NEW LACTOBACILLUS CURVATUS STRAINS USEFUL FOR INHIBITION OF LISTERIA

Abstract

The Lactobacillus curvatus strain deposited as DSM18775 is used as a biopreservative culture in a wide range of meat products due to its production of bacteriocin. The present invention relates to Lactobacillus curvatus strains having an extended lag phase of at least 24 hours at 30° C. relative to DSM18775. In a presently preferred embodiment, the strains are mutants of DSM18775, such as the Lactobacillus curvatus strain deposited as DSM32590 and the Lactobacillus curvatus strain deposited as DSM 32591. Further, the invention relates to a method for inhibiting Listeria in a food product comprising adding bacteria of a Lactobacillus curvatus strain according to the invention to a food product in a concentration of at least 10.sup.5 CFU/g.

Claims

1. A mutant Lactobacillus curvatus strain which is a mutant of the Lactobacillus curvatus strain deposited at the Deutsche Sammlung von Mikroorganismen and Zellkulturen wherein (DSMZ) under accession number DSM 18775, wherein the strain has an extended lag phase of at least 24 hours at 30° C. relative to DSM 18775.

2. A Lactobacillus curvatus strain according to claim 1 wherein the extended lag phase is at least 48 hours at 30° C. relative to DSM 18775.

3. (canceled)

4. A mutant Lactobacillus curvatus strain according to claim 1, deposited at the DSMZ under accession number DSM 32590.

5. A Lactobacillus curvatus strain according to claim 1, deposited at the DSMZ under accession number DSM 32591.

6. A composition comprising bacteria of a mutant Lactobacillus curvatus strain according to claim 1.

7. The composition according to claim 6, comprising bacteria of the mutant Lactobacillus curvatus strain DSM 32590 and bacteria of the mutant Lactobacillus curvatus strain DSM 32591.

8. The composition according to claim 6, wherein the mutant Lactobacillus curvatus strain is present in the composition at a concentration of at least 10.sup.5 CFU/g.

9. The composition according to claim 6, wherein the bacteria of the mutant Lactobacillus curvatus strain are the only bacteria present in the composition.

10. The composition according to claim 6, wherein the composition further comprises bacteria of one or more species selected from Pediococcus acidilactici, Lactobacillus sakei, Lactococcus lactis, Leuconostoc carnosum, and Staphylococcus carnosus.

11. The composition according to claim 10, wherein the bacteria of one or more of the species selected from Pediococcus acidilactici, Lactobacillus sakei, Lactococcus lactis, Leuconostoc carnosum, and Staphylococcus carnosus is selected from Pediococcus acidilactici deposited at the DSMZ under accession number DSM 28307, Lactobacillus sakei deposited at the DSMZ under accession number DSM 14022, Lactococcus lactis deposited at the DSMZ under accession number DSM 11037, and Leuconostoc carnosum LC1043.

12. A method for inhibiting the amount of spoilage or pathogenic bacteria in a food product, comprising adding bacteria of a mutant Lactobacillus curvatus strain according to claim 1 to a food product at a concentration of at least 10.sup.5 CFU/g.

13. The method according to claim 12, wherein the pathogenic bacteria is Listeria.

14. A method for inhibiting the amount of spoilage or pathogenic bacteria in a food product, comprising adding a composition according to claim 6 to a food product in an amount to provide the mutant Lactobacillus curvatus strain at a concentration of at least 10.sup.5 CFU/g.

15. The method according to claim 14, wherein the pathogenic bacteria is Listeria.

Description

LEGEND TO FIGURES

[0058] FIG. 1

[0059] Growth performance of TpG3

[0060] Right plate, TpG3 after 24 hours incubation at 30° C.; left plate, DSM 18775 incubated under the same conditions. Inoculation was performed with equal amount of cells (200 μl: 0.25 OD.sub.600; Petri dish diameter 14 cm).

[0061] FIG. 2

[0062] pH evolution of the cooked ham samples (n=3) during storage at +7° C.

