium sulfate and 80 μΐ 1 mg / ml L-amino acid oxidase in 3.2 M ammonium sulfate.

Z int u i gel i j k <s_ t e s.t.

A mixture containing 2 ml of 10% (w / v) of a tryptic degradation product of casein in 25 mM potassium phosphate pH 7 and 0.5 mg of protein from cell extract was incubated for 6 hours at 30 ° C. The bitterness of the casein product was evaluated organoleptically by a panel of 6 persons. No change in bitterness was termed bitter, residual bitterness was termed slightly debittered, and no bitter taste was termed completely debittered.

Partial purification of the. different peptidases

The conditions for purification on Mono-Q ion exchanger chromatography by FPLC were determined as follows: 4 mg of cell-free extract (100 μΐ) was applied to a Mono-Q HR 5/5 column (Pharmacia LKB, Uppsala, Sweden). Enzyme activity was eluted with a linear gradient from 0.15 to 0.5 M NaCl in 20 mM Tris hydrochloride (pH 8.0). The flow rate was 1 ml / min. and 1 ml fractions were collected and tested for peptidase activity with different (chromogenic) peptides.

Protein Beoaling

Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, Cal., United States) with bovine serum albumin as standard.

Chemicals and reagents

Unless otherwise stated, all amino acids and peptide derivatives used in this study contain the L anomers of amino acids. All chemicals were "reagent grade" and obtained from commercial sources.

RESULT1E.H

Screening with 178 Lactobacillus and Lactococcus strains

Cell-free extracts of 178 Lactobacillus and Lactococcus strains, divided into 13 different species, were tested for their proteolytic activity using the following substrates: Lys pNA and Arg pNA (for aminopeptidase in general), Glu-pNA (for glutamyl aminopeptidase ), Pro pNA (for prolydase), Ala-Pro pNA (for X-poly dipipididyl aminopeptidase) and Suc-Phe-pNa and MeO-Suc-Arg-Pro-Tyr pNA (for endopeptidase / proteinase).

In Figure 1, the mean proteolytic activities (indicated in the figure as black dots) for three major substrates (Lys pNA (Fig. IA), Ala-Pro pNA (Fig. 1B) and MeO-Suc-Arg-Pro Tyr-pNA (Fig. 1C)) shown for each species. Also shown are the maximum and minimum values. The results clearly show a wide variation of proteolytic activity for each species. High activities have been found, but also very low activities. The same was true for the assays with the other substrates (results not shown). The results also show that Lactobacillus helveticus has the highest mean activity with respect to all three chromogenic peptides. The same is true for other peptides, except Suc-Phe pNA (results not shown).

To determine the debittering properties of the Lactobacilli, experiments were performed with a bitter tryptic digest of casein (see material and method). Fig. 2 shows a number of strains capable of debittering a tryptic digest of casein within 6 hours (fully debittered, black bar; partially debittered, gray bar; and unbittered, white bar). In particular Lactobacillus helveticus strains (20 strains) and also a number of other strains (in particular Lactococcus species (2 strains) and Lactobacillus casei (5 strains)) show significant debittering activity.

Comparison between the proteolytic activity of Lactobacillus helveticus SBT 2171 and other lactic acid bacteria

Lactobacillus helveticus SBT 2171, 8 other highly proteolytic Lactobacillus strains and 2 Lactococcus strains have been further investigated for their peptidase and proteinase activity.

The results of the hydrolysing activity on 21 substrates are summarized in Table 2. The highest proteolytic activities are in bold. Lactobacillus helveticus SBT 2171 has the highest activity for 12 of the 21 substrates, including some interesting carboxypeptidase substrates. Furthermore, the SBT 2171 strain is also one of the highest active strains for a number of other substrates. In particular, the activities of peptidases are highest for Lactobacillus helveticus SBT 2171: Leu-Leu-Leu, Lys-pNA, Leu-Leu, Lys-pNA, Leu-Leu and MeO-Suc-Arg-Pro-Tyr-PNA ( S2586). Ala-Pro pNA and resorufin casein are also very well hydrolysed by this strain.

Gly-Phe-βΝΑ, Lys-Ser-4MPNA, Phe-Pro-Ala-βΝΑ, Pro-Arg-βΝΑ and Ser-Met-βΝΑ are also best hydrolyzed by Lactobacillus helveticus STB.

When comparing the proteolytic activity of Lactobacillus helveticus SBT 2171 with the activity of starter culture Lactococci (L. lactis ssp. Cremoris Wg2 and L. lactis ssp. Lactis SK112), some significant differences stand out. For example, Lactobacillus helveticus SBT 2171 shows a five- to seven-fold higher activity for Lys pNA, a more than 90-fold higher activity for Pro-pNA and an almost 10-fold higher activity for MeO-Suc-Arg-Pro- Tyr pNA. Furthermore, except Glu-Glu substrates, all other substrates are best hydrolyzed by strain SBT 2171.

The results clearly show that Lactobacillus helveticus SBT 2171 contains the highest proteolytic activity of all strains tested, including a number of Lactococci starter cultures.

Bittering experiments have been performed with a number of high proteolytic strains. The results show that all strains have some debittering activity, the highest of Lactobacillus helveticus SBT 2171 (Fig. 3).

