CN116064333A - Breast milk source lactobacillus plantarum, and product and application thereof - Google Patents
Breast milk source lactobacillus plantarum, and product and application thereof Download PDFInfo
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Abstract
The invention provides a breast milk source plantLactobacillus plantarum, and products and applications thereof relate to the field of biotechnology. The invention provides lactobacillus plantarumLactobacillus plantarum) The strain is preserved in China general microbiological culture Collection center (China Committee) with the preservation number: CGMCC No.23655. The strain is derived from human breast milk, has good safety, has stronger acid resistance and bile salt resistance, can reduce uric acid level of animals, and is beneficial to preventing and treating various diseases such as hypertension, insulin resistance, obesity, metabolic syndrome, nonalcoholic fatty liver disease, cardiovascular diseases and the like.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to breast milk-derived lactobacillus plantarum, a product and application thereof.
Background
Probiotics are increasingly consumed worldwide, and in particular in the dairy industry, their safety is also of great concern. Current safety studies of probiotics should have established a scientific consensus based on strain level. The safety of probiotics is clearly defined in many countries such as germany and the european union. The world health organization (World Health Organization, WHO) has given in 2002 the main content and basic guidelines for the safety assessment of food lactic acid bacteria products. Along with the release of the plan outline of "health China 2030", not only is food safety paid attention to, but also the attention of healthy functional food is higher and higher. The safety of probiotics is becoming a focus of attention, so safety problems during use can be avoided by evaluating the safety before commercial application.
In view of this, the present invention has been made.
Disclosure of Invention
The first aim of the invention is to provide a lactobacillus plantarumLactobacillusplantarum) To solve the problem ofAt least one of the above problems is solved.
The second object of the present invention is to provide a microbial agent.
A third object of the present invention is to provide the above Lactobacillus plantarum [. Sup.Lactobacillusplantarum) Or the application of the microbial inoculum in preparing products for reducing uric acid.
A fourth object of the present invention is to provide a feed.
A fifth object of the present invention is to provide a medicament.
A sixth object of the present invention is to provide the use of the above feed or medicament.
In order to solve the technical problems, the following technical scheme is adopted:
in a first aspect, the invention provides a lactobacillus plantarumLactobacillusplantarum) The lactobacillus plantarum is [ ]Lactobacillus plantarum) The microbial strain is preserved in China general microbiological culture Collection center (China Committee) with the preservation number: CGMCC No.23655.
As a further technical proposal, the lactobacillus plantarum isLactobacillusplantarum) Derived from human breast milk.
In a second aspect, the invention provides a microbial inoculum comprising the lactobacillus plantarumLactobacillus plantarum）。
In a third aspect, the invention provides the lactobacillus plantarumLactobacillusplantarum) Or the application of the microbial inoculum in preparing products for reducing uric acid.
As a further technical solution, the product comprises feed and medicine.
In a fourth aspect, the invention provides a feed comprising the Lactobacillus plantarumLactobacillus plantarum) Or the microbial inoculum.
In a fifth aspect, the present invention provides a medicament comprising said Lactobacillus plantarumLactobacillus plantarum) Or the microbial inoculum.
As a further technical scheme, the medicament further comprises auxiliary materials.
In a sixth aspect, the invention provides the use of a feed or medicament as described above in any one of the following a-f:
a. preparing a product for preventing or treating hypertension;
b. preparing a product for preventing or treating insulin resistance;
c. preparing a product for preventing or treating obesity;
d. preparing a product for preventing or treating metabolic syndrome;
e. preparing a product for preventing or treating the non-alcoholic fatty liver disease;
f. preparing a product for preventing or treating cardiovascular diseases.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides lactobacillus plantarumLactobacillusplantarum) The product is derived from human breast milk, has good safety, has strong acid and bile salt resistance, can reduce uric acid level of animals, and is beneficial to preventing and treating various diseases such as hypertension, insulin resistance, obesity, metabolic syndrome, nonalcoholic fatty liver disease, cardiovascular diseases and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the body weight changes of mice with different concentrations of Lactobacillus plantarum;
FIG. 2 is a graph showing the cumulative weekly feed intake change for each group of mice with different concentrations of Lactobacillus plantarum intervention;
FIG. 3 shows a comparison of strain survival in 3 hours at pH 3.0.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but it will be understood by those skilled in the art that the following embodiments and examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The specific conditions are not specified, and the process is carried out according to conventional conditions or conditions suggested by manufacturers. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In a first aspect, the invention provides a lactobacillus plantarum, the classification of which is named: lactobacillus plantarum, latin Wen Xueming:Lactobacillus plantarumthe method is preserved in China general microbiological culture Collection center, and has the preservation address: beijing city, chaoyang district, north Chen Xili No. 1,3, date of preservation: 2021, 10 and 25 days, deposit number: CGMCC No.23655.
