Rapid screening method of cholesterol-lowering lactic acid bacteria

Abstract

The invention discloses a rapid screening method of cholesterol-lowering lactic acid bacteria, and belongs to the technical field of biology. Comprising the following steps: (1) coating a fermented food sample solution into an MRS-maize germ oil solid culture medium for preliminary screening culture, and screening single colonies with acid producing circles; (2) carrying out catalase inspection and gram staining microscopy on the strains obtained by primary screening, and screening out bacterial colonies with positive gram staining results and negative catalase experimental results; and (3) inoculating the primarily screened strain into a liquid culture medium containing cholesterol micelles for secondary screening culture, measuring the cholesterol concentration before and after culture, calculating the degradation rate, and selecting the strain with the strongest cholesterol degradation capability as a target strain. According to the method, in the screening process of the cholesterol-reducing lactic acid bacteria, the lactic acid bacteria can be effectively screened, experimental steps are simplified, and the cholesterol-reducing lactic acid bacteria can be primarily screened, so that the overall screening efficiency is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a rapid screening method of a cholesterol-lowering lactic acid bacteria. BACKGROUND

[0002] Cardiovascular and cerebrovascular diseases are mainly caused by the deposition of high blood lipids (especially low-density liprotein cholesterol) at the damaged site of the arterial intima to form atherosclerotic plaques and increase the viscosity of blood. High blood lipids refer to the excessive content of lipids in the blood of the human body. These lipids are mainly divided into three types: (1) neutral fat: triglyceride (TG), which is the main energy storage substance in the body and is involved in energy metabolism. (2) Cholesterol: total cholesterol (TC) includes high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and very low-density lipoprotein cholesterol (VLDL-C). TC is an essential lipid for the human body, mainly synthesized in the liver, which is essential for maintaining the integrity of the cell membrane, synthesizing hormones and vitamin D, and producing bile acids. HDL-C can transport cholesterol from peripheral tissues back to the liver for metabolism, which helps prevent atherosclerosis, while LDL-C is responsible for transporting cholesterol from the liver to peripheral tissues, and its elevated levels are a major risk factor for atherosclerosis and cardiovascular and cerebrovascular diseases. VLDL-C has a small content and has a small impact on the human body. (3) Lipids: including phospholipids, glycolipids, sterols and steroids, etc. These lipids exist in the form of lipoproteins in the blood, and lipoproteins are divided into high-density lipoprotein (HDL), low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) according to their density.

[0003] Currently, clinical treatment for lowering cholesterol primarily relies on drug intervention, supplemented by dietary adjustments. This typically involves statins, cholesterol ester transporter inhibitors, and cholesterol absorption inhibitors. Statins alter the lipid composition of hepatocyte membranes, leading to decreased membrane stability and leakage of liver enzymes, resulting in elevated serum transaminase levels and adverse liver reactions. Furthermore, the products of their metabolism via cytochrome P450 oxidase (CYP450 enzyme) are hepatotoxic. Cholesterol ester transporter inhibitors cause an acute increase in plasma aldosterone levels, activating mineralocorticoid receptors, leading to hypermineralocorticoid levels and electrolyte changes, thereby causing elevated blood pressure. Cholesterol absorption inhibitors selectively inhibit the activity of the specific transporter NPC1L1 at the brush border of the small intestinal mucosa, reducing cholesterol transported into intestinal epithelial cells via vesicular endocytosis. However, while inhibiting cholesterol absorption, they also affect the absorption of other substances in the intestine, causing digestive system reactions. These reactions include diarrhea, abdominal pain, and flatulence. Taking lactic acid bacteria can regulate serum cholesterol levels without adverse liver reactions (such as elevated transaminase levels) or increased risk of hypertension, and its metabolites are non-toxic. Compared to medication, using lactic acid bacteria as a means of lowering cholesterol does not lead to drug dependence and is more economical.