Additional APIZYM experiments were performed with selected Lactobacilli and Lactococci. 59 substrates were examined for this experiment. Most strains show hydrolytic activity for a wide spectrum of substrates. Both APIZYM experiments show that Lactobacillus helveticus SBT 2171 has a hydrolytic activity for a wide spectrum of substrates (Table 3).

Detection of peptidase activity of Lactobacillus helveticus SBT 2171 on polyacrylamide gel (zymogram colorinai

Zymogram studies were performed to determine how many exopeptidases are present in Lactobacillus helveticus SBT 2171. Lactococcus lactis ssp. lactis Wg2 was used as the reference strain. Table 4 shows the Rp value of the hydrolysis activity, which were found on a 7.5% polyacrylamide gel after electrophoresis of cell-free extract of both strains with different substrates. Similar results were found for Lactococcus lactis ssp. lactis Wg2. However, the Rp values differ significantly from those of Lactobacillus helveticus SBT 2171. In addition, Lactococcus lactis ssp. cremoris Wg2 only 6 out of 11 substrates, while Lactobacillus helveticus SBT 2171 hydrolyses all substrates in the gel. These results indicate that different proteolytic enzymes are present in Lactobacillus helveticus SBT 2171 and Lactococcus lactis ssp. lactis Wg2.

SUIVEfilNg

The current experiments show a proteolytic activity of different Lactobacillus species. Comparative studies for peptidases have previously been performed for Lactobacillus casei subspecies (1, 2, 9). Only a few strains of Lactobacillus casei and a few enzyme activities have been described, while in the present experiments 178 strains of 12 Lactobacillus species have been compared for proteolytic activity. In addition, about 13 different peptidase and proteinase activities were measured for each strain using more than 20 different substrates and 60 additional substrates were used in a microenzyme APIZYM system. The results of this study clearly show that there is a wide variation in proteolytic activity in the different strains of Lactobacillus tested. Highly proteolytically active strains were present, but strains with hardly any activity were also found. In addition, the proteolytic activity of strains of the same species differs significantly (For example, most Lactobacillus helveticus strains are highly proteolytic, while Lactobacillus helveticus SBT 0351 shows little or no proteolytic activity (results not shown). It can be concluded that of the Lactobacillus tested and Lactococcus species, Lactobacillus helveticus is the most proteolytic species for a wide spectrum of substrates, but also the decrease in bitterness, as seen after incubation of the tryptic β casein digest with cell extracts of Lactobacillus helveticus, is significantly higher than with other lactic acid bacteria species. correlation between a specific peptidase activity and debittering activity can be made, this phenomenon is probably explained by a decrease in bitter peptides in the mixture.

Further study with a selection of a number of highly proteolytic Lactobacillus strains clearly shows that Lactobacillus helveticus SBT 2171 is the most proteolytic.

This strain has the highest proteolytic activity for: Lys-pNA, Arg-pNA, Glu-pNA, His-βΝΑ, Leu-Leu, Leu-Leu-Leu, propNA and Pro-Ala, MeO-Suc-Arg-Pro Tyr pNA, Bzl-Cys pNA, Z-Phe-Ala, Z-Ala-Ile and Hyp-Lys. In addition, this strain has measured very high activities for other substrates such as Ala-Pro pNA and resorufine casein. It appears that the general aminopeptidase of strain SBT 2171 is also capable of hydrolyzing Pro pNA. This fact was previously unknown to Lactobacillus helveticus.

It can be concluded that Lactobacillus helveticus SBT 2171 shows a higher proteolytic activity than the tested Lactococci strains. This is an interesting conclusion since most ripening cheeses (except Emmenthaler and Gruyère) (19) use mainly Lactococcus starter cultures, which implies a large contribution of these strains to proteolysis. Obviously, the development of a strain with superior proteolytic activities such as Lactobacillus helveticus will significantly influence cheese ripening. The high proteolytic activity of Lactobacillus helveticus SBT 2171 may be due to individual enzymes, but unknown expression or regulatory factors may also be involved.

Claims (14)

1. Lactobacillus Helveticus SBT 2171 or a mutant thereof, having the following properties; high proteolytic activity and debiting a tryptic hydrolyzate of casein in six hours. 2. Lactobacillus Helveticus SBT 2171 as deposited under the number CBS404.93. Use of a microorganism according to claim 1 or 2 in a fermentation process. Use according to claim 3, wherein the fermentation process is used in the ripening of cheese. A recombinant DNA molecule encoding at least a portion of a microorganism according to claim 1 or 2, of which recombinant DNA molecule hybridizes the encoding portion under stringent conditions with the DNA of the deposited microorganism number CBS404.93 . Use of a microorganism according to claim 1 or 2 in the proteolysis of biologically active peptides. A pharmaceutical formulation comprising a microorganism according to claim 1 or 2 and a pharmaceutically acceptable carrier. Proteolytic enzyme from a microorganism according to claim 1 or 2. Use of at least one enzyme according to claim 8 for the proteolysis of biologically active peptides. Pharmaceutical formulation comprising at least one enzyme according to claim 8 as well as a pharmaceutically acceptable carrier. A method of fermenting a milk-derived preparation, wherein a microorganism according to claim 1 or 2 is grown in culture in the presence of the preparation under normal growth conditions for Lactobacillus. The method of claim 11, wherein the composition is fresh cheese curd. Antibodies raised against one or more components of a micro-organism according to claim 1 or 2. Antibodies according to claim 13, which are specific for an organism according to claim 1 or 2. LITERATURE