Phytobacterium plantarum FMBL L23180 CLL capable of metabolizing pectic polysaccharides, fungicide and application of phytobacterium plantarum FMBL L23180 CLL

Abstract

The invention relates to the technical field of biology, in particular to a plant lactobacillus FMBL L23180CLL capable of metabolizing pectic polysaccharides, a fungicide and application of the fungicide, the plant lactobacillus FMBL L23180CLL is preserved in China Center for Type Culture Collection on August 15, 2024, and the preservation number is CCTCC NO: M 20241793; the strain has good pectin metabolism capacity, pectin can be effectively used as a carbon source to achieve rapid proliferation, pectin can be fermented to generate a large amount of short-chain fatty acid, and the strain has good carbon source utilization capacity and acid-resistant and cholate-resistant performance; the strain can be used for preparing fermented food, health care products, food additives and feeds, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a plant lactobacillus FMBLL23180CLL capable of metabolizing pectin polysaccharide, a bacterial agent, and application thereof. Background Art

[0002] Prebiotics generally refer to organic substances that are not digested and absorbed by the host but can selectively promote the metabolism and proliferation of beneficial bacteria in the body, thereby improving the health of the host. Pectin is a natural polysaccharide polymer and an important component of the cell wall of higher plants. It is widely present in the primary cell wall and middle cell of fruits, vegetables, grains, and bran, accounting for about two-thirds of the dry weight of the main cell wall of plants. It is an important component for maintaining the integrity of the cell wall structure, strength and flexibility, and is a new type of prebiotic. It is mainly composed of galacturonic acid (GalA), and contains a large amount of L-rhamnose (Rha), D-arabinose (Ara), D-galactose (Gal) and 13 other different monosaccharides. It has physiological functions such as lowering cholesterol, anti-tumor, anti-oxidation, lowering blood sugar, and promoting intestinal health. It is widely used in food, medicine and other fields; it can improve the disorder of intestinal flora caused by high-fat diet, promote the growth of beneficial bacteria, inhibit the proliferation of harmful bacteria, and maintain intestinal homeostasis. Under the action of intestinal flora, pectin can be converted into metabolites with lipid-lowering activity, such as short-chain fatty acids (SCFA), which play an important role in regulating the body's metabolism. Pectin can also improve type 2 diabetes by regulating sugar metabolism, regulating blood sugar and blood lipid levels, improving glucose tolerance and insulin resistance, and repairing organ damage.

[0003] However, recent studies have found that pectin and its derivatives usually have little molecular structure change when passing through the stomach and small intestinal fluid, and can be fermented and utilized by microorganisms in the large intestine. In other words, the human body cannot directly digest and utilize pectin in the stomach and small intestine. It can only be utilized in the large intestine, and it needs to be utilized with the help of probiotics. Pectin that is not fully utilized may combine with certain minerals (such as calcium, iron, zinc, etc.) in the intestine to form insoluble complexes, thereby interfering with the absorption of these minerals. Long-term human mineral deficiency may occur, especially for those who are prone to mineral deficiency, such as children, pregnant women, the elderly, etc. In some cases, pectin may be combined with certain drugs to affect the absorption and bioavailability of drugs. For example, some drugs for treating chronic diseases such as cardiovascular disease and diabetes may be affected by pectin, reducing their therapeutic effects. Therefore, screening probiotics that can ferment pectin has become a technical problem that needs to be solved urgently by those skilled in the art.

[0004] Lactobacillus plantarum is a recognized probiotic with beneficial effects such as maintaining the balance of human intestinal flora, regulating immunity, lowering blood lipids and regulating blood pressure. Compared with other lactic acid bacteria, Lactobacillus plantarum has a strong ability to utilize carbohydrates. This characteristic plays an important role in the colonization of Lactobacillus plantarum in the gastrointestinal tract and exerting its beneficial effects. Carbohydrates play a pivotal role in bacterial growth. The intermediates produced by carbohydrate metabolism are components of bacterial cell structure, and at the same time provide energy for bacterial survival and promote the growth of microorganisms. However, there are few reports on Lactobacillus plantarum that can directly utilize pectin.

