<i>Lactococcus lactis </i>subsp. <i>lactis </i>CCFM1018 and application thereof in preparation of food and medicine for excreting plasticizer

Abstract

The disclosure discloses Lactococcus lactis subsp. lactis CCFM1018 and application thereof in preparation of food and medicine for excreting a plasticizer, and belongs to the technical field of microorganisms. The Lactococcus lactis subsp. lactis CCFM1018 not only is significantly better than the intestinal resident bacteria Escherichia coli and Enterococcus faecalis in terms of the effect of promoting the excretion of DEHP and MEHP, but also is better than the commercial strain Lactobacillus rhamnosus LGG. Therefore, the Lactococcus lactis subsp. lactis CCFM1018 of the disclosure can be used as an effective means to prevent and alleviate body damage caused by DEHP and MEHP, and does not have toxic or side effects of drugs. Lactococcus lactis subsp. lactis CCFM1018 can be used to prepare pharmaceutical compositions and fermented food for alleviating and preventing the toxicity of DEHP and metabolites thereof, and has a very broad application prospect.

Claims

1. A microbial composition, the composition comprising live Lactococcus lactis subsp. lactis CCFM1018 cells deposited on Feb. 11, 2018, at the Guangdong Microbial Culture Collection Center, Guangdong Institute of Microbiology, under the GDMCC number 60332, wherein: (a) the composition is a fermented food product prepared by fermentation of the Lactococcus lactis subsp. lactis CCFM1018 cells, wherein the fermented food product comprises dairy products, bean products, or fruit and vegetable products, wherein the dairy products comprise milk, sour cream, or cheese; and wherein the fruit and the vegetable products comprise cucumber, carrot, beet, celery, or cabbage, or (b) the composition is a microbial preparation in which the Lactococcus lactis subsp. lactis CCFM1018 cells are fermented, collected, mixed with a cytoprotective agent, and freeze-dried to obtain a freeze-dried preparation containing live cells of the Lactococcus lactis subsp. lactis CCFM1018.

2. The composition of claim 1, wherein the composition (a) is a liquid preparation.

3. The composition of claim 2, wherein the composition further comprises a sucrose solution.

4. A method of preparing a fermented product, the method comprising adding live Lactococcus lactis subsp. lactis CCFM1018 cells to fermentation raw materials to ferment for a period of time to obtain the fermented product.

5. The method of claim 4, wherein the fermented product is a food.

6. The method of claim 4, wherein the fermented product is a fermented beverage.

7. The method of claim 6, wherein the fermented beverage is a fermented dairy product prepared by inoculating milk with the live Lactococcus lactis subsp. lactis CCFM1018 cells and fermenting the milk at 30° C.-37° C. for at least 4 hours.

8. The method of claim 6, wherein the fermented beverage is a fermented fruit and vegetable product prepared by inoculating a fruit and a vegetable paste, ora fruit and a vegetable juice with the live Lactococcus lactis subsp. lactis CCFM1018 cells for fermentation.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1 is a schematic diagram showing comparison in the adsorption capacity of DEHP and MEHP in vitro by the strain of the present application and control strains Lactobacillus rhamnosus GG (LGG), Escherichia coli and Enterococcus faecalis.

(2) FIG. 2 is a schematic diagram of influence of the strain of the present application on the DEHP and MEHP content in serum and feces.

(3) FIG. 3 is a schematic diagram of improvement of the strain of the present application on the level of testosterone in the serum of a DEHP-exposed model rat.

(4) FIG. 4 is a schematic diagram of influence of the strain of the present application on the content of malondialdehyde (MDA) in the testis of a DEHP-exposed model rat.

(5) FIG. 5 is a schematic diagram of improvement of the strain of the present application on tissue damage of the testis of a DEHP-exposed model rat.

(6) FIG. 6 is a schematic diagram of influence of the strain of the present application on SOD activity in the liver of a DEHP-exposed model rat.