The invention provides lactobacillus plantarumLactobacillusplantarum) The product is derived from human breast milk, has good safety, has strong acid and bile salt resistance, can reduce uric acid level of animals, and is beneficial to preventing and treating various diseases such as hypertension, insulin resistance, obesity, metabolic syndrome, nonalcoholic fatty liver disease, cardiovascular diseases and the like.
In a second aspect, the invention provides a microbial inoculum comprising the lactobacillus plantarumLactobacillus plantarum）。
The lactobacillus plantarum provided by the invention is included in the microbial inoculum, so that the microbial inoculum has all technical effects of the lactobacillus plantarum provided by the invention.
In a third aspect, the invention provides the lactobacillus plantarumLactobacillusplantarum) Or the application of the microbial inoculum in preparing products for reducing uric acid.
The inventors have found that the Lactobacillus plantarum provided by the invention can reduce uric acid level in animals, so that the Lactobacillus plantarum can be used for preparing uric acid-reducing products.
In some preferred embodiments, the products include, but are not limited to, feeds and pharmaceuticals.
In a fourth aspect, the invention provides a feed comprising the Lactobacillus plantarumLactobacillus plantarum) Or the microbial inoculum.
In a fifth aspect, the present invention provides a medicament comprising said Lactobacillus plantarumLactobacillus plantarum) Or the microbial inoculum.
In some preferred embodiments, the medicament further comprises an adjuvant. The choice of the auxiliary materials in the invention is not particularly limited, and the auxiliary materials of medicines known to those skilled in the art can be adopted.
The feed and the medicine provided by the invention contain the lactobacillus plantarum provided by the invention, so that the feed and the medicine have the effect of reducing the uric acid level of animals and have all the beneficial effects of the lactobacillus plantarum.
In a sixth aspect, the invention provides the use of a feed or medicament as described above in any one of the following a-f:
a. preparing a product for preventing or treating hypertension;
b. preparing a product for preventing or treating insulin resistance;
c. preparing a product for preventing or treating obesity;
d. preparing a product for preventing or treating metabolic syndrome;
e. preparing a product for preventing or treating the non-alcoholic fatty liver disease;
f. preparing a product for preventing or treating cardiovascular diseases.
Uric acid is one of indexes for evaluating renal function conditions, has important connection among different organ systems, and can be used for preventing and treating various diseases such as hypertension, insulin resistance, obesity, metabolic syndrome, nonalcoholic fatty liver disease, cardiovascular diseases and the like by reducing uric acid level. The feed and the medicine provided by the invention have the effect of reducing the uric acid level of animals, so that the feed and the medicine can be used for preparing products for treating or preventing the diseases.
The invention is further illustrated by the following specific examples and comparative examples, however, it should be understood that these examples are for the purpose of illustration only in greater detail and should not be construed as limiting the invention in any way.
EXAMPLE 1 isolation of Breast milk-derived Lactobacillus plantarum HM-P18 (i.e., lactobacillus plantarum, abbreviated as P18) provided by the present invention
Fresh breast milk samples were selected, serially diluted with sterile PBS buffer (available from Jiete Biofiltration Co., ltd.) and applied to a sample containing 1% CaCO 3 Culturing for 48 hours in an anaerobic incubator at 37 ℃, picking up white or milky single colony with obvious transparent circle, carrying out gram staining and microscopic examination, selecting a strain which is rod-shaped and gram positive, selecting a strain with better growth vigor from a plurality of strains, identifying, obtaining lactobacillus plantarum P18, and then carrying out enrichment culture on the strain in the anaerobic incubator at 37 ℃; the bacterial liquid and the sterilized 30% glycerol are mixed according to the proportion of 1:1 and then stored in a refrigerator at the temperature of minus 80 ℃ for standby.
