CN116004428A - Lactobacillus plantarum with cholesterol reducing function and application thereof - Google Patents
Lactobacillus plantarum with cholesterol reducing function and application thereof Download PDFInfo
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- CN116004428A CN116004428A CN202211056437.8A CN202211056437A CN116004428A CN 116004428 A CN116004428 A CN 116004428A CN 202211056437 A CN202211056437 A CN 202211056437A CN 116004428 A CN116004428 A CN 116004428A
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- 240000006024 Lactobacillus plantarum Species 0.000 title claims abstract description 54
- 235000013965 Lactobacillus plantarum Nutrition 0.000 title claims abstract description 54
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- 238000002360 preparation method Methods 0.000 claims description 10
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- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
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- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 1
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses lactobacillus plantarum with cholesterol reducing function and application thereof, and belongs to the field of microorganisms. The lactobacillus plantarum (Lactobacillus plantarum) HJ-YB6 is obtained by separating and screening lactobacillus plantarum HJ-S2 from the intestinal tract of the coral by ARTP mutagenesis. The lactobacillus plantarum (Lactobacillus plantarum) HJ-YB6 has the cholesterol reducing capability reaching 65%, has good tolerance to acid and high bile salts, has good safety, and has a certain effect on maintaining the balance of intestinal microbial flora.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to lactobacillus plantarum with cholesterol reducing function and application thereof.
Background
Cardiovascular and cerebrovascular diseases are the head killers of human beings, and the high and low cholesterol levels in blood are related to the pathogenesis of the cardiovascular and cerebrovascular diseases, and the excessive cholesterol levels in blood are easy to cause atherosclerosis and other related diseases. The grease in the food can make the food more delicious, but can also enable people to consume more cholesterol when eating, and the long-term high-cholesterol diet can increase the cholesterol content in blood and increase the incidence risk of cardiovascular and cerebrovascular diseases.
Lactobacillus plantarum is often used in the manufacture of fermented foods, and can impart a unique flavor to the fermented foods by metabolizing carbohydrates, producing lactic acid, and the like during the manufacture of the fermented foods. Studies have shown that lactic acid bacteria can adsorb cholesterol in the environment to the cell membrane via a membrane adsorption pathway, resulting in a decrease in cholesterol concentration in the environment. Reducing cholesterol levels in foods by lactic acid bacteria is a safe and effective way.
Disclosure of Invention
The invention aims to provide lactobacillus plantarum with cholesterol reducing function and application thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
lactobacillus plantarum (Lactobacillus plantarum) HJ-YB6 with cholesterol reducing capability is preserved in China general microbiological culture Collection center (CGMCC) No.25278, the preservation date is 2022, 07, 11 months and the preservation address is Beicheng Kogyo area Beicheng Xiyu No. 1, 3.
The invention relates to application of lactobacillus plantarum (Lactobacillus plantarum) HJ-YB6 in preparation of a preparation for degrading cholesterol.
The microbial inoculum prepared from the lactobacillus plantarum (Lactobacillus plantarum) HJ-YB 6.
The microbial inoculum disclosed by the invention is applied to the preparation of products for degrading cholesterol.
The beneficial effects are that:
(1) The inventor separates and screens lactobacillus plantarum HJ-S2 from the intestinal tract of the whale, and the lactobacillus plantarum is identified by sequencing (Lactobacillus plantarum). HJ-S2 is subjected to ARTP mutagenesis to obtain lactobacillus plantarum (Lactobacillus plantarum) HJ-YB6, and the cholesterol reducing capability reaches 65%.
(2) The lactobacillus plantarum (Lactobacillus plantarum) HJ-YB6 has good tolerance to acid and high bile salt, good safety and a certain effect on maintaining the balance of intestinal microbiota.
Drawings
FIG. 1 shows colony morphology of Lactobacillus plantarum (Lactobacillus plantarum) HJ-YB 6.
FIG. 2 shows the result of hemolysis of Lactobacillus plantarum (Lactobacillus plantarum) HJ-YB 6.
FIG. 3 shows the passaging results of Lactobacillus plantarum (Lactobacillus plantarum) HJ-YB6 in example 1.
FIG. 4 shows a cholesterol standard curve measured by OPA method.
Detailed Description
The lactobacillus plantarum HJ-YB6 is classified and named as lactobacillus plantarum (Lactobacillus plantarum), the preservation number is CGMCC No.25278, and the lactobacillus plantarum is preserved in China general microbiological culture collection center (CGMCC for short, address: beicheng Kogyo Chen Xiyun No. 1, 3, and post code 100101) in the year 07 and the month 11 of 2022.
The lactobacillus plantarum (Lactobacillus plantarum) HJ-YB6 is obtained by ARTP mutagenesis, and the lactobacillus plantarum treated by the ARTP identifies the cholesterol reducing capability of the screening strain by a phthalaldehyde method. And then, re-screening is carried out by measuring the acid resistance and the bile salt resistance, and whether the strain tolerates the gastrointestinal tract environment is determined, so that the probiotic effect is achieved.