DETAILED DESCRIPTION

(7) Lactococcus lactis subsp. lactis CCFM1018 of the disclosure was deposited on Feb. 11, 2018 at the Guangdong Microbial Culture Collection Center, Guangdong Institute of Microbiology, the address is 5.sup.th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC No. 60332.

(8) The Lactococcus lactis Subsp. Lactis CCFM1018 has the Following Biological Characteristics:

(9) (1) Bacterial characteristics: The bacterium is gram-positive, non-spore forming and non-moving, has oval bacterial cells, and appears in pairs and clusters.

(10) (2) Colony characteristics: Obvious colonies are formed on a culture medium after 36 h of cultivation. The colonies are small, round on the front and protruding on the side, have neat edges, are milky white and opaque, are moist and smooth on the surface, and produce no pigment.

(11) (3) Growth characteristics: The bacterium is a facultative anaerobic bacterium, the optimum growth temperature is 30° C., the growth is good at 32-38° C., and the growth is slow at 45° C. or above. The optimum initial pH is 6-7. The bacterium grows well in a culture solution containing glucose.

(12) (4) The bacterium has good tolerance to artificial simulated gastrointestinal fluid.

(13) (5) The bacterium can alleviate the damage of DEHP and the metabolite MEHP to the testis.

(14) (6) The bacterium can significantly increase the excretion of DEHP and MEHP in feces.

(15) (7) The bacterium can significantly reduce the levels of DEHP and MEHP in serum, and reduce the accumulation of DEHP and MEHP in the body.

(16) (8) The bacterium can significantly improve the activity of superoxide dismutase (SOD) in the liver.

(17) Extraction Method of the Strain:

(18) (I) Isolation and Screening of Lactic Acid Bacteria, Including the Following Steps:

(19) (1) collecting a plurality of fermented vegetable samples, and enriching the samples in GM17 culture media containing sorbitol at 30° C. for 12 h;

(20) (2) carrying out gradient dilution on the enriched samples, spreading the diluted samples on GM17 solid plates supplemented with 0.02% bromcresol purple, and performing culturing for 24-48 h;

(21) (3) selecting individual colonies with obvious color-changing zones and conforming to the basic morphology of lactic acid bacteria, performing plate streaking purification, and screening and isolating lactic acid bacteria; and

(22) (4) culturing the above individual colonies in liquid GM17 culture solutions for 24 h, then performing gram staining, and selecting gram-positive bacteria for performing subsequent experiments.

(23) (II) Preliminary Identification of Lactococcus lactis: Calcium-Dissolving Zone Determination Method, Including the Following Steps:

(24) (1) culturing the lactic acid bacteria screened in step (I) in a liquid sorbitol GM17 culture solution for culturing the lactic acid bacteria for 24 h, then taking 1 mL of the culture and centrifuging the culture at 8,000×g for 2 min;

(25) (2) washing bacterial cells twice with a 0.05 M KH.sub.2PO.sub.4 solution;

(26) (3) resuspending the obtained bacterial paste, streaking the bacterial paste on a sorbitol GM17-0.75% CaCO.sub.3 solid culture medium, and culturing the bacteria for 24 h; and

(27) (4) selecting colonies with obvious calcium-dissolving zones and being convex, round, fine and white without mycelia, and after gram staining, preliminarily judging the bacteria as Lactococcus if the bacterial cells are spherical by microscopic observation.