Example 2
Inoculating Lactobacillus plantarum P18 into MRS liquid culture medium, activating for three generations, inoculating the activated third generation bacterial liquid into MRS liquid culture medium at 2% inoculum size, culturing at 37deg.C for 12 hr, and diluting bacterial liquid to 10 after 12 hr -1 -10 -7 Concentration gradients, 100 mu L of each concentration gradient bacterial liquid is coated on MRS solid culture medium, and the culture is carried out for 48 hours at 37 ℃. And finally, selecting a flat plate with the calculated colony number of 30-300, and calculating and preparing bacterial solutions with different concentrations by using a flat plate counting method. The viable count was calculated using the following formula:
CFU/ml= (colony number average Í dilution)/coating weight.
Example 3
The experimental strain P18 is isolated from the breast milk of healthy people and identified as lactobacillus plantarum. SPF grade C57BL/6J Nifdc female mice with age of 6-8 weeks are used as test animals (the mice are used as test animals in the following examples), the mice are adaptively fed for one week, can drink and eat freely, and the day and night light and shade alternation is carried out for 12h/12h, the room temperature of the facility is 22+/-2 ℃, and the relative humidity is 50% -60%.
With reference to the method of maximum limit in GB15193.3-2014 acute oral toxicity test, mice are fed adaptivelyAfter one week of culture, the mice were randomly divided into a control group and an experimental group, each group of 10 mice, the control group was perfused with 0.85% physiological saline, and the experimental group was perfused with high concentration Lactobacillus plantarum (1 Í 10) 10 cfu/mL) of bacteria solution, the gastric lavage amount is 0.2 mL per mouse, the continuous gastric lavage is 7 d, sufficient feeding and drinking water are ensured during the period, and the physiological state and the death condition of the mouse are observed and recorded so as to push out half-lethal dose LD50.
Results: after the high-dose Lactobacillus plantarum 7 d is continuously infused, daily observation indexes of mice are normal, meanwhile, the mice are not poisoned and have no death phenomenon, and LD50 is calculated to be more than 10 g/kg.
TABLE 1 mice poisoning performance observations
Example 4
Toxicity studies were performed with reference to GB15193.22-2014, 28d oral toxicity test, mice were randomly divided into control groups after one week of adaptive rearing, low dose Lactobacillus plantarum intervention groups (1 Í 10) 8 cfu/mL), medium dose Lactobacillus plantarum intervention group (1 Í 10) 9 cfu/mL), high dose Lactobacillus plantarum intervention group (1 Í 10) 10 cfu/mL), 10 mice per group. The control group was perfused with 0.85% physiological saline, lactobacillus plantarum was used to intervene in the group to perfuse with bacteria of different concentrations, the amount of the perfused stomach was 0.2 mL/mouse, the continuous lavage was 28d, the body weight and feeding amount of the mice were recorded weekly during this period, fasted at 28d, and dissected sacrificed at 29d morning 8.
Results: during the test period, the weights of the mice in each group grew steadily, and there was no significant difference in weight between each experimental group and the Control group (P > 0.05), as shown in FIG. 1 (note: control group; P18-L low dose Lactobacillus plantarum intervention group (1 Í) 8 cfu/mL); P18-M medium dose Lactobacillus plantarum intervention group (1 Í 10) 9 cfu/mL); P18-G high dose Lactobacillus plantarum intervention group (1 Í 10) 10 cfu/mL)). There was no significant difference in cumulative weekly food intake (p > 0.05) between mice in each experimental group and Control group, as shown in FIG. 2 (note: control group;P18-L Low dose Lactobacillus plantarum intervention group (1 Í 10) 8 cfu/mL); P18-M medium dose Lactobacillus plantarum intervention group (1 Í 10) 9 cfu/mL); P18-G high dose Lactobacillus plantarum intervention group (1 Í 10) 10 cfu/mL)).
Example 5
Following the 29d dissecting sacrifice of the mice from example 4, the heart, liver, spleen, lung and kidney colors and whether there was a significant change were observed, the heart, liver, spleen, lung and kidney were removed with a sterile scalpel and placed on a sterile plate, rinsed with sterile saline and blotted with filter paper, and the individual organs were weighed to calculate the mice viscera ratio according to the formula. The heart, liver, spleen, lung and kidney were each spread on MRS solid medium, and after culturing at 37℃for 48 hours, the medium was observed for the growth of colonies.