The invention will now be described in further detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The experimental procedures and reagents not shown in the formulation of the examples were all in accordance with the conventional conditions in the art.
Example 1
1. Preparation of original Strain HJ-S2 sample
MRS culture medium preparation:
10% of peptone, 10% of beef powder, 5% of yeast powder, 20% of glucose, 2% of diammonium hydrogen citrate, 5% of sodium acetate trihydrate, 2% of dipotassium hydrogen phosphate, 0.5% of magnesium sulfate heptahydrate and 0.2% of manganese sulfate monohydrate, 18% of agar is added to a solid flat plate on the basis of a liquid culture medium, and the solid flat plate is subjected to high-pressure sterilization at 121 ℃ for 20min.
2. Freezing the frozen tube of HJ-S2 strain at room temperature, inoculating the frozen tube into MRS liquid culture medium, culturing at 37 ℃ for 24 hours, re-inoculating the frozen tube into the MRS liquid culture medium according to 1% of inoculum size, culturing at 37 ℃ for 24 hours, repeating for three times, inoculating the obtained bacterial liquid into the MRS liquid culture medium according to 1% of inoculum size, and culturing at 37 ℃ to logarithmic phase to obtain bacterial liquid for mutagenesis.
2. ARTP mutagenesis
1. The bacterial liquid is collected after centrifugation, resuspended in 1mL of sterile physiological saline, 10 mu L of the bacterial liquid is coated on a glass slide, and the bacterial liquid is placed in an ARTP mutation breeding instrument for mutagenesis. The mutagenesis parameters were set as: the air flow is 10L/min, the power of the power supply is 120W, and the distance between the emission source and the slide glass is 2mm; the time for mutagenesis treatment was 0s, 20s, 40s, 60s, 80s and 100s.
2. Post-mutagenesis treatment
Immediately after mutagenesis, the slide glass is placed into a centrifuge tube filled with 1mL of sterile physiological saline, and the thallus is eluted by shaking vigorously for 5 min. Diluting the eluent to 10 times -5 And will 10 -2 、10 -3 、10 -4 、10 -5 Four gradients of bacterial liquid were plated onto MRS solid plates at 100. Mu.L, incubated at 37℃for 48h, and lethality was calculated using non-mutagenized strain-plated plates as controls. After mutagenesis, the strain is subcultured.
Selecting a strain with a higher degradation rate than the original strain HJ-S2 cholesterol for subculturing. After serial passages, a strain that can stably maintain the cholesterol degradation level was selected as the target strain.
3. Screening of mutant strains with cholesterol-lowering function
1. Drawing of standard curve of phthalic dicarboxaldehyde (OPA)
Cholesterol standard solutions 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6mg/mL (using glacial acetic acid)Solvent), 1mL of cholesterol standard solution is accurately sucked, and mixed acid (concentrated sulfuric acid: glacial acetic acid=1:1) 10mL,1.0mg/mL OPA 0.5mL reagent (absolute ethanol as solvent) were thoroughly mixed and allowed to stand for 10min. The blank uses 1mL absolute ethyl alcohol to replace cholesterol solution, the absorbance value is measured at 550nm wavelength, the cholesterol concentration is taken as the abscissa, the absorbance value is taken as the ordinate, a standard curve is drawn, the linear regression equation of the blank is calculated as y=0.9824x+0.0231, and the correlation coefficient is R 2 = 0.9942, standard curve is shown in fig. 4.
2. Determination of cholesterol-lowering Capacity of lactic acid bacteria
Cholesterol-containing MRS medium configuration: weighing 1g of cholesterol, adding into 5mL of Tween 80, heating in boiling water bath to dissolve, adding into MRS culture medium preheated to 60 ℃, packaging after uniform distribution, and autoclaving at 121 ℃ for 20min for use.
Selecting groups with mortality rate greater than 95%, picking single colonies from corresponding plates, streaking on an MRS plate, and culturing at 37 ℃ for 24 hours to obtain a primary plate.
Single bacterial colony is selected on a primary flat plate, three bacterial colonies are randomly selected from each flat plate, inoculated into an MRS liquid culture medium, after culturing for 24 hours, the concentration of bacterial cells is detected, the OD600 value is 0.6-0.8, bacterial liquid is inoculated into the MRS-CHOL liquid culture medium according to the inoculum size of 1 percent, the bacterial liquid is cultured for 24 hours at 37 ℃, the bacterial liquid is centrifuged for 5000r/min and 4 ℃ for 10 minutes, the supernatant is taken to measure the cholesterol content, the unvaccinated MRS-CHOL culture medium is taken as a blank control group, the cholesterol degradation rate of the lactobacillus is measured by adopting a phthalic aldehyde (OPA) method, and the experiment is repeated for 3 times to obtain the average value. The strain with positive mutation on cholesterol degradation ability was selected as the strain for the subsequent passage experiment.