(28) (III) Molecular Biological Identification of Lactococcus lactis Subsp. Lactis, Including the Following Steps:

(29) (1) Performing Extraction of a Genome of a Single Bacterium:

(30) A. culturing the lactic acid bacteria screened in step (II) overnight, taking 1 mL of the bacterial suspension cultured overnight in a 1.5 mL centrifuge tube, centrifuging the bacterial suspension at 8,000×g for 2 min, and discarding the supernatant to obtain the bacterial cells;

(31) B. after purging the bacterial cells with 1 mL of sterile water, centrifuging the bacterial cells at 8,000×g for 2 min, and discarding the supernatant to obtain the bacterial cells;

(32) C. adding 200 μL of SDS lysate and placing the mixed solution in a water bath at 80° C. for 30 min;

(33) D. adding 200 μL of phenol-chloroform solution to the bacterial cell lysate, wherein the compositions in volume ratio of the phenol-chloroform solution are Tris saturated phenol:chloroform:isoamyl alcohol=25:24:1; after mixing by inversion, performing centrifuging at 12,000×g for 5-10 min, and taking 200 μL of supernatant;

(34) E. adding 400 μL of absolute alcohol or absolute isopropanol to the 200 μL of supernatant, allowing the mixed solution to stand for 1 h at −20° C., performing centrifuging at 12,000×g for 5-10 min, and discarding the supernatant;

(35) F. adding 500 μL of 70% (volume percentage) absolute alcohol to resuspend the precipitate, performing centrifuging at 12,000×g for 1-3 min, and discarding the supernatant;

(36) G. drying the precipitate in an oven at 60° C., or drying the precipitate naturally; and

(37) H. re-dissolving the precipitate with 50 μL of ddH.sub.2O for PCR;

(38) (2) Performing 16S rDNA PCR:

(39) A. bacterial 16S rDNA 50 μL PCR reaction system:

(40) 10×Taq buffer, 5 μL; dNTP, 5 μL; 27 F, 0.5 μL; 1492R, 0.5 μL; Taq enzyme, 0.5 μL; template, 0.5 μL; ddH.sub.2O, 38 μL.

(41) B. PCR Conditions:

(42) 95° C. 5 min; 95° C. 10 s; 55° C. 30 s; 72° C. 30 s; step 2-4 30×; 72° C. 5 min; 12° C. 2 min;

(43) (3) preparing a 1% agarose gel, then mixing the PCR product with 10× loading buffer, performing loading at an amount of 5 μL, performing running at 120 V for 30 min, and then performing gel imaging; and

(44) (4) performing sequencing analysis on the PCR product of 16S rDNA, using BLAST to search and compare the similarity in GeneBank with the sequence results, selecting a new Lactobacillus strain with the sequencing result identified as belonging to Lactococcus lactis subsp. lactis, and storing the new Lactobacillus strain at −80° C. for later use.

Example 1: Tolerance of Lactococcus lactis Subsp. Lactis CCFM1018 to Simulated Gastrointestinal Fluid

(45) A solid culture medium was inoculated with cryopreserved Lactococcus lactis subsp. lactis CCFM1018 by streaking, and culturing was performed statically and aerobically for 24 h at a temperature of 30° C. After subculturing 2-3 times in a liquid culture solution, the Lactococcus lactis subsp. lactis CCFM1018 culture solution was taken and centrifuged at 8,000×g for 5 min, and bacterial cells were collected. According to the ratio of 1:1 (m/v), the bacterial cells were resuspended in artificial simulated gastric juice with the pH of 2.5 (an MRS culture medium containing 1% pepsin, pH=2.5), and the solution was mixed and then aerobically cultured at 37° C. Samples were taken at the beginning (0 h), 1 h, 2 h and 3 h respectively. Plate colony counting was performed by pouring culture using an MRS agar culture medium, the number of viable bacteria was determined, and the survival rate was calculated. The survival rate is the ratio of the number of viable bacteria in the culture solution to the number of viable bacteria at 0 h, expressed in %.

