CN113598375A - Application of lactobacillus plantarum with function of reducing cholesterol - Google Patents
Application of lactobacillus plantarum with function of reducing cholesterol Download PDFInfo
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- CN113598375A CN113598375A CN202110967660.7A CN202110967660A CN113598375A CN 113598375 A CN113598375 A CN 113598375A CN 202110967660 A CN202110967660 A CN 202110967660A CN 113598375 A CN113598375 A CN 113598375A
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A61K35/74—Bacteria
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
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Abstract
Application of lactobacillus plantarum with a function of reducing cholesterol belongs to the field of probiotic functions. In order to relieve the overhigh blood fat caused by excessive intake of cholesterol, further various cardiovascular diseases are caused, and certain threats are caused to human health. The invention evaluates the cholesterol-reducing capability of Lactobacillus plantarum J26 through in vitro experiments and animal experiments, proves that the Lactobacillus plantarum J26 has good cholesterol-reducing capability in vitro, can reduce the contents of cholesterol and triglyceride in the serum of a high-fat mouse in vivo, and can reduce the liver weight ratio of the mouse. The Lactobacillus plantarum can be used for preparing health care products or medicines for reducing blood fat.
Description
Technical Field
The invention belongs to the field of probiotic functions, and particularly relates to application of lactobacillus plantarum with a function of reducing cholesterol.
Background
The normal life of human body can be affected by the high content of cholesterol in the human body, cardiovascular diseases are caused, and health hidden troubles are caused. High serum cholesterol levels are more likely to cause cardiovascular and cerebrovascular diseases. Every 1% increase of serum cholesterol content, the incidence rate of cardiovascular and cerebrovascular diseases increases by 3%. Most of the cholesterol in the human body comes from food, except for a small amount of its own synthesis. The us diet and health research council proposed in 1989 to limit cholesterol in food. However, with the improvement of living standard of people, people often pursue high-nutrition diet, which causes unreasonable diet and excessive diet intake of cholesterol, and causes the incidence rate of cardiovascular diseases to rise year by year. Therefore, there is a need to study how to reduce the cholesterol level in the diet and to develop cholesterol-lowering foods.
The hyperlipemia and the hypertension can cause a plurality of organs of an organism to be damaged, so as to cause cardiovascular diseases, the incidence rate of the cardiovascular diseases is rising year by year, and the reduction of the blood fat and the blood pressure becomes a research hotspot in recent years. The lactobacillus is used as a food safe microorganism, researches show that the lactobacillus has the capacity of reducing cholesterol, the lactobacillus can be used for reducing cholesterol, so that side effects caused by drug treatment can be avoided, intestinal flora imbalance caused by cholesterol-reducing drugs can be treated, certain advantages are realized in clinical application, and meanwhile, the lactobacillus also has various probiotic functions and is beneficial to human body health.
Disclosure of Invention
In order to relieve the overhigh blood fat caused by excessive intake of cholesterol, further various cardiovascular diseases are caused, and certain threats are caused to human health. The invention provides an application of Lactobacillus plantarum with a cholesterol reducing function in preparation of a health-care product or a medicine for reducing blood fat, wherein the Lactobacillus plantarum is Lactobacillus plantarum J26.
In one embodiment of the invention, lactobacillus plantarum is activated to obtain a bacterial liquid, bacterial sludge is collected after centrifugation, the bacterial sludge is washed with normal saline for 2-3 times, and the number of bacterial colonies is adjusted to obtain a bacterial suspension.
Further, the culture medium selected for activation is any one of an MRS culture medium, an LB culture medium, and an NB culture medium.
Further defined, the bacterial suspension has a concentration of 108cfu/mL～109cfu/mL。
The invention has the beneficial effects that:
the invention evaluates the cholesterol-reducing capability of lactobacillus plantarum J26 through in vitro experiments and animal experiments, proves that the lactobacillus plantarum J26 has good cholesterol-reducing capability in vitro, can reduce the contents of cholesterol and triglyceride in the serum of a high-fat mouse in vivo, and can reduce the liver weight ratio of the mouse. In addition, the lactobacillus plantarum J26 has resistance to various antibiotics, has no antibiotic transfer risk, has the advantage of not generating harmful substances such as tyramine, cadeverine, putrescine, histamin and the like, and can be used for preparing health-care products or medicines for reducing blood fat.
