A plant lactobacillus fmbbl l23180 cll metabolizing pectin polysaccharide, a microbial inoculum, and application thereof

Abstract

The present application relates to the field of biotechnology, in particular to a plant lactobacillus FMBL L23180CLL capable of metabolizing pectin polysaccharide, bacterial agent and application thereof, the plant lactobacillus FMBL L23180CLL is preserved in China typical culture preservation center on August 15, 2024, and the preservation number is CCTCC NO:M 20241793;The strain of the application has good metabolic ability of pectin, can effectively utilize pectin as carbon source to realize 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 tolerance performance;It can be used for preparing fermented food, health products, food additives and feed, and has wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a plant lactobacillus fermentum FMBLL23180CLL metabolizing pectin polysaccharide, a bacterial agent and application thereof. BACKGROUND

[0002] Prebiotics generally refer to some organic substances which 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, which widely exists in the primary cell wall and middle layer of fruits, vegetables, cereals and bran, and accounts for two-thirds of the dry weight of the main cell wall of plants. It is an important component for maintaining the integrity, strength and flexibility of the cell wall, 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 other 13 different monosaccharides. It has physiological functions such as reducing cholesterol, anti-tumor, antioxidant, reducing blood sugar, benefiting intestinal health, etc., and is widely used in food, medicine and other fields; it can improve the intestinal flora structure disorder caused by high-fat diet, promote the growth of beneficial bacteria, inhibit the proliferation of harmful bacteria, and maintain the 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 plays an important role in regulating the metabolism of the body. Pectin can also regulate sugar metabolism, regulate blood sugar and lipid levels, improve glucose tolerance and insulin resistance, repair organ damage, and improve type II diabetes.

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

[0004] Lactiplantibacillus plantarum is a recognized probiotic, which has the functions of maintaining the balance of human intestinal flora, regulating immunity, reducing blood lipids and regulating blood pressure. Compared with other lactic acid bacteria, Lactiplantibacillus plantarum has strong carbohydrate utilization ability. This characteristic plays an important role in the colonization of Lactiplantibacillus plantarum in the gastrointestinal tract and the exertion of its probiotic functions. Carbohydrates play a crucial role in bacterial growth. Carbohydrate metabolism produces intermediates that are components of bacterial cell structure, and also provides energy for bacterial survival and promotes microbial growth. However, there are few reports on Lactiplantibacillus plantarum that can directly utilize pectin.

[0005] The inventors isolated a Lactiplantibacillus plantarum FMBL L23180 CLL from human intestinal tract, which can better metabolize pectin, effectively utilize pectin as a carbon source to achieve rapid proliferation, ferment pectin to produce a large amount of short-chain fatty acids, has good carbon source utilization ability and acid and bile salt tolerance; can be used for preparing fermented food, health products, food additives and feed, and has broad application prospects. SUMMARY

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

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

[0008] The third object of the present application is to provide a freeze-dried agent containing the Lactiplantibacillus plantarum FMBL L23180 CLL.

[0009] The fourth object of the present application is to provide the use of the Lactiplantibacillus plantarum FMBL L23180 CLL and / or the bacterial agent and / or the freeze-dried agent in the preparation of functional products, which have one or more of the following functions:

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

[0011] (2) acid tolerance;

[0012] (3) bile salt tolerance;

[0013] (4) pectin metabolism ability;

[0014] (5) production of short-chain fatty acids by utilizing pectin.

[0015] Preferably, the functional product is one or more of a food, a food additive, a dietary supplement, a health product, a pharmaceutical product, a feed or a feed additive.

[0016] A fifth object of the present application is to provide the use of the Lactiplantibacillus plantarum FMBL L23180 CLL and / or the bacterial agent and / or the freeze-dried agent in the preparation of a fermented dairy product, a fermented legume product or a fermented fruit and vegetable product.

[0017] A sixth object of the present application is to provide a synbiotic comprising the Lactiplantibacillus plantarum FMBL L23180 CLL or the bacterial agent or the freeze-dried agent.

[0018] Preferably, the synbiotic further comprises one or more of pectin, pecticoligosaccharides, resistant starch, D-trehalose, soy oligosaccharides, galactooligosaccharides, raffinose, maltodextrin, fructooligosaccharides, isomaltooligosaccharides, xylooligosaccharides, stachyose and inulin.

[0019] The present application provides a Lactiplantibacillus plantarum FMBL L23180 CLL, which was deposited with the China Center for Type Culture Collection on August 15, 2024, and has the accession number CCTCC NO: M 20241793. The Lactiplantibacillus plantarum FMBL L23180 CLL has good pectin metabolism ability, 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 tolerance. It can be used for the preparation of microecological preparations, fermented foods, health products, food additives and feeds, and has a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Colony characteristics and microscope characteristics of Lactiplantibacillus plantarum FMBL L23180 CLL

[0021] Figure 2 Phylogenetic tree constructed based on the GroEL gene sequence of Lactiplantibacillus plantarum FMBL L23180 CLL

[0022] Figure 3 Growth curves of 6 Lactobacillus strains in pectin-only carbon source medium

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

[0024] The following embodiments facilitate a better understanding of the present application, but are not limited to the present application. The experimental methods in the following examples are all conventional laboratory methods unless otherwise specified, and the experimental materials used in the following examples are all conventional biochemical reagents unless otherwise specified. The quantitative tests in the following examples are all triplicate tests, and the results are averaged.