(46) The Lactococcus lactis subsp. lactis CCFM1018 culture solution was taken and centrifuged at 8,000×g for 5 min, and bacterial cells were collected. The bacterial cells (1:1) were resuspended in artificial simulated intestinal juice (an MRS culture medium containing 0.3% bovine bile salt and 1% trypsin, pH=8.0), and then aerobically cultured at 37° C. Samples were taken at the beginning (0 h), 1 h, 2 h, 3 h and 4 h respectively. Plate colony counting was performed by pouring culture using an MRS agar culture medium, the number of viable bacteria was determined, and the survival rate was calculated. The survival rate is the ratio of the number of viable bacteria during sampling in the culture solution to the number of viable bacteria at 0 h, expressed in %. The experimental results are shown in Table 1 and Table 2. It can be seen that Lactococcus lactis subsp. lactis CCFM1018 has good tolerance to artificial gastric juice and intestinal juice.

(47) TABLE-US-00001 TABLE 1 Tolerance of Lactococcus lactis subsp, lactis CCFM1018 in artificial simulated gastric juice Artificial simulated gastric juice Treatment time (h) 1 2 3 Survival rate (%) 93.27 ± 2.04 90.88 ± 3.25 85.39 ± 4.14

(48) TABLE-US-00002 TABLE 2 Tolerance of Lactococcus lactis subsp, lactis CCFM1018 in artificial simulated intestinal juice Artificial simulated intestinal juice Treatment time (h) 1 2 3 4 Survival rate (%) 89.65 ± 6.29 82.55 ± 5.58 70.32 ± 5.57 60.67 ± 6.36

Example 2: Lactococcus lactis Subsp. Lactis CCFM1018 has Good Adsorption Capacity for DEHP and MEHP in an Aqueous Solution Containing Plasticizer DEHP or MEHP In Vitro

(49) Adsorption of bacterial cells: After purification and activation culture of experimental bacteria, MRS liquid culture media were inoculated with the bacteria according to an inoculum concentration of 1% (v/v), and culturing was performed at 30° C. for 20 h (E. coli was cultured in an LB culture medium at 37° C. with shaking; E. faecalis was cultured at 37° C. in an MRS liquid culture medium). Then the culture solutions were centrifuged at 8,000×g for 20 min, and the supernatants were discarded. After resuspension with ultrapure water, centrifugation was performed at 8,000×g for 20 min, and the supernatants were discarded to obtain viable bacterial cells, namely wet bacterial cells. The wet bacterial cells were resuspended in a 50 mg/L DEHP or 10 mg/L MEHP aqueous solution, and the final bacterial cell concentration reached 1 g wet bacterial cells/L. As a blank control, wet bacterial cells were resuspended in ultrapure water without DEHP and MEHP. The volume of each group is 1 mL. The mixed solutions were incubated with shaking at 37° C. for 4 h, and then centrifugation was performed at 3,500×g for 10 min. After the supernatants were collected and passed through a 0.22 μm filter membrane, the content of DEHP or MEHP in the filtrate was determined by UPLC-MS, and the average value was taken for 3 parallel experiments.

(50) Determination of DEHP and MEHP adsorption capacity: The content of remaining DEHP or MEHP in filtrates was determined by UPLC-MS of a Waters EYNAPT MS system, a C18 column (2.1×100 mm, 1.7 μm, Waters Co.) was used, the column temperature was 30° C., and the injection volume was 1 μL. Eluents A and B were 100% methanol and 0.1% (v/v) formic acid aqueous solution respectively, gradient elution was adopted, and the flow rate was 0.3 mL/min. The gradient elution conditions are shown in Table 3.

(51) TABLE-US-00003 TABLE 3 Gradient elution conditions t/min 0-0.5 0.5-7.0 7.0-7.5 7.5-10.0 Proportion of 60% 60-100% 100-60% 60% eluent A