[0063] The y-axis shows the pH. A pH drop of 0.3 units is generally considered sensorially acceptable.

[0064] FIG. 3

[0065] Total cell count in the cooked ham samples (n=3) during storage at +7° C.

[0066] The y-axis shows the total cell count in cfu/g. The detection limit is 1.0E+02 cfu/g.

[0067] FIG. 4

[0068] Lactic acid bacteria cell count in the cooked ham samples (n=3) during storage at +7° C.

[0069] The y-axis shows the lactic acid bacteria cell count in cfu/g. The detection limit is 1.0E+02 cfu/g.

[0070] FIG. 5

[0071] Listeria spp. cell count in the cooked ham samples (n=3) during storage at +7° C.

[0072] The y-axis shows the Listeria spp. cell count in cfu/g. The detection limit is 1.0E+02 cfu/g.

DEPOSIT and EXPERT SOLUTION

[0073] The Lactobacillus curvatus strain TpG3 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) under the accession number DSM 32590 with a deposit date of Aug. 16, 2017 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0074] The Lactobacillus curvatus strain TpG57 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) under the accession number DSM 32591 with a deposit date of Aug. 16, 2017 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0075] The Pediococcus acidilactici strain HP has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) under the accession number DSM 28307 with a deposit date of Jan. 30, 2014 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0076] The Lactobacillus sakei strain BJ-33 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) under the accession number DSM 14022 with a deposit date of Jan. 31, 2001 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0077] For the above-identified deposited microorganisms, the following additional indications apply:

[0078] As regards the respective Patent Offices of the respective designated states, the applicants request that a sample of the deposited microorganisms stated above only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn.

[0079] The Lactobacillus curvatus strain DSM 18775 is referred to in European patent EP2132297.

[0080] The Lactococcus lactis strain DSM 11037 is referred to in granted European patent EP928333.

[0081] The Pediococcus acidilactici strain DSM 10313 is referred to in European patent EP1716258.

[0082] The Leuconostoc carnosum LC1043 is available from the Danish Meat Research Institute or commercially available from Chr. Hansen A/S (Denmark).

[0083] The Listeria innocua strain Seeliger has been deposited as ATCC 33090.

[0084] The Micrococcus luteus strain has been deposited as DSM 1790.

[0085] Embodiments of the present invention are described below, by way of non-limiting examples.

EXAMPLES

[0086] Materials and Methods:

[0087] ALOA agar (Oxoid)

[0088] Lactobacilli MRS Broth™ (Difco)

[0089] Lactobacilli MRS Agar Agar™ (Difco)

[0090] Brain Heart Infusion (BHI) agar (Oxoid CM375)

[0091] BHI medium, Merck

[0092] Bacto™ Heart Infusion broth (BD) Difco

[0093] Microtitre plates (MTP) NUNC, Denmark

[0094] M17 agar (Oxoid)

[0095] MRS agar (Oxoid)

[0096] MRS medium (Merck)

Example 1

[0097] Inducing Mutants of Lactobacillus curvatus DSM 18775 Having an Extended Lag Phase at 30° C.

[0098] Adaptive Laboratory Evolution (ALE) of DSM 18775

[0099] For the ALE experiment, Lactobacillus curvatus cells were grown in MRS broth (10 ml) and incubated for 2×24 hours at 15° C. at which time a transfer (2%) was made to fresh MRS broth and continuous incubation at the permissive temperature 15° C. This cell transfer was repeated every 48 hours over a period of 8 weeks. Every 2.sup.nd week along the incubation period, a sample of the cells was subjected to UV-irradiation to speed up putative temperature sensitive (TS) mutants. Instead of the 2% transfer, an aliquot of 7 ml of exponentially growing cells were diluted to 0.25 OD.sub.600 in a Petri dish. UV mutagenesis was performed by exposing the cell layer to UV at 70 mJ/cm.sup.2 for 10 min. One ml of the UV-treated cells was then transferred to 10 ml fresh MRS broth and continuously incubated at 15° C. till next transfer. At the end of the eight weeks period a sample was removed and screened for TS variants as described below.