[0004] However, current technologies for screening lactic acid bacteria are slow, and their cholesterol-lowering ability is relatively limited. Therefore, rapid screening methods for cholesterol-lowering lactic acid bacteria need further improvement. Summary of the Invention

[0005] The purpose of this invention is to provide a rapid screening method for cholesterol-lowering lactic acid bacteria to address the problems existing in the prior art. This invention aims to provide a rapid screening method for cholesterol-lowering lactic acid bacteria, wherein the strain possesses significant screening ability and time-saving characteristics in the screening process. Addressing the problems of slow screening speed and potentially limited cholesterol-lowering ability of lactic acid bacteria in the prior art, this invention, through a specific screening and optimization process, obtains lactic acid bacteria strains with a certain cholesterol-lowering ability, capable of maintaining efficient cholesterol degradation under simulated in vitro gastrointestinal environment conditions.

[0006] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is a method for screening cholesterol-lowering lactic acid bacteria, comprising the following steps: (1) The fermented food sample liquid was spread onto MRS-corn germ oil solid medium for initial screening and culture to screen single colonies with acid-producing zones; (2) The strains obtained from the initial screening were subjected to catalase test and Gram staining microscopy to screen out colonies that were positive for Gram staining and negative for catalase test. (3) The primary screening strains were inoculated into a liquid culture medium containing cholesterol micelles for secondary screening culture. The cholesterol concentration before and after culture was measured, the degradation rate was calculated, and the strain with the strongest cholesterol degradation ability was selected as the target strain.

[0007] Based on the above technical solution, the present invention has the following technical effects: (1) This invention provides a convenient and economical screening method. Traditional screening methods often use cholesterol or cholesterol micelles to screen lactic acid bacteria, but solvents such as cholesterol and glacial acetic acid are expensive and pose certain risks. Using corn germ oil instead of cholesterol can reduce the use of expensive chemical additives while meeting the need for rapid screening of cholesterol-lowering lactic acid bacteria.

[0008] (2) The screening method of the present invention can effectively screen lactic acid bacteria with cholesterol-degrading ability under limited time conditions, reduce the occurrence of lactic acid bacteria with low cholesterol tolerance, and reduce the workload of subsequent rescreening of cholesterol-lowering lactic acid bacteria.

[0009] (3) The cholesterol-lowering ability of the strain helps regulate serum cholesterol levels and prevent and treat cardiovascular diseases. Attached Figure Description

[0010] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0011] Figure 1 The acid-producing zone of the colony.

[0012] Figure 2 This describes the colony morphology of the bacterial strain.

[0013] Figure 3 This is a Gram staining morphology diagram.

[0014] Figure 4 This represents the cholesterol degradation rate.

[0015] Figure 5 This is the growth curve.

[0016] Figure 6 This refers to the ability to produce acid. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0023] This invention provides a method for screening cholesterol-lowering lactic acid bacteria, comprising the following steps: (1) The fermented food sample liquid was spread onto MRS-corn germ oil solid medium for initial screening and culture to screen single colonies with acid-producing zones; (2) The strains obtained from the initial screening were subjected to catalase test and Gram staining microscopy to screen out colonies that were positive for Gram staining and negative for catalase test. (3) The primary screening strains were inoculated into a liquid culture medium containing cholesterol micelles for secondary screening culture. The cholesterol concentration before and after culture was measured, the degradation rate was calculated, and the strain with the strongest cholesterol degradation ability was selected as the target strain.

[0024] In some specific implementations, the concentration of corn germ oil in the MRS-corn germ oil solid culture medium is 5-20 g / L.

[0025] In some specific implementations, the initial screening culture is carried out at 37°C and an initial pH of 7.0 for 24-48 hours.

[0026] In some specific embodiments, the concentration of cholesterol micelles in the liquid culture medium containing cholesterol micelles is 10 ml / L.

[0027] In some specific implementation schemes, the concentration of cholesterol before and after culture is determined by the phthalaldehyde method.

