<i>Lactobacillus plantarum </i>ZUST49 and bacterial agent, and use thereof

Abstract

The present disclosure discloses a Lactobacillus plantarum ZUST49 and a bacterial agent, and use thereof, and belongs to the technical field of microorganisms. An aspect of the present disclosure provides a Lactobacillus plantarum ZUST49 and a bacterial agent containing the Lactobacillus plantarum ZUST49, and another aspect provides use of the Lactobacillus plantarum ZUST49 and the bacterial agent containing the Lactobacillus plantarum ZUST49. The Lactobacillus plantarum ZUST49 of the present disclosure may convert glucoraphanin into sulforaphane, especially in a human or animal intestinal environment. After an enteric probiotic preparation prepared from the strain of the present disclosure is orally taken, the Lactobacillus plantarum ZUST49 may efficiently convert the glucoraphanin into the sulforaphane in an intestinal tract, which not only solves a problem that stability of the sulforaphane cannot be improved in the prior art, but also effectively exerts a probiotic effect.

Claims

1. A method of preparing an enteric probiotic preparation, comprising: preparing a glucoraphanin capsule comprising 100 M of glucoraphanin; preparing a bacterial agent capsule comprising 10.sup.10 CFU/mL of Lactobacillus plantarum ZUST49; and assembling the bacterial agent capsule and the glucoraphanin capsule to obtain an enteric capsule containing the Lactobacillus plantarum ZUST49 and the glucoraphanin, wherein the enteric probiotic preparation comprises the enteric capsule, wherein the Lactobacillus plantarum ZUST49 has a deposit number of CGMCC No. 26892.

2. The method according to claim 1, wherein the enteric probiotic preparation is for providing sulforaphane to a human body.

3. A method of preparing an enteric probiotic preparation, comprising: preparing a glucoraphanin capsule comprising of glucoraphanin; preparing a bacterial agent capsule comprising a bacterial agent; and assembling the bacterial agent capsule and the glucoraphanin capsule to obtain an enteric capsule containing the bacterial agent and the glucoraphanin, wherein the enteric probiotic preparation comprises the enteric capsule, and wherein the bacterial agent comprises Lactobacillus plantarum ZUST49 having a deposit number of CGMCC No. 26892.

4. The method according to claim 3, wherein the enteric probiotic preparation is for providing sulforaphane to a human body.

5. The method according to claim 3, wherein a content of the Lactobacillus plantarum ZUST49 in the bacterial agent is 110.sup.5 CFU/g.

6. The method according to claim 3, wherein the bacterial agent further comprises glucoraphanin and/or a raw material containing the glucoraphanin.

7. The method according to claim 6, wherein in the bacterial agent, the raw material comprising the glucoraphanin is one of dried broccoli, cabbage, and cabbage mustard or a mixture thereof.

8. The method according to claim 6, wherein a content of the glucoraphanin in the bacterial agent is 0.2-10 mg/g.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a 16S rRNA phylogenetic tree of Lactobacillus plantarum ZUST49;

(2) FIG. 2 shows characteristic graphs of colony morphology and microscopic morphology of Lactobacillus plantarum ZUST49;

(3) FIG. 3 shows a glucoraphanin conversion rate of Lactobacillus plantarum ZUST49;

(4) FIG. 4 is a flow chart of a manufacturing process of an enteric capsule; and

(5) FIG. 5 shows a comparison of the content of sulforaphane in mouse plasma.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(6) The present disclosure will be further described below with reference to the accompanying drawings and embodiments, but is not thereby limited to the scope of the described examples. The experimental methods in the following examples without specified specific conditions should be selected according to conventional methods and conditions, or according to instructions of products.

Example 1 Separation, Screening, and Identification of Lactobacillus plantarum ZUST49

(7) (1) Culture Medium:

(8) MRS culture medium: 10 g of peptone, 8 g of beef powder, 4 g of yeast powder, 20 g of glucose, 2 g of dipotassium phosphate, 2 g of diammonium hydrogen citrate, 5 g of sodium acetate, 0.2 g of magnesium sulfate, 0.04 g of manganese sulfate, 1 mL of Tween 80, and 15 g of agar were weighed and added to 1,000 mL of distilled water. The materials were heated and dissolved, a constant volume was set, a pH was adjusted to 6.0-6.4, and a high-pressure sterilization was performed at 121 C. for 15 min.