The cholesterol degradation rate was calculated according to the following formula:
cholesterol degradation rate (%) = (1-a/B) ×100
Wherein: a is the concentration of cholesterol in the fermentation supernatant of the experimental group;
b is the concentration of cholesterol in the fermentation supernatant of the control group.
The degradation capacity of the primary strain HJ-YB6 cholesterol reaches 68 percent. The results are shown in FIG. 3.
4. Hemolysis experiment of mutant Strain HJ-YB6
1. The mutagenized strain is streaked and activated on an MRS plate for 3 times continuously for standby.
2. Single colonies were picked, streaked on platelets, cultured at 37℃for 24 hours, and observed for hemolysis.
3. As a result, there was no hemolysis (FIG. 2).
5. Acid and bile salt resistance experiments
1. Acid resistance experiment: preparing MRS culture medium with pH 2 and pH 3, inoculating activated bacterial liquid strain HJ-YB6 according to 1% of inoculation amount, culturing at 37 ℃ and 180rpm for 2 hours, coating on an MRS flat plate, and calculating the survival rate of the strain by taking the flat plate coating result of the common MRS liquid culture medium with bacterial liquid HJ-YB6 according to 1% of inoculation amount as a control.
The survival rate after 2 hours was 82.4%.
2. Bile salt resistance experiment: the activated bacterial liquid HJ-YB6 is inoculated into 100mL of MRS culture medium containing 0.3% of bile salt according to the inoculation amount of 1%, the bacterial liquid is coated on an MRS flat plate after being cultured for 2 hours at 37 ℃ and 180rpm, and the survival rate of the bacterial strain is calculated by taking the flat plate coating result of the common MRS liquid culture medium inoculated with the bacterial liquid HJ-YB6 according to the inoculation amount of 1% as a control.
The survival rate after 2 hours is 85.6%.
Example 2
Preparation of cholesterol-reducing bacterial powder by using lactobacillus plantarum HJ-YB6
1. Preparation of lactobacillus plantarum HJ-YB6 bacterial mud
The colony of the lactobacillus plantarum HJ-YB6 is picked into 10mL MRS liquid culture medium, and shake culture is carried out for 24 hours at 37 ℃ for activation. The activated bacterial liquid is inoculated into 15mL MRS liquid culture medium according to the inoculation amount of 3% to prepare germplasm liquid, and the bacterial liquid is subjected to shaking culture at 37 ℃ for 24 hours. The germplasm liquid is inoculated into 2L of MRS liquid culture medium with 3 percent of inoculation amount for expansion culture, and shaking culture is carried out for 24 hours at 37 ℃. Centrifuging the obtained thallus fermentation liquor for 10min at 10000r and 4 ℃, discarding supernatant, collecting thallus precipitate, and rinsing thallus with sterile 0.9% physiological saline for 2 times to obtain lactobacillus plantarum HJ-YB6 bacterial mud.
2. Preparation of protective agent
The lyoprotectant comprises 25% skimmed milk powder, 5% polydextrose, and 5% inulin. Dissolving the protective agent in water, and sterilizing at 110deg.C for 15 min.
3. Preparation of lactobacillus plantarum HJ-YB6 bacterial powder
Fully mixing the prepared lactobacillus plantarum HJ-YB6 bacterial mud with a protective agent solution according to the proportion of 1:5, pre-freezing for 8 hours at the temperature of-80 ℃ to uniformly freeze the lactobacillus plantarum HJ-YB6 bacterial mud on the inner wall of a container, and then performing vacuum freeze drying for 24-36 hours to obtain lactobacillus plantarum HJ-YB6 bacterial powder. The obtained bacterial powder is rehydrated by normal saline, and the viable count in the lactobacillus plantarum HJ-YB6 bacterial powder is measured to be 1.0x10 9 ～6.0×10 9 CFU/g。
Claims (6)
1. Lactobacillus plantarum (Lactobacillus plantarum) HJ-YB6 with cholesterol reducing capability is preserved in China center for type culture collection, with a preservation number of CGMCC No.25278 and a preservation date of 2022, 07 and 11 days.
2. Use of lactobacillus plantarum (Lactobacillus plantarum) HJ-YB6 according to claim 1 for the preparation of a cholesterol-degrading formulation.
3. A microbial agent prepared from lactobacillus plantarum (Lactobacillus plantarum) HJ-YB6 of claim 1.
4. A microbial agent according to claim 3, wherein: a lactobacillus plantarum (Lactobacillus plantarum) HJ-YB6 having cholesterol-lowering ability as claimed in claim 1 cultured in MRS liquid medium at 37 ℃.
5. Use of the microbial agent of claim 3 for the preparation of a cholesterol-degrading product.
6. The use according to claim 5, characterized in that: the product is a food.
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