(52) Mass spectrometry conditions: The ionization source was an ESI source; MRM detection (DEHP: MS+; MEHP: MS−) was used; the Capillary was 3.0 KV; the Conc was 40.00 V; the source temperature (radiation source temperature) was 120° C.; the desolvation temperature was 400° C.; the Conc gas flow was 50 L/h; and the desolvation gas flow was 700 L/h. The gas flow rate was 0.1 mL/min; the mass-to-charge ratio scanning range was 100-2,000; the scanning time was 1 s, and the interval was 0.061 s. The results were analyzed by MassLynx V4.1 (Waters Co.). In the study, the minimum detection limits of DEHP and MEHP were 0.05 ppm and 0.1 ppm, respectively. The adsorption rate of lactic acid bacteria was calculated based on the difference in the DEHP or MEHP concentration before and after adsorption, and the adsorption rate was calculated by the following formula:
Adsorption rate (%)=[(The content of plasticizer in an aqueous solution before
adsorption−the content of plasticizer in ultrapure water)−(The content of plasticizer in a
supernatant after adsorption−the content of plasticizer in a supernatant in the blank
control)]/(The content of plasticizer in an aqueous solution before adsorption−the content of
plasticizer in ultrapure water)×100.

(53) The determination results are shown in FIG. 1. It is obvious from FIG. 1 that compared with a commercial bacterium LGG (DEHP: 13.75%, MEHP: 15.60%) and intestinal resident bacteria E. coli (DEHP: 5.86%, MEHP: 0.28%) and E. faecalis (DEHP: 1.75, MEHP: 0.17%), the adsorption rates (DEHP: 57.67%, MEHP: 67.36%) of the strain Lactococcus lactis subsp. lactis CCFM1018 of the disclosure are significantly higher than those of the control bacteria, and far higher than the previously reported adsorption rate of 9.62% for DEHP. Therefore, the Lactococcus lactis subsp. lactis CCFM1018 has good adsorption capacity for DEHP and MEHP.

Example 3: Lactococcus lactis Subsp. Lactis CCFM1018 has No Acute Toxic or Side Effects on SD Rats

(54) Lactococcus lactis subsp. lactis CCFM1018 was resuspended in a 2% (w/v) sucrose solution with a bacterial cell density of 1.0×10.sup.9 CFU/mL. 10 healthy male SD rats weighing about 100 g were taken, given 2 mL of suspension of the above concentration through gavage daily, observed for a week, and recorded for the death and body weight.

(55) The experimental results are listed in Table 4. The results showed that feeding Lactococcus lactis subsp. lactis CCFM1018 with a concentration of 1.0×10.sup.9 CFU/mL did not cause significant effects on the rats, and the rats had no significant change in the body weight or death. The rat had no obvious pathological symptoms.

(56) TABLE-US-00004 TABLE 4 Changes in body weight and death of rats Time (d) 1 2 3 4 5 6 7 Body 100.213 ± 108.547 ± 117.149 ± 128.023 ± 137.643 ± 146.07 ± 159.68 ± weight (g) 3.85 3.63 5.32 3.72 4.92 6.64 4.41 Death — — — — — — — Note: — represents no rats died.

Example 4: Influence of Lactococcus lactis Subsp. Lactis CCFM1018 on the DEHP and MEHP Content in Serum and Feces of DEHP-Exposed Rats

(57) 18 healthy male SD rats weighing about 100 g were randomly divided into 3 groups: a blank control group (control), a DEHP-exposed model group (DEHP), and a Lactococcus lactis subsp. lactis CCFM1018 intervention group (DEHP+CCFM1018), and each group contains 6 rats. On days 1-7 of the experiment, the blank control group and the DEHP-exposed model group were given 2 mL of 2% (w/v) sucrose solution through gavage daily, and the rats in the Lactococcus lactis subsp. lactis CCFM1018 intervention group were given 2 mL of the Lactococcus lactis subsp. lactis CCFM1018 suspension with a concentration of 1.0×10.sup.9 CFU/mL prepared according to Example 3 of this specification through gavage. On day 8, the drinking water of the blank control group was changed to an aqueous solution containing 0.05% (m/V) sucrose fatty acid ester. DEHP was dissolved in the 0.05% (m/V) sucrose fatty acid ester aqueous solution, and except the blank group, exposure was performed via the drinking water at a dose of 3,000 mg/kg body weight every day. Gavage with the probiotic and the control 2% (w/v) sucrose solution continued during the exposure period. After four weeks of continuous exposure, the feces were collected and the animals were euthanized. Testes, liver and serum were collected, and the DEHP and MEHP content in the serum and feces were determined. The results of the determination are shown in FIG. 2. In FIG. 2, * indicates significance compared to the DEHP content in the control group; *P<0.05, **P<0.01, ***P<0.005, ****P<0.001; #P<0.05, ##P<0.01, ###P<0.005, ####P<0.001.