Description of the drawings:
FIG. 1 is a standard graph of cholesterol;
FIG. 2 shows the results of comparing the cholesterol removal rate of Lactobacillus plantarum J26 with that of another 24 strains of Lactobacillus stored in the laboratory;
FIG. 3 is an electrophoretogram of the extraction of the plasmid of Lactobacillus plantarum J26; wherein M is Mark, and 1-3 are all lactobacillus plantarum J26;
FIG. 4 is a graph showing the results of measurement of nitroreductase activity;
FIG. 5 is a graph showing the results of the detection of the amino acid decarboxylase activity;
FIG. 6 is a graph showing the results of a hemolysis test;
FIG. 7 is a graph showing the results of the indigo substrate test.
Detailed Description
The present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.
The materials, reagents, methods and apparatus used in the following examples, which are not specifically illustrated, are conventional in the art and are commercially available to those skilled in the art.
Lactobacillus plantarum J26 is described in Yicha H, Xuesong Li, Xinyu Liu, Yashuo Zhang, WeiZhang, Chaoxin Man, Yujun Jiang.2019.Transcriptomic responses of Caco-2 cells to Lactobacillus rhamnosus GG and Lactobacillus plantarum J26 againstigative scientific. journal of Dairy science.
Example 1: cholesterol lowering function of lactobacillus plantarum J26
The determination steps of the cholesterol-lowering efficacy of the lactobacillus plantarum J26 are as follows:
(1) taking lactobacillus plantarum J26, other 24 strains of lactobacillus preserved in a laboratory, and E.coli ATCC 25922, S.aureus ATCC 25923 and Salmonella ATCC 14028 preserved in the laboratory, respectively inoculating the lactobacillus plantarum J26, the other 24 strains of lactobacillus preserved in the laboratory, the E.coli ATCC 25922, the S.aureus ATCC 25923 and the Salmonella ATCC 14028 to an MRS culture medium, an LB culture medium and an NB culture medium, separating a single colony after the growth of the strain reaches a logarithmic phase, carrying out subculture for two generations to obtain a working solution, mixing the working solution and glycerol uniformly, and preserving the strain at-80 ℃.
(2) Drawing a cholesterol standard curve
Respectively putting 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL and 0.6mL of cholesterol stock solution in a clean test tube, sequentially adding o-phthalaldehyde and concentrated sulfuric acid after nitrogen blowing, uniformly mixing, standing, measuring the light absorption value at 550nm, drawing a standard curve, and obtaining a labeled curve as shown in figure 1, wherein the linear regression equation is as follows: y is 0.0074x +0.0044 and the correlation coefficient R is2A linear relationship is better illustrated at 0.9951, and a standard curve can be used for cholesterol concentration determination.
(3) The cholesterol concentration was obtained from the standard curve, and the cholesterol removal rate of 25 strains of lactobacillus including lactobacillus plantarum J26 was calculated according to the following formula.
In the formula: c1Mass concentration of cholesterol in supernatant after strain inoculation to MRS-CHOL-THIO for 24h
C0Mass concentration of Cholesterol in the initial Medium
The experiment takes 25 strains including lactobacillus plantarum J26, which are separated from traditional dairy products in the west Tibetan region and lactobacillus preserved in a laboratory as research objects, and comprises 8 strains. The removal rate of cholesterol by the strains was determined by the o-phthalaldehyde method, and the results are shown in fig. 2, wherein 25 lactic acid bacteria have certain cholesterol-lowering ability, but the removal rates of cholesterol are different. The higher the removal rate is, the stronger the cholesterol reducing capability of the strain in vitro is. And selecting the L.plantarum J26 with the highest cholesterol removal rate as a subsequent experimental study object.