Visceral volume ratio = visceral mass (g)/mouse body weight mass (g).
Results: after dissection, the mice were observed for normal and non-diseased organs, and heart, liver, spleen, lung and kidney were collected and weighed to calculate their corresponding organ indices as shown in table 2. The organ indexes of mice in the interference groups of the lactobacillus plantarum with different concentrations have no significant difference (P is more than 0.05) compared with the control group, which indicates that the lactobacillus plantarum P18 has no influence on the organ indexes of the mice.
The heart, liver, spleen, lung and kidney are coated on an MRS culture medium, and the culture medium is sterile and can grow, so that the lactobacillus plantarum P18 does not have translocation phenomenon in a mouse body and can not bring corresponding safety problems to the body.
TABLE 2 organ index of mice
Note that: p is more than 0.05, and the experimental group and the Control group have no significant difference; P18-L Low dose Lactobacillus plantarum intervention group (1 Í 10) 8 cfu/mL); P18-M medium dose Lactobacillus plantarum intervention group (1 Í 10) 9 cfu/mL); P18-G high dose Lactobacillus plantarum intervention group (1 Í 10) 10 cfu/mL)。
Example 6
Following the 28d oral toxicity test, mice were fasted overnight and blood was collected the next day for biochemical analysis of blood and serum using the method of eyeball blood collection. 150. Mu.L of whole blood from mice was placed in a centrifuge tube containing an anticoagulant and the blood parameters of the mice were measured using a fully automated blood cell analyzer. The remaining blood was centrifuged at 3000 rpm for 10 min at 4℃to obtain serum, and the liver, kidney, blood glucose, blood lipid and cholesterol levels of mice were measured using a full-automatic biochemical analyzer, and the results were expressed as mean.+ -. Standard deviation.
Results: the hematopoietic system of experimental animals and human beings is one of the most sensitive targets of toxic substances, and is an important index of the healthy physiological state of human beings and experimental animals. After 28 days of gastric lavage mice, blood routine results showed small increases or decreases in blood parameters of lactobacillus plantarum-intervention mice compared to control mice, but no significant differences (p > 0.05), as shown in table 3. Lactobacillus plantarum P18 thus has no adverse effect on the blood parameters of the mice. The biochemical detection result of mouse serum shows that Uric Acid (UA) level of a high-dose lactobacillus plantarum intervention group (P18-G) is obviously reduced (P < 0.05) compared with that of a control group, and all indexes of the other groups are normal without obvious difference, as shown in table 4. Uric acid is also one of the indicators for evaluating renal function status, has important links between different organ systems, and reduces uric acid levels to be beneficial for preventing and treating various diseases such as hypertension, insulin resistance, obesity, metabolic syndrome, nonalcoholic fatty liver disease, cardiovascular diseases, and the like. From this, it was deduced that lactobacillus plantarum P18 might affect metabolic pathways, and is expected to be a potential probiotic strain that could regulate uric acid levels and thereby improve various diseases.
TABLE 3 influence of experimental strains on blood parameters of mice
Note that: p is more than 0.05, and the experimental group and the Control group have no significant difference; P18-L Low dose Lactobacillus plantarum intervention group (1 Í 10) 8 cfu/mL); P18-M medium dose Lactobacillus plantarum intervention group (1 Í 10) 9 cfu/mL); P18-G high dose Lactobacillus plantarum intervention group (1 Í 10) 10 cfu/mL)。
WBC: white blood cells; RBC: red blood cells; HGB: hemoglobin; HCT: hematocrit; MCV: average volume of red blood cells; MCH: average red blood cell hemoglobin amount; PLT: platelets.
TABLE 4 influence of experimental strains on the biochemical parameters of mouse serum
Note that: the same shoulder letters represent no significant difference (p > 0.05), and the different shoulder letters represent significant difference (p)<0.05 Control group; P18-L Low dose Lactobacillus plantarum intervention group (1 Í 10) 8 cfu/mL); P18-M medium dose Lactobacillus plantarum intervention group (1 Í 10) 9 cfu/mL); P18-G high dose Lactobacillus plantarum intervention group (1 Í 10) 10 cfu/mL)。
ALT: alanine aminotransferases; TC: killer T cells; TG: triglycerides; TBIL: total bilirubin; DBIL: direct bilirubin; BUN: serum urea nitrogen; UA: serum uric acid; CREA: a creatinine material; GLU: glucose.