[0100] Screening for Temperature-Sensitive Mutants of DSM 18775

[0101] Screening for temperature-sensitive (TS) mutants was carried out by plating aliquots of TS cells on MRS agar in an appropriate dilution to give approximately 150 to 200 colonies per plate after incubation at 15° C. for 3×24 hours. Single colonies were then transferred to individual wells of MTP (96 well plates) containing 100 μl fresh MRS broth and incubated for another 3×24 hours at 15° C. TS colonies were identified by replica plating into another MTP with 100 μl MRS broth and incubated aerobically for 24 hours at 30° C. at which temperature TS mutants are identified as showing growth with a longer lag phase than wild type. Screening for TS mutants was also performed by replica plating individual colonies from MTP onto MRS agar followed by aerobically incubation for 24 hours at 30° C. Finally, the TS colonies were cultured in MRS broth at 20° C.

[0102] Results

[0103] After eight weeks of transfer of Lactobacillus curvatus at 15° C. and four times UV-treatments approximately 4,000 mutants were screened for their inability to grow at 30° C. One such mutant, TpG3, demonstrated a reduced growth rate compared to the mother strain over a period of 24 hours at 30° C. The same reduced growth rate was observed by re-testing the putative TS strain in a bigger volume of 10 ml MRS broth over a period of 72 hours at 30° C. On MRS agar plates, no growth at all was observed after 24 hours incubation, whereas good growth was observed for DSM 18775 (FIG. 1).

Example 2

[0104] Selection of Further Mutants of Lactobacillus curvatus DSM 18775 Having an Extended Lag Phase at 30° C.

[0105] Second Generation Mutants Derived from TpG3

[0106] To further extend the lag phase the Lactobacillus curvatus DSM 18775 mutant TpG3 was selected and subjected to continuously ALE at 15° C. over a period of 2 weeks with cell transfers (2%) every other day in addition to one UV-mutagenic treatment as described under Example 1. Out of approximately 500 colonies screened, only 2 mutants were isolated showing deferred growth at 30° C. with an extended lag phase of more than 48 hours relative to the mother strain, DSM 18775. It was decided to continue with one of the two mutants, TpG57 (DSM 32591).

Example 3

[0107] Detection of the Antimicrobial Product of Mutants Having an Extended Lag Phase at 30° C.

[0108] Bacteriocin Activity Test

[0109] The bacteriocin sensitive indicator strain, Micrococcus luteus DSM 1790, was amplified by incubation at 30° C. in BHI (BD) broth under vigorous shaking (125 rpm). To test for production of bacteriocins, a 2-fold dilution series of the supernatants of pre-cultures (spent medium) was made in MTP and an aliquot of these dilutions (50 μl) was applied to the indicator strain, Micrococcus luteus DSM 1790. The optical density [OD] at 600 nm was recorded every 30 minutes over a period of 20 hours at 30° C. Activity is reported in arbitrary units (AU/ml) (Table 1), and defined as the reciprocal of the highest two-fold dilution showing 50% growth inhibition of the sensitive strain.

[0110] Results

[0111] The results provided in Table 1 show the inhibition of the bacteriocin-sensitive strain, Micrococcus luteus (DSM1790), with spent medium from Lactobacillus curvatus strains DSM 32590 and DSM 18775 and by positive control Pediococcus acidilactici DSM 10313 grown for 20 hours at 20° C. OD.sub.600 is the cell density of Micrococcus after incubation with various dilutions of spent medium (bacteriocin).