[0005] The inventors isolated a strain of Lactobacillus plantarum FMBL L23180 CLL from the human intestine, which can metabolize pectin well, effectively use pectin as a carbon source to achieve rapid proliferation, ferment pectin to produce a large amount of short-chain fatty acids, and has good carbon source utilization ability and acid and bile salt resistance. It can be used to prepare fermented foods, health products, food additives and feed, and has broad application prospects. Summary of the invention

[0006] In view of the above technical problems, the primary object of the present invention is to provide a Lactobacillus plantarum FMBL L23180 CLL, wherein the Lactobacillus plantarum FMBL L23180 CLL was deposited in the China Center for Type Culture Collection on August 15, 2024, with a deposit number of CCTCC NO: M 20241793.

[0007] The second object of the present invention is to provide a bacterial agent containing the Lactobacillus plantarum FMBL L23180 CLL.

[0008] The third object of the present invention is to provide a lyophilized agent containing the Lactobacillus plantarum FMBL L23180CLL.

[0009] The fourth object of the present invention is to provide the use of the Lactobacillus plantarum FMBL L23180 CLL and / or the bacterial agent and / or the lyophilized agent in the preparation of functional products, wherein the functional products have one or more of the following effects:

[0010] (1) Have good carbon source metabolism ability;

[0011] (2) Acid resistance;

[0012] (3) Tolerance to bile salts;

[0013] (4) Ability to metabolize pectin;

[0014] (5) Use pectin to produce short-chain fatty acids.

[0015] Preferably, the functional product is one or more of food, food additives, dietary supplements, health products, medicines, feed or feed additives.

[0016] The fifth object of the present invention is to provide the use of the Lactobacillus plantarum FMBL L23180 CLL and / or the bacterial agent and / or the lyophilized agent in the preparation of fermented dairy products, bean products or fruit and vegetable products.

[0017] The sixth object of the present invention is to provide a synbiotic, wherein the synbiotic contains the Lactobacillus plantarum FMBL L23180 CLL or the bacterial agent or the lyophilized agent.

[0018] Preferably, the synbiotics also include one or more of pectin, pectin oligosaccharides, resistant starch, D-trehalose, soybean oligosaccharides, galacto-oligosaccharides, raffinose, maltodextrin, fructo-oligosaccharides, isomaltooligosaccharides, xylooligosaccharides, stachyose and inulin.

[0019] The beneficial effects of the present invention are as follows: the present invention provides a plant lactobacillus (Lactiplantibacillus plantarum) FMBL L23180 CLL, which was deposited in the China Center for Type Culture Collection on August 15, 2024, with a deposit number of CCTCC NO: M 20241793. The plant lactobacillus FMBL L23180 CLL has a good ability to metabolize pectin, can effectively utilize pectin as a carbon source to achieve rapid proliferation, and can ferment pectin to produce a large amount of short-chain fatty acids; it has good carbon source utilization ability and acid and bile salt resistance; it can be used to prepare microecological preparations, fermented foods, health products, food additives and feed, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Colony characteristics and microscopic features of Lactobacillus plantarum FMBL L23180 CLL

[0021] Figure 2 Phylogenetic tree based on the CLLGroEL gene sequence of Lactobacillus plantarum FMBL L23180

[0022] Figure 36 Growth curve of Lactobacillus strains in medium with pectin as the only carbon source

[0023] Figure 4 Changes of total sugar content during the fermentation of Lactobacillus plantarum FMBL L23180 CLL DETAILED DESCRIPTION

[0024] The following embodiments are for better understanding of the present invention, but are not limited to the present invention. The experimental methods in the following examples, unless otherwise specified, are all routine laboratory methods, and the experimental materials used in the following examples, unless otherwise specified, are all conventional biochemical reagents. The quantitative tests in the following examples are all repeated three times, and the results are averaged.

[0025] The apple pectin in the following examples was purchased from MacLean's reagent.