(58) Experimental results showed that after intervention of the Lactococcus lactis subsp. lactis CCFM1018, the DEHP and MEHP content in serum was significantly reduced (by 30.47% and 63.63% respectively compared to the blank control group), and at the same time the DEHP and MEHP content in feces was increased (by 58.90% and 21.42% respectively compared to the blank control group). This shows that the Lactococcus lactis subsp. lactis CCFM1018 can effectively promote the excretion of DEHP and the metabolite MEHP from the body.

Example 5: Alleviating Effect of Lactococcus lactis Subsp. Lactis CCFM1018 on the Reproductive Toxicity of DEHP-Exposed Rats

(59) The serum obtained in Example 4 was taken, and the testosterone content in the serum was determined according to the method shown in the ELISA kit. The results are shown in FIG. 3. In FIG. 3, * indicates significance compared to the control group, and # indicates significance compared to the model group; *P<0.05, #P<0.05.

(60) The testicular tissue obtained in Example 4 was weighed, normal saline was added at a ratio of 1:9 (m/m), and the testicular tissue was broken in a tissue homogenizer to obtain 10% testicular tissue homogenate. The level of malonaldehyde (MDA) in the homogenate was determined. The determination results are shown in FIG. 4. In FIG. 4, * indicates significance compared to the control group, and # indicates significance compared to the DEHP model group; *P<0.05, #P<0.05.

(61) Testicular tissue was taken for performing paraffin sectioning, and conventional H&E staining was performed. The staining results are shown in FIG. 5.

(62) By comparing the testosterone content in serum, the MDA level in the testis and the testicular pathological indexes of the DEHP-exposed model group and the Lactococcus lactis subsp. lactis CCFM1018 intervention group, it was found that Lactococcus lactis subsp. lactis CCFM1018 of the disclosure can alleviate the abnormality in testosterone content in rat serum caused by DEHP intake (DEHP-exposed model group: 19.65 mmol/L, CCFM1018 intervention group: 64.75 mmol/L), reduce the MDA content in the testicular tissue (DEHP-exposed model group: 50.28 nmol/mg, CCFM1018 intervention group: 28.66 nmol/mg), alleviate testicular tissue damage, and play a significant role in alleviating the reproductive damage caused by DEHP intake.

Example 6: Lactococcus lactis Subsp. Lactis CCFM1018 Improves SOD Activity in the Liver of DEHP-Exposed Rats

(63) The liver tissue obtained in Example 4 was weighed, normal saline was added at a ratio of 1:9 (m/m), and the liver tissue was broken in a tissue homogenizer to obtain 10% liver tissue homogenate. The activity level of superoxide dismutase (SOD) in the homogenate was determined. The determination results are shown in FIG. 6. In FIG. 6, * indicates significance compared to the control group, and # indicates significance compared to the DEHP model group; *P<0.05, **P<0.01, #P<0.05. By comparing the SOD activity in the liver of the DEHP-exposed model group and the Lactococcus lactis subsp. lactis CCFM1018 intervention group (33.29 U/mg and 49.43 U/mg, respectively), it is found that the Lactococcus lactis subsp. lactis CCFM1018 of the disclosure can effectively improve the SOD activity in liver decreased by the intake of DEHP.