TABLE-US-00001 TABLE 1 Dilution factor OD.sub.600 after 20 hours growth of spent medium DSM 32590 DSM 18775 DSM 10313 Undiluted 0 0 0 ½ 0 0.216 0 ¼ 0 0.483 0 ⅛ 0 0.612 0 1/16 0 0.713 0 1/32 0 0.734 0 1/64 0 0.744 0 1/128 0.256 0.744 0.309 1/256 0.554 0.743 0.679 1/512 0.677 0.732 0.703 1/1024 0.677 0.732 0.707 No Bacteriocin 0.698 0.732 0.698 Activity of the 2560 40 2560 sample (AU/ml)

[0112] The assay for production of bacteriocin demonstrated that the TS strain produced substantially more bacteriocin than the mother strain DSM 18775 over a period of 20 hours at 20° C. For DSM 32590 and DSM 10313 (a bacteriocin positive control strain) 50% inhibition was recorded at 1/128-fold dilution over a “no bacteriocin” sample without spent medium added to the Micrococcus luteus DSM 1790 inoculate. In Table 1 DSM 18775 shows 50% inhibition at ½-fold dilution with a total of 40 AU/ml [2/0.05]. The AU/ml for TpG3 was recorded to 2560 AU/ml [128/0.05]. Thus the activity for TpG3 is recorded 64-fold higher than the mother strain [AU-TpG3/AU-DSM 18775=2560/40=64].

Example 4

[0113] Challenge Test of TpG3 and TpG57 on Cooked Ham

[0114] TpG3 and TpG57 were tested to check both strains in regard to their ability to inhibit Listeria spp. as well as sensorial attributes and benefit on the shelf-life of cooked ham.

[0115] Preparation of the Listeria innocua Pre-Culture

[0116] The L. innocua strain which was used for the preparation of the spraying culture for inoculation was stored at −50° C. 3 days before the start of the challenge test one cryobead of the L. innocua strain was put into a tube which contained 9 ml BHI medium and was incubated at 37° C. overnight. The following day 0.1 ml of the overnight culture was transferred into 9 ml fresh BHI medium and incubated at 37° C. overnight. The following morning the pre-culture was stored at 4° C. until use. In the meantime, the L. innocua cell count of the final pre-culture was enumerated on ALOA agar. The plate was incubated at 37° C. overnight and enumerated for calculation of the cell count of the spraying solution to be used.

[0117] Preparation of the Lactobacillus curvatus Pre-Cultures

[0118] Pre-cultures of TpG3 and TpG57, respectively, were prepared in 150 ml MRS medium and incubated at 30° C. overnight (without shaking). The active cell count of the overnight cultures was enumerated with a flow-cytometer and an appropriate dilution made in salt-peptone solution in order to obtain a spraying solution.

[0119] Samples

[0120] 100 g of sliced cooked ham was packed into a vacuum pouch for preparation of a sample. All pouches were inoculated by spraying on the surface of the cooked ham slices with 100 cfu/g of Listeria innocua and with one of the test strains (except for the control batch) in a cell count of 1.0E+07 cfu/g or 1.0E+05 cfu/g as follows:

[0121] Batch 1—Control

[0122] Batch 2—Lactobacillus curvatus DSM 18775 (1.0E+07 cfu/g)

[0123] Batch 3—TpG3 (1.0E+07 cfu/g)

[0124] Batch 4—TpG57 (1.0E+07 cfu/g)

[0125] Batch 5—TpG3 (1.0E+05 cfu/g)

[0126] Batch 6—TpG57 (1.0E+05 cfu/g)

[0127] Then the vacuum pouches were packed under modified atmosphere (70% N.sub.2/30% CO.sub.2) and stored at +7° C. for at least 28 days.

[0128] Analyses

[0129] Listeria spp. enumeration and pH measurement of each batch were carried out each week in triplicate. Both total cell count enumeration as well as lactic acid bacteria enumeration was carried out each second week also in triplicate. Cell count of the test strains was enumerated on MRS agar and the Listeria cell count was enumerated on ALOA agar in accordance with the corresponding ISO methods (Total plate count: ISO 4833-2:2013, Lactic acid bacteria: 15015214:1998, Listeria spp.: ISO 11290-2:2017). In order to measure the pH-value of the cooked ham the sample material was grinded and homogenized with a household mixer (Moulinette from Moulinex). Furthermore, a sensorial evaluation with focus on smell and taste of the samples was done every week in addition and to support the microbiological analyses.