(4) Centrifugally collecting bacterial sludge from the bacteria liquid after lactobacillus plantarum J26 is activated for two generations, washing the bacterial sludge for three times by using normal saline, and respectively adjusting the total number of bacterial colonies to be 108cfu/mL and 109cfu/mL, as bacterial suspension for gastric lavage.
(5) And (5) performing intracholesterol reduction experiments by using the fresh bacterial suspension obtained in the step (4) through intragastric gavage every day.
The experimental animals are 40 Kunming-line mice, half of the mice are female and male, the age of the mice is about 4 weeks, the weight of the mice is 20 +/-2 g, the 40 mice are divided into four groups, each group comprises 10 mice, and the specific grouping and feeding modes are shown in table 1. After the mice are purchased, the experiment is carried out for the first week, the environment is adapted, and four groups freely eat the basic feed. In the second week of the experiment, three groups except the control group were fed with high fat diet to establish a high fat model. From the third week of the experiment, four groups of mice were gavaged daily for 28 consecutive days at a dose of 1mL/100g per day per body weight.
TABLE 1 Experimental grouping and feeding methods for in vivo experiments
(6) On the 28 th day of gavage, four groups of mice in step (5) were weighed, eyeballs were bled, then the necks were cut off and sacrificed, livers were dissected out, jejunums were taken out, washed clean, liquid nitrogen-frozen and stored at-80 ℃ for future use.
(7) Centrifuging the blood collected in the step (6), putting serum into a clean centrifugal tube, measuring serum Total Cholesterol (TC), Triglyceride (TG) and high density lipoprotein cholesterol (HDL-C), and calculating an Atherosclerosis Index (AI) by using a formula as follows:
the blood lipid profile of each group of mice is shown in Table 2.
TABLE 2 variation of blood lipid (mmol/L) in different groups of mice
Note: a, b, c, d represent the variability within different groups at the same time (p < 0.05).
(8) The liver weight ratio of each group of mice after 28 days of gastric perfusion was calculated, and the liver weight ratio is the liver weight/body weight, and the change of the liver weight ratio of the different groups of mice is shown in table 3.
TABLE 3 variation of liver weight ratio (mg/g) in different groups of mice
Note: a, b, c, d represent the variability within different groups at the same time (p < 0.05).
Safety evaluation of Lactobacillus plantarum J26
(1) Antibiotic susceptibility assay
Measurement of Minimum Inhibitory Concentration (MIC)
Six clinical common antibiotics of chloramphenicol, gentamicin, vancomycin, erythromycin, ampicillin and tetracycline are selected to carry out sensitivity analysis on the strains, and MRS culture media containing the following antibiotics with different concentrations are respectively prepared: 0.1, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024 μ g/mL. L.plantarum J26 after two generations of activation was inoculated at 2% inoculum size to the above antibiotic-containing medium, and after 24 hours of culture at 37 ℃, the minimum inhibitory concentration was determined and evaluated in comparison with the antibiotic resistance regulation of bacteria established by the European Food Safety Agency (EFSA). The MIC values of lactobacillus plantarum J26 for the different antibiotics are shown in table 4.
TABLE 4 MIC values results (μ g/mL) for Lactobacillus plantarum J26 for different antibiotics
Comparing the assay results with the microbial cut-off, it was found that l.plantarum J26 showed no resistance, i.e. sensitivity, to ampicillin and showed resistance to the other 5 antibiotics.
Extraction and detection of L.plantarum J26 plasmid
Extracting L.plantarum J26 strain plasmids, performing electrophoresis on the extracted plasmid solution in agarose gel for 150V and 20min, detecting whether a plasmid band exists, wherein a plasmid extraction electrophoresis chart is shown in figure 3, as can be seen from figure 3, when plasmid detection is performed on L.plantarum J26, a band does not appear, so that it can be presumed that L.plantarum J26 does not contain a drug-resistant plasmid or a drug-resistant gene exists on bacterial DNA, resistance transfer does not occur in the two cases, and the primary judgment that L.plantarum J26 does not have the risk of resistance transfer is carried out.