[0028] This invention discloses a rapid screening method for cholesterol-lowering lactic acid bacteria. This method not only effectively screens lactic acid bacteria and simplifies experimental procedures, but also performs preliminary screening of cholesterol-degrading lactic acid bacteria, thereby improving overall screening efficiency. The method includes the following steps: 1) preparation of a culture medium specifically designed to promote the growth and preliminary screening of lactic acid bacteria; 2) further screening of the strains to ensure their activity and stability in the cholesterol micelle environment; 3) assessment of cholesterol degradation by measuring changes in cholesterol in the culture medium during the culture process.

[0029] Lactic acid bacteria, as the most common probiotics, are widely found in fermented foods. Fermented foods, a representative food unique to Asian diets, contribute a large number of foodborne microbial strains to the host's gut microbiota, and the lactic acid bacteria screened from them are characterized by high safety. In recent years, with in-depth research on lactic acid bacteria, it has been discovered that various lactic acid bacteria, such as *Lactobacillus plantarum*, *Lactobacillus fermentum*, and *Lactobacillus rhamnosus*, have beneficial effects on cholesterol metabolism and gut microbiota, and are considered effective strategies for preventing and improving cardiovascular and cerebrovascular-related chronic diseases, possessing broad market application prospects and significant social health implications.

[0030] The lactic acid bacteria strains screened by this invention have the following characteristics and application value: Rapid screening capability: The method of the present invention exhibits rapid screening capability during the screening process, quickly screening lactic acid bacteria with cholesterol-lowering ability and inhibiting the growth of harmful microorganisms.

[0031] Highly efficient cholesterol degradation: The lactic acid bacteria screened in this invention can effectively reduce cholesterol levels, regulate human serum cholesterol levels, and prevent and treat hypercholesterolemia.

[0032] Wide range of applications: The lactic acid bacteria strains screened in this invention can be widely used in the production of fermented vegetables, dairy products, meat products and other foods to improve the nutritional value of these foods.

[0033] Biosafety: Using the lactic acid bacteria strains of this invention as a biotechnology means to replace drug intervention and treatment reduces the adverse reactions of drug treatment, which is in line with the pursuit of modern treatment.

[0034] Economic and social benefits: Taking lactic acid bacteria can regulate serum cholesterol levels without adverse liver reactions (such as elevated transaminase levels) or increased risk of hypertension, and its metabolites are non-toxic. Compared with drugs, using lactic acid bacteria as a means of lowering cholesterol does not lead to drug dependence and is more economical.

[0035] The lactic acid bacteria strains screened in this invention can degrade at least 50% of cholesterol within 48 hours under conditions of 37°C and pH 7.0.

[0036] This invention provides a method for applying lactic acid bacteria strains, including: (1) Adding lactic acid bacteria during food processing can improve human cholesterol levels by utilizing the degradation ability of the strains.

[0037] (2) Dietary supplements made from cholesterol-lowering lactic acid bacteria in the form of microcapsules, such as probiotic powder and probiotic tablets. People can take them directly, which is convenient and quick to supplement probiotics and play a role in lowering cholesterol.

[0038] (3) After extracting, separating and purifying cholesterol-lowering lactic acid bacteria or their metabolites, they are made into pharmaceutical preparations, such as capsules, tablets, oral liquids, etc., for the treatment of diseases such as hypercholesterolemia.

[0039] (4) Adding cholesterol-lowering lactic acid bacteria to the feed of livestock and poultry can reduce the cholesterol content in their blood, improve their meat quality, enhance their immunity, and promote their growth and development.

[0040] Example 1 Isolation and purification of lactic acid bacteria Take 1 mL of fermented food sample solution and serially dilute it to 10-fold using physiological saline. -7 Take samples of each diluted 10... -4 10 -5 10 -6 10 -7 100 μL of bacterial suspension was spread onto 15–20 mL of MRS-corn germ oil (5–20 g / L) solid medium, cooled and solidified, and then incubated at 37 °C for 24 h. Single colonies were picked according to the size of the acid-producing zone, and purified by streaking repeatedly on MRS solid medium 2–3 times and examined under a microscope to obtain pure colonies.