[0026] In the following examples, the culture medium formula used is as follows:

[0027] MRS medium: peptone 10.0 g; beef extract 10.0 g; yeast extract 5.0 g; glucose or pectin 20.0 g; Tween 80 1.0 mL; K2HPO4 2.0 g; sodium acetate 5.0 g; diammonium hydrogen citrate 2.0 g; MgSO4·7H2O 0.58 g; MnSO4·4H2O 0.25 g, agar powder 20.0 g, deionized water 1000 mL, pH 6.8.

[0028] MRS liquid culture medium: peptone 10.0 g; beef extract 10.0 g; yeast extract 5.0 g; glucose or pectin 20.0 g; Tween 80 1.0 mL; K2HPO4 2.0 g; sodium acetate 5.0 g; diammonium hydrogen citrate 2.0 g; MgSO4·7H2O 0.58 g; MnSO4·4H2O 0.25 g, deionized water 1000 mL, pH 6.8.

[0029] Nutrient agar medium: 5.0 g peptone, 3.0 g beef extract, 5.0 g sodium chloride, 20.0 g agar powder, and 1000 mL deionized water.

[0030] PYG medium: peptone 10.0 g, yeast extract 5.0 g, glucose 1.0 g, agar powder 20.0 g, deionized water 1000 mL;

[0031] TSA medium: 15.0 g tryptone, 5.0 g soy peptone, 5.0 g sodium chloride, 20.0 g agar powder, 1000 mL deionized water;

[0032] Example 1: Isolation and identification of strains

[0033] 1. Strain isolation and purification

[0034] Fresh stool samples were collected from Kazakh children in Yili Prefecture, Xinjiang, and the strains were isolated by culture under anaerobic conditions (80% nitrogen, 10% hydrogen, and 10% carbon dioxide) using the gradient dilution plate method.

[0035] Take 1g of fecal sample and dissolve it in 9mL MRS liquid culture medium. Mix thoroughly and dilute the fecal sample to 10 -3 , 10 -4 , 10 -5 , and spread on MRS agar medium and cultured at 37°C for 24-48 hours. After picking the suspected lactic acid bacteria colonies and purifying them three times, the isolates were stored in MRS liquid medium supplemented with 30% glycerol and stored at -20°C. A strain was isolated from them and named FMBL L23180 CLL.

[0036] 2. Strain Identification

[0037] The DNA of the strain was extracted using a kit, and the groEL gene of the strain was amplified by PCR, as shown in Table 1. After the PCR reaction, the product was subjected to 1.2% agarose gel electrophoresis to observe the molecular size, and the appropriate PCR product was selected and sent to the company for sequencing. The returned sequencing results were uploaded to the NCBI database for BLAST comparison. After the comparison, the sequence of the corresponding genus was obtained from the database, and the phylogenetic tree was established using MEGA11.0.

[0038] Table 1 PCR amplification of groEL gene

[0039]

[0040] The bacterial strain FMBL L23180 CLL has the following colony characteristics and microscopic characteristics: Figure 1 The phylogenetic tree is shown in Figure 2 As shown, the strain FMBL L23180 CLL was identified as Lactobacillus plantarum, with a Latin name of Lactiplantibacillus plantarum. It was named Lactiplantibacillus plantarum FMBL L23180 CLL, and was deposited in the China Center for Type Culture Collection on August 15, 2024, with a deposit number of CCTCC NO: M 20241793. The deposit address is Wuhan University, Wuhan, China, and the telephone number is (027)-68752319.

[0041] In the following examples, Lactobacillus plantarum FMBL L23180CLL is abbreviated as Lactobacillus plantarum FMBL L23180 CLL.

[0042] Example 2: Metabolic effects of Lactobacillus plantarum FMBLL23180 CLL on pectin

[0043] 1. Growth curve of Lactobacillus plantarum FMBL L23180 CLL in medium with pectin as carbon source

[0044] Experimental strains: Lactobacillus plantarum FMBL L23180 CLL, Lactobacillus plantarum FMBL L23181 CLL, Lactobacillus plantarum FMBL L23190 CLL, Lactobacillus rhamnosus FMBLL23136YWT, and Lactobacillus rhamnosus FMBL L23139 YWT were all isolated from feces of healthy people and preserved in the Food Microbiology and Biotechnology Research Center, School of Food Science and Technology, Shihezi University; Lactobacillus rhamnosus LGG was purchased from the China Industrial Microbiological Culture Collection Management Center.