(9) MRS liquid culture medium: no agar was added during the preparation of the MRS culture medium, other components were completely the same, and a high-pressure sterilization was performed at 121 C. for 15 min.

(10) M17 culture medium: 5 g of soytone, 2.5 g of peptone, 2.5 g of casein peptone, 5 g of beef extract powder, 5 g of lactose, 0.5 g of sodium ascorbate, 19 g of -sodium glycerophosphate, 0.25 g of magnesium sulfate, and 12.75 g of agar, 1,000 mL of distilled water was added, the materials were heated and dissolved, a constant volume was set, a pH was adjusted to 7.0-7.4, and a high-pressure sterilization was performed at 121 C. for 15 min.

(11) M17 liquid culture medium: no agar was added during the preparation of the M17 liquid culture medium, other components were completely the same, and a high-pressure sterilization was performed at 121 C. for 15 min.

(12) Crude thioglycoside culture medium: 100 g of broccoli seeds were taken and crushed into fine powder, 400 mL of distilled water was added, the material was boiled for 30 min, filtered with 3 layers of gauze to remove a residue, centrifugation was performed at 10,000 r/min for 10 min to remove a precipitate, a constant volume was set to 500 mL, and sterilization was performed at 121 C. for 30 min.

(13) (2) Isolation and Purification of Bacterial Solution

(14) Chinese cabbages purchased from market were shredded, seasoning and purified water were added, and the material was sealed and naturally fermented for one week. A pickled vegetable solution was immediately transferred into an anaerobic operation box. Sterile plates of the MRS, M17, and crude thioglycoside culture mediums were prepared, cooled, and solidified, and 0.1 mL of a bacterial solution was dripped by using a pipette, and ed. Types, time, and numbers of streaked culture dishes were marked, and the culture dishes were taken out after the marking was all completed, and put into a constant-temperature anaerobic incubator at 37 C. for 24 h.

(15) After a 1st culture was completed, a single colony was picked to a corresponding liquid culture medium, types, numbers, and dates were recorded, and the culture mediums were put into the constant-temperature anaerobic incubator at 37 C. for 24 h after the recording was all completed.

(16) (3) Screening of Strain

(17) In a sterile operating station, a glucoraphanin solution was added to the liquid culture medium after the high-temperature sterilization via a20 m filter membrane to a final concentration of glucoraphanin of 1 mM, and the culture medium was sub-packaged into test tubes. 25 L of a bacterial solution after a 2nd culture was added into the test tubes of the corresponding culture mediums by using a pipette, and the bacteria were put into a constant-temperature incubator at 37 C. for culture for 12 h. After the culture was completed, a concentration of the glucoraphanin in each test tube was measured by a glucose detection kit method, a conversion rate was calculated, and a bacterial solution with a relatively high degradation rate was screened, recorded, and numbered. A colony morphology of Lactobacillus plantarum ZUST49 on the MRS culture medium was milky white, smooth, and round with a bulge, and neat in edges. A microscopic morphology was in rod-like, paired or chain-like arrangements (FIG. 2).

(18) (4) Deposit and Identification of Strain

(19) 500 L of the bacterial solution after 2 times of the liquid culture was taken and added into 500 L of 40% glycerol (final concentration of 20%), the materials were shaken and mixed uniformly, and the mixture was cryopreserved in a refrigerator at 20 C.

(20) The Lactobacillus plantarum ZUST49 obtained by screening using a modern molecular biology identification technology was subjected to a 16S rRNA sequence identification. A sequencing result was shown in SEQ ID NO. 1 of a sequence listing. A molecular biology rapid identification was performed by a 16S rDNA gene sequence analysis method. An identification result was Lactobacillus plantarum with a homology similarity of 100%, and the strain was named ZUST49. A 16S rRNA phylogenetic tree of the strain was shown in FIG. 1. The strain was deposited in China General Microbiological Culture Collection Center (CGMCC) located at No. 3, Yard 1, West Beichen Road, Chaoyang District, Beijing, Institute of Microbiology of Chinese Academy of Sciences on Mar. 23, 2023, and had the deposit number of CGMCC No. 26892.