Example 7 Preparation of Lactococcus lactis Subsp. Lactis CCFM1018 Fermentation Agent

(64) An MRS culture medium was inoculated with Lactococcus lactis subsp. lactis CCFM1018, and culturing was performed at 30-37° C. for at least 12 h to obtain a fermentation agent containing the Lactococcus lactis subsp. lactis CCFM1018.

(65) Optionally, the bacterial cells in the fermentation solution were collected and mixed with a 3% sucrose solution to prepare a bacterial suspension.

(66) Optionally, the bacterial suspension containing the Lactococcus lactis subsp. lactis CCFM1018 is mixed with a cytoprotective agent, and the mixture was freeze-dried at a low temperature to prepare a Lactococcus lactis subsp. lactis CCFM1018 freeze-dried preparation.

Example 8: Production of Milk Containing Lactococcus lactis Subsp. Lactis CCFM1018 Using Same

(67) Raw skimmed milk was sterilized with heat at 95° C. for 20 min, and then cooled to 4° C. Then the Lactococcus lactis subsp. lactis CCFM1018 fermentation agent was added, and the concentration reached 10.sup.6 CFU/ml or above. The milk product was stored under refrigeration at 4° C., and thus, milk containing live bacteria of the Lactococcus lactis subsp. lactis CCFM1018 of the disclosure was obtained.

Example 9: Preparation of Fermented Food Using Lactococcus lactis Subsp. Lactis CCFM1018 by Fermentation

(68) The Lactococcus lactis subsp. lactis CCFM1018 was used to prepare fermented dairy products, fermented bean products, or fermented fruit and vegetable products. The dairy products include milk, sour cream, and cheese; and the fruit and vegetable products include cucumber, carrot, beet, celery, and cabbage products. The Lactococcus lactis subsp. lactis CCFM1018 can be resistant to gastric acid and bile salts, promote excretion of a plasticizer and metabolites thereof from the body, reduce the content of the plasticizer and the metabolites thereof in serum, and significantly reduce the damage of the plasticizer and the metabolites thereof to testicular tissue and liver tissue.

(69) The Lactococcus lactis subsp. lactis CCFM1018 of the disclosure has good resistance to gastric acid and bile salts, can promote excretion of the plasticizer DEHP and the metabolite MEHP thereof from the body, reduces the content of the plasticizer and the metabolite thereof in serum, and significantly reduces the damage of the plasticizer and the metabolite thereof to testicular tissue and liver tissue.

(70) The Lactococcus lactis subsp. lactis CCFM1018 not only is significantly better than the intestinal resident bacteria Escherichia coli (DEHP: 5.86%, MEHP: 0.28%) and Enterococcus faecalis (DEHP: 1.75%, MEHP: 0.17%) in terms of in vitro adsorption of DEHP and MEHP (57.67% and 67.36% respectively), but also is better than the commercial strain Lactobacillus rhamnosus LGG (DEHP: 13.75%, MEHP: 15.60%). In rats, the Lactococcus lactis subsp. lactis CCFM1018 can significantly reduce the DEHP and MEHP content in serum (by 30.47% and 63.63% respectively compared to the blank control group), and at the same time increase the DEHP and MEHP content in feces (by 58.90% and 21.42% respectively compared to the blank control group). Therefore, the Lactococcus lactis subsp. lactis CCFM1018 of the disclosure can be used as an effective means to prevent and alleviate body damage caused by DEHP and MEHP, and does not have toxic or side effects of drugs. The Lactococcus lactis subsp. lactis CCFM1018 can be used to prepare pharmaceutical compositions and fermented food for alleviating and preventing the toxicity of DEHP and the metabolites thereof, and has a very broad application prospect.

(71) Although the disclosure has been disclosed above in the preferred examples, it is not intended to limit the disclosure. Anyone skilled in the art can make various changes and modifications without departing from the spirit and scope of the disclosure. Therefore, the protection scope of the disclosure should be defined by the claims.