[0045] After the strain was activated for 2 generations in MRS liquid medium, the bacterial solution was centrifuged at 8000rpm for 10min to remove the supernatant, the bacterial sludge was washed twice with sterile saline and resuspended into a bacterial solution (OD600 = 1.0 ± 0.05), and added to MRS medium with pectin (addition amount 2.0%) as the sole carbon source at a 2% inoculum, and fermented in a 37°C incubator for 24h. The ultraviolet absorbance value (OD value) at 600nm was measured every 2h, and the growth curve of the strain was drawn.

[0046] Growth curve Figure 3 As shown in the figure, at the end of fermentation, the absorbance values ​​of Lactobacillus rhamnosus FMBL L23136 YWT, Lactobacillus rhamnosus FMBL L23139 YWT and Lactobacillus rhamnosus LGG were all <0.35, indicating that the three strains could not effectively utilize pectin. At the end of fermentation, the absorbance values ​​of Lactobacillus plantarum FMBL L23180CLL, Lactobacillus plantarum FMBL L23181 CLL and Lactobacillus plantarum FMBL L23190 CLL were >1.0, indicating that the three strains of Lactobacillus plantarum could utilize pectin, but Lactobacillus plantarum FMBL L23180 CLL had the strongest ability to utilize pectin. Lactobacillus plantarum FMBL L23180 CLL entered the logarithmic growth phase after 4 hours of fermentation and entered the stable phase after 12 hours. The OD value at the end of fermentation was about 1.3, which was significantly higher than that of other strains. This also shows that Lactobacillus plantarum FMBL L23180 CLL has a good ability to metabolize pectin and can effectively use pectin as a carbon source to achieve rapid proliferation.

[0047] 2. Changes in total sugar content and pH during pectin fermentation by Lactobacillus plantarum FMBL L23180 CLL

[0048] After activating Lactobacillus plantarum FMBL L23180 CLL and Lactobacillus rhamnosus LGG for 2 generations, the bacterial solution was centrifuged at 8000 rpm for 10 min to remove the supernatant, the bacterial slurry was washed twice with sterile saline and resuspended into a bacterial solution (OD600=1.0±0.05), and added to MRS medium with pectin (added amount 2.0%) as the sole carbon source according to an inoculum amount of 2%, and fermented in a 37°C incubator for 24 h, and the changes in total sugar content at 0 h, 4 h, 8 h, 12 h, 18 h, and 24 h of fermentation were measured respectively, and the pH at the end of fermentation was measured using a pH meter.

[0049] The total sugar content was determined by the anthrone-sulfuric acid method. Pipette 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mL of glucose standard solution into a 10 mL test tube, add water to 2 mL, add 6 mL of anthrone sulfate solution, boil in water for 15 min, take out, cool rapidly for 15 min, and measure OD 620 , draw a standard curve. Dilute the lactobacillus fermentation liquid 20 times with distilled water, determine the absorbance value of the lactobacillus fermentation liquid according to the above method, and substitute it into the standard curve to calculate the content of total sugar.

[0050] Combination Figure 3 and Figure 4 It can be seen that during the fermentation process of Lactobacillus plantarum FMBL L23180 CLL, pectin was used as a carbon source for rapid reproduction, which caused a rapid decrease in total sugar and a decrease in pH to 4.01; while during the fermentation process of Lactobacillus rhamnosus LGG, the total sugar content changed little and the pH did not decrease significantly. During the fermentation process, pectin was rapidly utilized by Lactobacillus plantarum FMBL L23180 CLL within 0-12 hours. After 12 hours, the growth of Lactobacillus plantarum FMBL L23180 CLL entered a stable period, and the total sugar consumption rate gradually decreased. At 24 hours of fermentation, the total sugar consumption rate reached more than 70%, which further demonstrated that Lactobacillus plantarum FMBL L23180 CLL could metabolize pectin well.