[0041] Example 2 like Figure 1Taking six lactic acid bacteria strains preserved in the laboratory as an example, their cholesterol degradation rates were 18.31%, 29.36%, 58.73%, 54.76%, 48.78%, and 52.44%, respectively. Lactic acid bacteria strains with degradation rates below 30% either did not grow or showed no acid-producing zone. Figure 2 The colonies are pale yellow, smooth, round, and raised with regular edges. The colony diameter is 1–3 mm. Figure 3 Its Gram staining result was positive, and its catalase test result was negative.

[0042] Example 3 Secondary screening of cholesterol-degrading lactic acid bacteria The purified and preserved bacterial strains were inoculated into MRS broth for activation twice. The activated bacterial solution was then inoculated into 50 mL of MRS broth containing cholesterol micelles (10 mL / L) at a volume fraction of 2%, and cultured at 37°C for 48 h. 1 mL of the supernatant was transferred to a new test tube, and 4 mL of 95% ethanol and 3 mL of 33% KOH were added. The tube was heated in a 60°C water bath for 15 min, followed by the addition of 5 mL of n-hexane and 4 mL of distilled water. After thorough shaking, the mixture was allowed to separate into layers. 3 mL of the upper layer was transferred to a small test tube and evaporated under nitrogen at 60°C. 5 mL of phthalaldehyde and 3 mL of concentrated sulfuric acid were added, and the mixture was thoroughly mixed. The OD550 was measured. The cholesterol removal rate of each strain was calculated using the following formula. The experiment was repeated five times, and the average value was calculated. Cholesterol removal rate (%) = (AB) / A*100%; In the formula: A: represents the OD value of the blank group at a wavelength of 550 nm; B: represents the OD value of the experimental group at a wavelength of 550 nm. The cholesterol degradation rate of lactic acid bacteria is as follows: Figure 4 .

[0043] Example 4 Further molecular biology techniques were used to identify the strain, extract its genomic DNA, amplify its 16S rDNA gene sequence by PCR, and sequence it. The sequencing results were then compared with BLAST homology sequences in NCBI. The results are shown in Table 1. 16S rDNA sequence analysis results of lactic acid bacteria.

[0044] Table 1. Results of 16S rDNA sequence analysis of lactic acid bacteria

[0045] Example 5 Determination of growth curve The strain was activated twice by inoculating it into MRS broth medium, and then inoculated into MRS broth at a volume fraction of 2%. The culture was incubated at 37°C, and the OD was measured every 3 hours.600 absorbance value (nm). Growth curve shown. Figure 5 .

[0046] Example 6 Determination of acid-producing capacity The activated bacterial strain was inoculated into MRS liquid medium at a 2% inoculum and incubated at 37°C for 12 hours. The pH after fermentation was measured every 3 hours, and the experiment was repeated 3 times. The pH value after lactic acid bacteria fermentation is shown in [reference needed]. Figure 6 .

[0047] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for screening cholesterol-lowering lactic acid bacteria, characterized in that, Includes the following steps: (1) The fermented food sample liquid was spread onto MRS-corn germ oil solid medium for initial screening and culture to screen single colonies with acid-producing zones; (2) The strains obtained from the initial screening were subjected to catalase test and Gram staining microscopy to screen out colonies that were positive for Gram staining and negative for catalase test. (3) The primary screening strains were inoculated into a liquid culture medium containing cholesterol micelles for secondary screening culture. The cholesterol concentration before and after culture was measured, the degradation rate was calculated, and the strain with the strongest cholesterol degradation ability was selected as the target strain.

2. The screening method according to claim 1, characterized in that, The concentration of corn germ oil in the MRS-corn germ oil solid culture medium is 5-20 g / L.

3. The screening method according to claim 1, characterized in that, The initial screening culture conditions were as follows: cultured at 37°C and an initial pH of 7.0 for 24-48 hours.

4. The screening method according to claim 1, characterized in that, The concentration of cholesterol micelles in the liquid culture medium containing cholesterol micelles is 10 ml / L.

5. The screening method according to claim 1, characterized in that, The concentration of cholesterol before and after culture was determined by the phthalaldehyde method.

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