Mutant strain of <i>Lactococcus lactis </i>and its application

Abstract

The present invention relates to a mutant strain of Lactococcus lactis and its application, and belongs to the field of food biotechnology. The mutant strain, Lactococcus lactis WH101, was screened for its better tolerance to harsh environmental factors, especially higher acid tolerance. The OD.sub.600 of Lactococcus lactis WH101 was increased by 5.5 times than that of the parent strain when cultured at pH 4.5. The survival rate of the mutant strain was 22.4 times higher than that of the parent strain after 3 hr treatment in pH 4.0 culture medium. The survival rate of the mutant strain was 5.2, 2.0 and 1.9 times over that of the parent strain treated with 15% ethanol for 4 hr, 15% NaCl for 6 hr and 1 mM H.sub.2O.sub.2 for 3 hr, respectively.

Claims

1. A mutant strain, named Lactococcus lactis WH101, with higher acid tolerance than that of its parent strain, which was deposited in the Chinese Type Culture Collection with the accession CCTCC NO: M 2016233.

2. The mutant strain of claim 1, wherein said mutant strain exhibits higher acid, ethanol, oxygen and osmotic pressure tolerance than that of its parent strain.

3. A method of for producing a fermentation food, comprising using the mutant strain of claim 1 as the fermentation strain to produce said fermentation food.

4. The method of claim 3 wherein said fermentation food is kimchi.

5. The method of claim 3 wherein said fermentation food is yoghurt.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1. Survival rates of different Lactococcus lactis strains cultured under different environmental stresses. Lactococcus lactis WH101 and NZ9000 strains were cultured under 15% ethanol for 4 hr (white bar), 15% NaCl for 6 hr (gray bar) and 1 mM H.sub.2O.sub.2 for 3 hr (black bar), respectively.

EXAMPLES

(2) Materials and Methods:

(3) Medium: GM17 medium: glucose 5.0 g/L, tryptone 5.0 g/L, soybean peptone 5.0 g/L, beef extract 5.0 g/L, yeast extract 2.5 g/L, vitamin C 0.5 g/L, magnesium sulfate 0.25 g/L, glycerophosphate disodium 19.0 g/L. The pH of GM17 medium was adjusted with 25% lactic acid.

Example 1. Breeding of Lactococcus lactis (CCTCC NO: M 2016233)

(4) Lactococcus lactis NZ9000 was used as the parent strain, which was cultured in GM17 medium until reaching the logarithmic growth phase. The concentration of bacterial solution was adjusted to 10.sup.7 CFU/ml, centrifuged at 5000 rpm for 10 min, washed and resuspended with 0.85% saline twice. An equal volume of GM17 medium containing 0.5% v/v diethyl sulfate (DES) was added to the L. lactis cells, and incubated at 30 C., 100 rpm for 30 min. The cells were immediately washed with 0.85% normal saline for 5 times, and resuspended in an equal volume of GM17 medium, and incubated at 30 C. for 1.5 hr.

(5) 1 ml GM17 (pH 5.0) medium was added to 96 deep well plates of 2.2 ml, and the L. lactis culture was inoculated into 96 deep well plates with the inoculation amount of 2%. The cells were incubated at 30 C. for 48 hours. The mutant strains were screened preliminarily according to the culture biomass, and then the mixed strains were diluted appropriately into pH 5.0 plates. The colonies were fermented at pH 5.0 and selected by the biomass. The OD.sub.600 of parent strain cultured at pH 5.0 was 0.076, and the OD.sub.600 of mutant strain Lactococcus lactis WH101 culture reached 0.612. The strain named Lactococcus lactis WH101 was deposited in the Chinese Type Culture Collection on Apr. 29, 2016 with the accession number CCTCC NO: M 2016233.

Example 2. Growth Performance of Lactococcus lactis (CCTCC NO: M 2016233) Under Acid Stresses

(6) Lactococcus lactis NZ9000 and Lactococcus lactis WH101, which were preserved in 80 C. glycerol tube, were inoculated into GM17 medium at a 2% inoculation rate and incubated at 30 C. for 12 hours.

(7) Lactococcus lactis NZ9000 and Lactococcus lactis WH101 were inoculated into GM17 medium with a 2% inoculation rate at pH 4.5 and were cultured at 30 C. for 48 hours. At the end of fermentation, the concentration of fermentation broth was determined. At pH 4.5, the OD.sub.600 of mutant strain culture was 0.527, which increased by 5.5 times than that of the parent strain. The results are shown in Table 1.

(8) TABLE-US-00001 TABLE 1 Growth performance of strains under acid stress Lactococcus Lactococcus Strain lactis NZ9000 lactis WH101 OD.sub.600 0.081 0.527

Example 3. Acid Tolerance Experiment

(9) Lactococcus lactis NZ9000 and Lactococcus lactis WH101, which were preserved in 80 C. glycerol tube, were inoculated into GM17 medium at a 2% inoculation rate and incubated at 30 C. for 12 hours.

(10) Lactococcus lactis NZ9000 and Lactococcus lactis WH101 were inoculated into GM17 medium at a 2% inoculation rate, respectively, and cultured at 30 C. until the logarithmic growth phase.

(11) The cells were harvested by centrifugation at 5000 rpm for 10 min. Then they were washed twice with 0.85% normal saline and resuspended in GM17 (pH 4.0) medium, the suspension was kept for 13 h. The cells were washed twice with the same physiological saline and resuspended in an equal volume of physiological saline. 100 l of the bacterial solution was applied to the plate after appropriate dilution and incubated at 30 C. for 24 hours to calculate the survival rate (Table 2). The survival rate of Lactococcus lactis WH101 was 4.1 times and 22.4 times than that of the parent strain after 2 hr and 3 hr under acid stress at pH 4.0, respectively.

(12) TABLE-US-00002 TABLE 2 Acid tolerance experiment Survival rate (%) Lactococcus Lactococcus Hours (h) lactis NZ9000 lactis WH101 0 100 100 1 65.08 74.34 2 0.99 4.04 3 0.05 1.11

Example 4. Other Environment Tolerance Experiments

(13) The cells of the logarithmic growth phase were obtained according to Example 3. For ethanol tolerance experiment, the cells were washed twice with 0.85% normal saline and resuspended in GM17 (15% ethanol v/v) medium, and the suspension was kept for 13 hr. For oxygen tolerance experiment, the cells were resuspended and kept in GM17 (1 mmol/L H.sub.2O.sub.2) medium for different times. Lastly, for osmotic pressure tolerance experiment, the cells were resuspended in GM17 (15% NaCl v/v) medium for different times. The survival rates of the two strains were calculated in these environmental tolerance experiments (FIG. 1). The survival rate of the mutant strain was 5.2, 2.0 and 1.9 times over that of the parent strain treated with 15% ethanol for 4 hr, 15% NaCl for 6 hr and 1 mM H.sub.2O.sub.2 for 3 hr, respectively.

(14) While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All figures, tables, appendices, patents, patent applications and publications, referred to above, are hereby incorporated by reference.