Nisin Resistant Strains of Lactobacilli and Methods for Reducing the Post Acidification in Food Products

Abstract

The invention relates to nisin-resistant lactobacilli and their use for preparing food products with reduced post-acidification.

Claims

1-22. (canceled)

23. A mutant strain of Lactobacillus bulgaricus selected from the group consisting of the L. bulgaricus strain deposited as CNCM Deposit Number I-4452, CNCM Deposit Number I-4453 and CNCM Deposit Number I-4454.

24. A fermented dairy product comprising the mutant strain of claim 23, wherein the mutant strain is the L. bulgaricus strain deposited as CNCM Deposit Number I-4452.

25. A fermented dairy product comprising the mutant strain of claim 23, wherein the mutant strain is the L. bulgaricus strain deposited as CNCM Deposit Number I-4453.

26. A fermented dairy product comprising the mutant strain of claim 23, wherein the mutant strain is the L. bulgaricus strain deposited as CNCM Deposit Number I-4454.

27. A method of preserving a dairy product comprising administering the mutant strain of claim 23 to dairy product.

28. The method of claim 27, wherein the mutant strain in contained in a food matrix when it is administered to the dairy product.

29. The method of claim 27, wherein the dairy product is a fermented dairy product and the mutant strain is administered the dairy product either before, during or after fermentation of the dairy product.

30. The method of claim 29, further comprising storing the fermented dairy product at a temperature of between about 0 C. and 25 C. after administration of the mutant strain.

31. The method of claim 29, wherein the fermentation is carried out in the presence of at least one additional living bacterium.

32. The method of claim 31, wherein the at least one additional living bacterium is selected from the group consisting of Streptococcus spp. and Lactobacillus spp.

Description

FIGURES

[0055] This invention is illustrated by the following figures:

[0056] FIG. 1: FIG. 1 represents the kinetics of acidification of the strains CNCM I-2836 and clones I-4452 and I-4453. The ordinate corresponds to the pH and the abscissa corresponds to the time (in hours). The line in black corresponds to the strains L. bulgaricus CNCM I-4452. The line in gray corresponds to the strains L. bulgaricus CNCM I-4453. The line with circle gray (which gives a tick line gray due to the large number or experimental points) corresponds to the strains L. bulgaricus CNCM I-2836.

[0057] FIG. 2: FIG. 2 represents the measure of pH and acidity of 3 fresh dairy products (with I-2836, 1-4453 or I-4452) after 28 days of storage. The histogram represents the acidity measured at 28 days (in D) and the curve represents the pH measured at 28 days.

EXAMPLES

[0058] The present invention will be explained with reference to examples which are given for illustration only and are not intended for limiting the invention.

Example 1

Obtaining Nisin Resistant Lactobacilli

[0059] Mother strains, I-2836 and I-3435, chosen in that application are industrial strains used for fermented milk manufacturing showing significant post-acidification during storage.

[0060] 1) Evaluation of the Sensitivity of a Lactobacillus bulgaricus and L. helveticus to Nisin

[0061] To assess the nisin sensibility of the strain L. bulgaricus I-2836, and L. helveticus I-3435 the strains are grown in a rank of MRS tubes containing increasing amounts of nisin. After 16 h at 37 C., the optical density of each culture is measured.

[0062] 2) Obtaining Nisin Resistant Lactobacilli

[0063] Strains L. bulgaricus I-2836, and L. helveticus I-3435 were grown in MRS medium with an inhibitory concentration of nisine. After 16 h at 37 C., 10 ml of culture were centrifuged. Pellets were suspended in 100 l of peptone-saline solution and sprayed on agar MRS medium in petri dishes. A piece of blotting paper was placed in the middle of the plate and then soaked with 15 l of a nisine solution 2.5 mg/ml. After 72 h incubation at 37 C., resistant clones appeared in the inhibition zone around the disc of blotting paper. The clones which were the closest to the disk were recovered for analysis. Notably, their sensibility to nisin was tested in liquid medium using increasing amounts of nisin concentration.

[0064] All the clones obtained in particular I-4452 and I-4453 and I-4454 have a minimum inhibitory concentration of at least 6.25 g/ml M. This MIC confirms resistance of clones to nisin.

[0065] The clones I-4452 and I-4453 obtained were then tested by fermentation in milk. The kinetics of acidification provided information about the milk-acidifying capacity of these clones.

[0066] 3) Acidification Kinetics in Milk with Clones Derived from Strain I-2836

[0067] a) Fresh Dairy Product Preparation

[0068] The starter culture inoculum consists of: [0069] Strain I-4452 [0070] Strain I-4453 [0071] Strain I-2836

[0072] Strains were grown twice in MRS medium containing 6.25 g nisin/ml.

[0073] Starter were prepared in 100 ml sterilized milk (135 g/L milk powder and 2 g/L yeast extract). The inoculation rate was 2% and milks were incubated at 44 C. till a Dornic acidity of 70 was reached. The fermented milks were stored one night at 4 C.

[0074] Dairy product were prepared from 100. ml sterilized milk, inoculate with the above mixed culture. The cultures were grown at 44 C. until they reach a pH value of about 4.75. They were then cooled to 4 C. and stored at 4 C. during 28 days.

[0075] b) Follow Up of Acidification Kinetics

[0076] The acidification kinetic was followed continuously with a CINAC system (Ysebaert, France) which allows continuous measurement of pH values.

[0077] It can be seen from FIG. 1 that the acidification kinetics are slower for clones than for mother strain I-2836;

[0078] 4) Measure of pH and Acidity of Fresh Dairy Product After 28 Days of Storage

[0079] The measure was done on 3 fresh dairy products containing 2 clones derived from strain I-2836 (I-4452 and I-4453) and one containing the strain I-2836.

[0080] There acidity was measured after 28 days of storage at 4 C.

TABLE-US-00001 pH at the Acidity Acidity Fermentation end of the ( D) pH ( D) Strain time fermentation day 0 day 28 day 28 I-2836 3 h 25 4.74 77 D 4.43 116 D I-4453 8 h 4.75 75 D 4.55 104 D I-4452 8 h 25 4.75 76 D 4.62 99 D

[0081] These results are shown in FIG. 2.

[0082] The products with nisin resistant lactobacilli have showed only weak post acidification after 28 days.