[0130] Results

[0131] The results of the pH evolution of the cooked ham samples, the total cell count, the lactic acid bacteria cell count and the Listeria spp. cell count are provided in FIGS. 3 to 6.

[0132] The results of the sensorial assessment of the cooked ham samples (n=2) during storage at +7° C. is provided in Table 2.

TABLE-US-00002 TABLE 2 Day 0 Day 7 Day 14 Day 21 Day 28 Batch Strain Taste Smell/Odor Taste Smell/Odor Taste Smell/Odor Taste Smell/Odor Taste Smell/Odor 1 Control ok ok ok Ok still ok 1 ok/ old Old old old 2 bad 2 DSM18775 ok ok ok Ok still ok slightly slightly still ok sour sour sour sour 3 TpG3 Ok ok ok ok/more ok Ok ok slightly sour sour (1.0E+07 cfu/g) intensive sour 4 TpG57 Ok ok ok slightly ok not good bitter slightly slightly slightly (1.0E+07 cfu/g) sour sour/fresh sour/dry sour/fresh 5 TpG3 Ok ok ok/ Ok ok ok/ taste+/ Ok slightly ok (1.0E+05 cfu/g) less taste more taste juicy sour/fresh 6 TpG57 Ok ok slightly Ok ok Ok ok ok/slightly slightly slightly (1.0E+05 cfu/g) sour sour sour sour

CONCLUSION

[0133] In all samples where TpG3 or TpG57 were applied, the flora of lactic acid bacteria was homogeneous and the corresponding strain was identified by its morphology with high probability. All batches reached a cell count of 1.0E+08-1.0E+09 cfu/g after 28 days at 7° C., also the batches with an inoculation level of 1.0E+05 cfu/g as well as the control batch. In regard to the inhibition effect on Listeria spp. no significant differences between the batches were observed. It is noteworthy that the batches with a lower inoculation level of 1.0E+05 cfu/g show an equal inhibition effect on the growth of Listeria spp. as the batches with an inoculation cell count of 1.0E+07 cfu/g. The Listeria spp. cell count of all batches to which TpG3 or TpG57 was applied stayed around the detection limit of 1.0E+02 cfu/g during the whole challenge test, whereas the Listeria spp. cell count of the control batch increased to approx. 1.0-5.0E+06 cfu/g after 28 days (FIG. 5).

[0134] The pH drop of the batches with the lower inoculation level of 1.0E+05 cfu/g was slower compared to the batches with the high inoculation cell count of 1.0E+07 cfu/g and reached a slightly higher pH-value after 28 days. Maybe due to the slower acidification and the slightly higher final pH-value of the batches inoculated with 1.0E+05 cfu/g, only less sensorial deviation respectively negative impact on sensorial characteristics of the product was observed during the whole challenge test (FIG. 2). Sensory-wise the TpG3 batch with an initial cell count of 1.0E+05 cfu/g gave the best results followed by the batch of TpG57 with an initial cell count of 1.0E+05 cfu/g and also the batch with TpG3 with a cell count of 1.0E+07 cfu/g was sensorially acceptable till day 28, the end of the study (Table 2).

[0135] Summarizing, when applying the mutants with an initial cell count of 1.0E+05 cfu/g the pH drop of the batches was slower and reached a slightly higher pH-value after 28 days which resulted in less negative impact on the sensorial characteristics of the product. These batches were sensory-wise much better than the ones with a higher inoculation cell count as well as both the batch inoculated with DSM 18775 and the control batch and nonetheless the inhibition effect on the growth of Listeria spp. was as good as for the batches inoculated with 1.0E+07 cfu/g.