Example 2 Conversion Rate of Glucoraphanin by Lactobacillus plantarum ZUST49

(21) A glucoraphanin solution was added to the MRS liquid culture medium after the high-temperature sterilization via a20 m filter membrane to a final concentration of glucoraphanin of 1 mM, and the culture medium was sub-packaged into test tubes. 50 L of activated Lactobacillus plantarum ZUST49 solution was taken using a pipette, and added into corresponding MRS liquid culture mediums, and the liquid culture mediums were put into a constant-temperature incubator at 37 C. for culture for 12 h and 24 h respectively. After the culture was completed, a concentration of the glucoraphanin in each test tube was measured by a glucose detection kit method, a conversion rate was calculated. The result showed a highest conversion rate of the glucoraphanin of the screened strain may reach 39.5% (FIG. 3).

(22) TABLE-US-00001 TABLE 1 Conversion rate of glucoraphanin by Lactobacillus plantarum ZUST49 Before Reduction of fermentation Culture for 24 h glucoraphanin Content of 1.000 0.605 0.10 0.395 glucoraphanin (mM)

(23) Therefore, the conversion rate of the glucoraphanin was 39.5%.

(24) $\mathrm{Note}:\mathrm{Conversion}\mathrm{rate}\mathrm{of}\mathrm{glucoraphanin}=\frac{Redu\mathrm{ction}\mathrm{of}\mathrm{glucoraphanin}}{Co\mathrm{ntent}\mathrm{of}\mathrm{glucoraphanin}\mathrm{in}\mathrm{culture}\mathrm{medium}\mathrm{before}\mathrm{fermentation}}100\%$

Example 3 Preparation of Enteric Capsule Containing Glucoraphanin

(25) (1) Obtaining of Bacteria

(26) The screened strain was inoculated into 30 mL of an MRS liquid culture medium at an amount of 1% (V/V), subjected to an anaerobic culture at a constant temperature of 37 C. for 24 h, and activated twice. Then the strain was inoculated into 1.50 L of the MRS liquid culture medium at an amount of 1%, subjected to an anaerobic culture at a constant temperature of 37 C. for 24 h, and centrifuged at 6,000 r/min for 20 min to obtain bacteria. The bacteria were washed twice with a PBS buffer solution.

(27) (2) Preparation of Glucoraphanin Capsule

(28) Glucoraphanin (100 M), 2% of xylooligosaccharide, 4% of whey protein, and 2% of sodium alginate were mixed, the mixture was dropwise added into a 2% calcium chloride solution to form calcium alginate gel beads, stood for 30 min and placed in a 0.5% chitosan solution for immobilization, redundant chitosan was washed off, and the gel beads were placed into an ultralow-temperature refrigerator for freezing overnight at 80 C. and freeze-dried in a freeze dryer for 24 h to obtain 1.36 kg of a glucoraphanin capsule with the glucoraphanin content of 6.57 mg/g.

(29) (3) Preparation of Bacterial Agent Capsule

(30) The Lactobacillus plantarum strain (10.sup.10 CFU/mL), 2% of xylooligosaccharide, 4% of whey protein, and 2% of sodium alginate were mixed, the mixture was dropwise added into a 2% calcium chloride solution to form calcium alginate gel beads, stood for 30 min and placed in a 0.5% chitosan solution for immobilization, redundant chitosan was washed off, and the gel beads were placed into an ultralow-temperature refrigerator for freezing overnight at 80 C. and freeze-dried in a freeze dryer for 24 h to obtain 1.31 kg of a glucoraphanin enteric capsule.