Example 7
Acid resistance test:
the washed medium was resuspended in MRS broth with pH 3.0, anaerobically cultured at 37℃and sampled for 0,1,3h, respectively, for gradient dilution, plated on MRS solid medium plates, anaerobically cultured at 37℃for 48h and counted.
Strain survival (%) = N1/N0 x 100;
wherein: n0 is the viable count (cfu/mL) of 0 h; n1 is the viable count (cfu/mL) of the strain after acid resistance for 1 h.
Results: as shown in Table 5, the survival rate after 1h of survival at pH 3.0 is not less than 129.7%, and the number of viable bacteria is 10 9 cfu/mL or more; the survival rate is more than or equal to 167.67 percent after survival for 3 hours under the condition of pH value of 3.0, and the number of viable bacteria is 10 9 Above cfu/mL, no significant decrease in viable count occurred within 1h and 3 h. Compared with the strains R2 and R9, the survival rate of the strain in P18 h is obviously increased, and the acid-resistant effect is highIn other strains, as shown in FIG. 3.
Wherein, R2 and R9 are two breast milk-derived lactobacillus plantarum with better effect which are screened before the inventor company, and the specific reference is as follows: yin Chunmei, li Zhen, jiang Tiemin, et al. Preliminary determination of screening and identification of human breast milk-derived lactic acid bacteria and their ability to reduce blood pressure [ J ]. Food science and technology, 2019 (8): 18-22.
TABLE 5 acid resistance test results
Bile salt resistance test:
the washed culture medium was resuspended in MRS broth medium containing 0.3% bile salts, cultured anaerobically at 37℃and sampled for 0,1,3h respectively for gradient dilution, plated on MRS solid medium plates, anaerobically cultured at 37℃for 48h and counted.
Strain survival (%) = N1/N0 x 100;
wherein: n0 is the viable count (cfu/mL) of 0 h; n1 is the viable count (cfu/mL) of the strain after 1h of cholate tolerance.
Results: as shown in Table 6, the survival rate of the strain after 1h of survival under the condition of 0.3% bile salt is more than or equal to 147.06%, and the number of viable bacteria is 10 9 cfu/mL or more; the survival rate is more than or equal to 30 percent after the bacteria survive for 3 hours under the condition of 0.3 percent bile salt, and the number of viable bacteria is 10 8 cfu/mL or more.
TABLE 6 results of experiments with bile salts
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (9)
1. Lactobacillus plantarumLactobacillus plantarum) Characterized in that the lactobacillus plantarum isLactobacillus plantarum) The microbial strain is preserved in China general microbiological culture Collection center (China Committee) with the preservation number: CGMCC No.23655.
2. Lactobacillus plantarum according to claim 1Lactobacillus plantarum) Characterized in that the lactobacillus plantarum isLactobacillus plantarum) Derived from human breast milk.
3. A microbial inoculum comprising the Lactobacillus plantarum strain of claim 1Lactobacillus plantarum）。
4. Lactobacillus plantarum according to claim 1Lactobacillus plantarum) Or the use of the microbial inoculum of claim 3 for preparing uric acid-reducing products.
5. The use according to claim 4, wherein the product comprises feed and pharmaceuticals.
6. A feed comprising the Lactobacillus plantarum of claim 1Lactobacillus plantarum) Or the microbial agent of claim 3.
7. A medicament comprising the Lactobacillus plantarum of claim 1Lactobacillus plantarum) Or the microbial agent of claim 3.
8. The medicament of claim 7, further comprising an adjuvant.
9. Use of the feed of claim 6 or the medicament of claim 7 in any of the following a-f:
a. preparing a product for preventing or treating hypertension;
b. preparing a product for preventing or treating insulin resistance;
c. preparing a product for preventing or treating obesity;
d. preparing a product for preventing or treating metabolic syndrome;
e. preparing a product for preventing or treating the non-alcoholic fatty liver disease;
f. preparing a product for preventing or treating cardiovascular diseases.
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