(2) Nitroreductase Activity assay
Inoculating activated L.plantarum J26 into nitroreductase detection culture medium, culturing at 37 deg.C for 24 hr, sequentially adding 3-5 drops of alpha-naphthylamine solution and sulfanilic acid solution, and observing color change. If the culture medium turns red, the culture medium is positive; otherwise, it is negative. Coli ATCC 25922 was used as a positive control strain. The detection result is shown in fig. 4, the nitroreductase culture medium inoculated with the positive control strain e.coli ATCC 25922 is red and is positive; whereas the medium inoculated with l.plantarum J26 did not undergo a color change. This indicates that l.plantarum J26 is unable to produce nitroreductase.
(3) Analysis of amino acid Deacidification enzyme Activity
L.plantarum J26 was tested for the production of tyramine (tyramine), histamine (cadeverine), cadaverine (putrescine) and putrescine (histamine) during growth. L.plantarum J26 after two generations of activation was inoculated in four amino acid decarboxylase detection media at an inoculum size of 2%, respectively, and after culturing at 37 ℃ for 72 hours, color change was observed. If the culture medium turns purple, the culture medium is positive; otherwise, it is negative. Salmonella ATCC 14028 was used as a positive control strain. The detection result is shown in fig. 5, the color of the detection culture medium inoculated with the cadaverine and putrescine of the positive control strain Salmonella ATCC 14028 is purple, and the detection result is a positive result; the tyramine, cadeverine, putrescine and histamine detection culture media inoculated with L.plantarum J26 all have yellow color, and the result is negative. Thus, L.plantarum J26 did not produce the harmful metabolites tyramine, cadeverine, putrescine, histamin during growth.
(4) Experimental analysis of hemolysis
L.plantarum J26 after two generations of activation is streaked and inoculated on a Columbia blood agar culture medium, and the culture is carried out for 48 hours at 37 ℃, if grass green hemolytic ring appears, the alpha-hemolysis is obtained; if colorless and transparent hemolytic rings appear, the beta-hemolysis is obtained; if no hemolytic ring exists, the gamma-hemolysis is obtained. S. aureus ATCC 25923 was used as a positive control strain. As shown in fig. 6, the colony of s.aureus ATCC 25923 shows a transparent circle and is subjected to β -hemolysis, while the colony of l.plantarum J26 shows no hemolysis ring and is γ -hemolysis.
(5) Indigo matrix assay
Inoculating L.plantarum J26 after two generations of activation into peptone water culture medium, culturing at 37 ℃ for 24h, adding a small amount of xylene, shaking the test tube to extract the indigo matrix, adding Kovacs reagent along the tube wall after the indigo matrix floats on the surface of the culture medium, and observing the color change of the xylene layer. If the xylene layer is rose red, the result is positive; otherwise, it is negative. Coli ATCC 25922 was used as a positive control strain. The detection result is shown in fig. 7, the liquid surface of the detection culture medium inoculated with the positive control strain e.coli ATCC 25922 shows rose red, which is a positive result; the liquid level of the medium inoculated with L.plantarum J26 had no rose color, and the result was negative. This indicates that L.plantarum J26 does not produce tryptophanase to break down tryptophan to produce indole.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (4)
1. The application of Lactobacillus plantarum with the function of reducing cholesterol in preparing health-care products or medicines for reducing blood fat is characterized in that the Lactobacillus plantarum J26 is used as the Lactobacillus plantarum.
2. The application of claim 1, wherein the lactobacillus plantarum is activated to obtain a bacterial liquid, the bacterial liquid is centrifuged to collect bacterial sludge, the bacterial sludge is washed with normal saline for 2-3 times, and the number of bacterial colonies is adjusted to obtain a bacterial suspension.
3. The use of claim 2, wherein the culture medium selected for activation is any one of MRS culture medium, LB culture medium and NB culture medium.
4. The use according to claim 2, wherein the bacterial suspension is at a concentration of 108cfu/mL～109cfu/mL。
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| CN101864375A (en) * | 2010-01-15 | 2010-10-20 | 吉林省农业科学院 | Lactobacillu plantarurn with function of reducing cholesterol and application thereof |
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