(31) (4) Assembly of Capsule

(32) Canning was performed using a semi-automatic capsule canning machine, the bacterial agent capsule and the glucoraphanin capsule were assembled to obtain 2.65 kg of a capsule containing Lactobacillus plantarum ZUST49 and glucoraphanin, wherein the content of ZUST49 was 8.710.sup.5 CFU/g. A manufacturing process of the enteric capsule was shown in FIG. 4.

Example 4 Preparation of Enteric Capsule Containing Freeze-Dried Broccoli Powder

(33) (1) Obtaining of Bacteria

(34) The screened strain was inoculated into 30 mL of an MRS liquid culture medium according to the amount of 1% (V/V), subjected to an anaerobic culture at a constant temperature of 37 C. for 24 h, and activated twice. Then the strain was inoculated into 1.50 L of the MRS liquid culture medium according to the amount of 1%, subjected to an anaerobic culture at a constant temperature of 37 C. for 24 h, and centrifuged at 6,000 r/min for 20 min to obtain bacteria. The bacteria were washed twice with a PBS buffer solution.

(35) (2) Preparation of Freeze-Dried Broccoli Powder

(36) 15.0 kg of broccoli buds were cleaned, drained, and cut into blocks with a size of 3 cm3 cm. The blocks were subjected to hot-air drying in a hot-air drying box at 70 C. for 7 h, and crushed to obtain 1.65 kg of a broccoli powder with the glucoraphanin content of 0.87 mg/g.

(37) (3) Preparation of Bacterial Agent Capsule

(38) The Lactobacillus plantarum (10.sup.10 CFU/mL), 2% of xylooligosaccharide, 4% of whey protein, and 2% of sodium alginate were mixed, the mixture was dropwise added into a 2% calcium chloride solution to form calcium alginate gel beads, stood for 30 min and placed in a 0.5% chitosan solution for immobilization, redundant chitosan was washed off, and the gel beads were placed into an ultralow-temperature refrigerator for freezing overnight at 80 C. and freeze-dried in a freeze dryer for 24 h to obtain 1.51 kg of a glucoraphanin capsule.

(39) (4) Assembly of Capsule

(40) Canning was performed using a semi-automatic capsule canning machine, the bacterial agent capsule and the glucoraphanin capsule were assembled to obtain 3.11 kg of a capsule containing Lactobacillus plantarum ZUST49 and glucoraphanin, wherein the content of ZUST49 was 6.210.sup.5 CFU/g.

Example 5 Production of Sulforaphane by Feeding Mice with Enteric Capsule

(41) C57BL/6 male mice of 6 to 8 weeks were pre-reared for 1 week and randomly divided into an enteric capsule group, a glucoraphanin group, and a blank group. Mice in the enteric capsule group were intragastrically administered daily with an enteric capsule aqueous solution containing Lactobacillus plantarum ZUST49 and glucoraphanin (150 mol of glucoraphanin/kg and ZUST49 content of 8.710.sup.5 CFU/g, and prepared according to the method of example 3). The mice in the glucoraphanin group were intragastrically administrated with a glucoraphanin aqueous solution (150 mol of glucoraphanin/kg). The mice were fed with food and water ad libitum. Food intake and weight were recorded daily. The mice were deeply anesthetized with an intraperitoneal injection of ketamine/xylazine (87 mg/mL and 13 mg/mL respectively, and 0.1 mL/100 g mice) at 0 h and 12 h (6 mice/group each time period), and put to death by cervical dislocation after blood collection by cardiac puncture. The sulforaphane content in plasma of the mice was determined by a cyclocondensation method. A result was shown in FIG. 5 and indicated that the sulforaphane content in the plasma of the mice fed with the enteric capsule reached 0.320.15 UM, which was significantly higher than that of the glucoraphanin group (0.0170.030 M) and the blank group (0 M).

(42) Finally, it should be noted that the above examples area only intended to explain, rather than to limit, the technical solutions of the present disclosure. Although the present disclosure is described in detail with reference to the preferred example, those of ordinary skill in the art should understand that modifications or equivalent substitutions made to the technical solutions of the present disclosure without departing from the spirit and scope of the technical solutions of the present disclosure should be included within the scope of the claims of the present disclosure.