[0051] Example 3: Production of short-chain fatty acids by fermentation of pectin by Lactobacillus plantarum FMBLL23180 CLL

[0052] The short-chain fatty acids (SCFA) in the lactobacillus fermentation broth were analyzed by gas chromatography. The fermentation broth was centrifuged at 10,000 r / min for 15 min to remove cells and impurities, and then filtered through a 0.22 μm filter membrane and the SCFA content produced by Lactobacillus plantarum FMBL L23180 CLL during the fermentation process was analyzed by Agilent 7890B gas chromatograph.

[0053] The chromatographic column used was a DB-WAX analytical column (60m×250μm×0.25μm), with an injection port temperature of 250°C; column oven conditions: initial temperature of 100°C for 30s, increased by 8°C per minute, maintained at 160°C for 1min, then increased at 20°C / min, and maintained at 200°C for 5min; injection volume was 1μL; carrier gas conditions: N2, flow rate was 1.2mL / min, and split ratio was 2:1; FID detector conditions: temperature of 250°C, tail gas: N2 (25mL / min), H2 (30mL / min), air (400mL / min).

[0054] Table 2 SCFA content produced by pectin fermentation by Lactobacillus plantarum FMBL L23180 CLL

[0055]

[0056] Short-chain fatty acids are the main postbiotic substances produced by probiotic fermentation of dietary fiber. They play an important role in the body. In addition to providing energy to the body, they can enhance the barrier function of the intestine and maintain the integrity of the intestinal barrier. They also have anti-inflammatory, anti-tumor and immunomodulatory effects, and can regulate glucose and fat metabolism, which plays an important role in intestinal function and the immune system. As shown in Table 2, a large amount of SCFA is produced during the fermentation of pectin by Lactobacillus plantarum FMBL L23180 CLL, among which valeric acid has the highest content, reaching 542.23 μg / mL, followed by acetic acid, which has a content of 109.96 μg / mL.

[0057] Example 4: Acid and bile salt resistance of Lactobacillus plantarum FMBLL23180 CLL

[0058] Activated Lactobacillus plantarum FMBL L23180 CLL (OD 600 :1.0±0.05) was inoculated into MRS liquid medium at a 2% inoculum, cultured at 37℃ for 24h, centrifuged at 8000rpm for 10min, the supernatant was discarded, and the bacterial precipitate was collected. Then, it was added into MRS liquid medium with different pH values ​​(2.5, 3, 3.5, 4) and different bile salt concentrations (0.05%, 0.1%, 0.5%), and placed at 37℃ for 4h. The bacterial liquid at 0h and 4h was counted on plates to calculate its survival rate under different pH conditions.

[0059] Table 3 Bile salt tolerance survival rate

[0060]

[0061] Table 4 Acid resistance survival rate

[0062]

[0063] As shown in Table 3, after being treated at 0.05% and 0.1% bile salt concentrations for 4 hours, the survival rates of Lactobacillus plantarum FMBL L23180CLL reached 27.11% and 17.38%, respectively, and it can still survive at 0.1% bile salt concentration; as shown in Table 4, Lactobacillus plantarum FMBL L23180 CLL still has a survival rate of 6.46% under the condition of pH 2.5. The above results show that Lactobacillus plantarum FMBL L23180 CLL has good acid and bile salt resistance, and the good acid and bile salt resistance can enable the strain to reach the colonization site, thereby surviving, growing and playing a probiotic role in the gastrointestinal tract.

[0064] Example 5: Carbohydrate metabolism experiment

[0065] Activated Lactobacillus plantarum FMBL L23180 CLL was added at 2% (v / v) (OD 600 : 1.0 ± 0.05) was inoculated into the modified MRS liquid medium with prebiotics: resistant starch, chitosan, D-trehalose, soybean oligosaccharides, galacto-oligosaccharides, raffinose, maltodextrin, fructo-oligosaccharides, isomalto-oligosaccharides, xylo-oligosaccharides, stachyose, and inulin as the sole carbon source, respectively. Glucose was used as the positive control, and no carbon source was added as the negative control. The OD at 0 h was measured. 600 The absorbance of OD1 was determined by culturing at 37℃ for 24h. 600 The absorbance is OD2, and the final OD 600 =OD1-OD2, the experiment was repeated three times and the average value was taken.

[0066] Prebiotics are substances that cannot be digested and absorbed by the human body, but can stimulate or promote the proliferation of probiotics. As shown in Table 5, the results of the carbohydrate metabolism experiment showed that the plant lactobacillus FMBL L23180 CLL strain could effectively utilize 11 carbon sources including resistant starch, D-trehalose, soybean oligosaccharides, galacto-oligosaccharides, raffinose, maltodextrin, oligofructose, oligomalto-oligosaccharides, oligoxylose, stachyose, and inulin, but could not effectively utilize chitosan.

[0067] Table 5 Ability of Lactobacillus plantarum FMBL L23180 CLL to utilize carbohydrates

[0068] soy oligosaccharides 1.460 Resistant Starch 0.308 Maltodextrin 1.376 Raffinose 1.441 Xylo-oligosaccharide 0.502 Galacto-oligosaccharides 1.220 Fructo-oligosaccharides 1.592 Stachyose 0.843 Isomaltooligosaccharide 1.406 Inulin 0.486 Chitosan Oligosaccharide 0.084 D-Trehalose 1.490 Positive Control 1.610 Negative control 0.200

[0069] In summary, the present invention provides plant lactobacillus FMBL L23180 CLL capable of metabolizing pectin polysaccharides, a bacterial agent and an application thereof. The plant lactobacillus FMBL L23180 CLL has good ability to metabolize pectin, can effectively utilize pectin as a carbon source to achieve rapid proliferation, can ferment pectin to produce a large amount of short-chain fatty acids, has good carbon source utilization ability and acid and bile salt resistance; can be used for preparing fermented foods, health products, food additives and feed, and has broad application prospects.

Claims

1. A Lactobacillus plantarum FMBL L23180 CLL, characterized in that: The plant lactobacillus FMBL L23180 CLL was deposited in the China Center for Type Culture Collection on August 15, 2024, with a deposit number of CCTCC NO: M 20241793.

2. A bacterial agent, characterized in that Contains the Lactobacillus plantarum FMBL L23180 CLL according to claim 1.

3. A lyophilized agent, characterized in that: Contains the Lactobacillus plantarum FMBL L23180 CLL according to claim 1.

4. Use of the plant lactobacillus FMBL L23180 CLL according to claim 1 and / or the bacterial agent according to claim 2 and / or the lyophilized agent according to claim 3 in preparing a functional product, characterized in that: The functional product has one or more of the following functions: (1) Have good carbon source metabolism ability; (2) Acid resistance; (3) Tolerance to bile salts; (4) Ability to metabolize pectin; (5) Use pectin to produce short-chain fatty acids.

5. The use according to claim 4, characterized in that The functional product is one or more of food, food additives, dietary supplements, health products, medicines, feed or feed additives.

6. Use of the plant lactobacillus FMBL L23180 CLL according to claim 1 and / or the bacterial agent according to claim 2 and / or the lyophilized agent according to claim 3 in the preparation of fermented dairy products, bean products or fruit and vegetable products.

7. A synbiotic, characterized in that: The synbiotic contains the Lactobacillus plantarum FMBLL23180 CLL according to claim 1 or the bacterial agent according to claim 2 or the lyophilized agent according to claim 3.

8. The synbiotic according to claim 7, characterized in that The synbiotics also include one or more of pectin, pectin oligosaccharides, resistant starch, D-trehalose, soybean oligosaccharides, galacto-oligosaccharides, raffinose, maltodextrin, fructo-oligosaccharides, isomalto-oligosaccharides, xylooligosaccharides, stachyose and inulin.

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