[0025] The apple pectin in the following implementation case is purchased from McLean reagent.

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

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

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

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

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

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

[0032] Example 1, strain isolation and identification

[0033] 1. Strain isolation and purification

[0034] Fresh fecal samples of Kazakh children in Yili State, Xinjiang were collected, gradient dilution plating method was used, and the strains were isolated and cultured under anaerobic conditions (80% nitrogen, 10% hydrogen, 10% carbon dioxide).

[0035] Dissolve 1 g of fecal sample into 9 mL of MRS liquid medium, mix well, dilute the fecal sample to 10 -3 -4 -5 , and coat on MRS agar medium, cultivate at 37℃ for 24-48 hours. Pick the suspected lactic acid bacteria colonies and purify for 3 times, then store the isolated strain in MRS liquid medium added with 30% glycerol and store at -20℃. A strain is isolated from it and named FMBL L23180 CLL.

[0036] 2. Strain identification

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

[0038] Table 1. PCR amplification of groEL gene

[0039]

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

[0041] In the following examples, Lactiplantibacillus plantarum FMBL L23180 CLL is simply written as Lactiplantibacillus plantarum FMBL L23180 CLL.

[0042] Example II. Metabolic effect of Lactiplantibacillus plantarum FMBL L23180 CLL on pectin

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

[0044] The experimental strains: Lactiplantibacillus plantarum FMBL L23180 CLL, Lactiplantibacillus plantarum FMBL L23181 CLL, Lactiplantibacillus plantarum FMBL L23190 CLL, Lacticaseibacillus rhamnosus FMBL L23136 YWT, Lacticaseibacillus rhamnosus FMBL L23139 YWT were isolated from the feces of healthy people and preserved in the Center for Food Microbiology and Biotechnology Research of the College of Food Science of Shihezi University; Lacticaseibacillus rhamnosus LGG was purchased from the China Industrial Microbial Culture Collection Center.

[0045] After the above strains were activated in MRS liquid medium for 2 generations, the supernatant was removed by centrifuging the bacterial liquid at 8000 rpm for 10 min, and the bacterial slurry was washed twice with sterile normal saline and resuspended as a bacterial liquid (OD600=1.0±0.05). According to the inoculation amount of 2%, it was added to the MRS medium with pectin (addition amount 2.0%) as the only carbon source, and fermented in a 37°C incubator for 24 h. Every 2 h, the ultraviolet absorbance value (OD value) at 600 nm was measured, and the growth curve of the strain was drawn.

[0046] The growth curve is shown in Figure 3 At the end of fermentation, the absorbance values of Lacticaseibacillus rhamnosus FMBL L23136 YWT, Lacticaseibacillus rhamnosus FMBL L23139 YWT and Lacticaseibacillus rhamnosus LGG were all <0.35, indicating that the three strains could not effectively utilize pectin. The absorbance values of Lactiplantibacillus plantarum FMBL L23180 CLL, Lactiplantibacillus plantarum FMBL L23181 CLL and Lactiplantibacillus plantarum FMBL L23190 CLL were all >1.0 at the end of fermentation, indicating that the three strains of Lactiplantibacillus plantarum could utilize pectin, but among them, Lactiplantibacillus plantarum FMBL L23180 CLL had the strongest ability to utilize pectin. Lactiplantibacillus plantarum FMBL L23180 CLL entered the logarithmic growth phase after 4 h of fermentation, and entered the stationary phase after 12 h. The OD value at the end of fermentation was about 1.3, which was significantly higher than that of other strains, indicating that Lactiplantibacillus plantarum FMBL L23180 CLL had good ability to metabolize pectin and could effectively utilize pectin as a carbon source to achieve rapid proliferation.

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

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

[0049] The determination of total sugar content adopts anthrone-sulfuric acid method. 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mL of glucose standard solution was taken into a 10 mL test tube, water was added to 2 mL, 6 mL of anthrone sulfuric acid solution was added, boiled in a water bath for 15 min, taken out, quickly cooled for 15 min, and the OD 620 was measured to draw a standard curve. The lactic acid bacteria fermentation broth was diluted 20 times with distilled water, and the absorbance value of the lactic acid bacteria fermentation broth was determined according to the above method, and the total sugar content was calculated by substituting the standard curve.

[0050] It can be seen that Figure 3 and Figure 4 the fermentation process of Lactobacillus plantarum FMBL L23180 CLL uses pectin as a carbon source to rapidly proliferate, causing the total sugar to rapidly decrease and the pH to decrease to 4.01; while the total sugar content of Lactobacillus rhamnosus LGG changes little during the fermentation process, and the pH does not decrease significantly. During the fermentation process, pectin is rapidly utilized by Lactobacillus plantarum FMBL L23180 CLL within 0-12h, and after 12h, the growth of Lactobacillus plantarum FMBL L23180 CLL enters a stable period, and the total sugar consumption rate gradually decreases, reaching more than 70% at 24h of fermentation, which further indicates that Lactobacillus plantarum FMBL L23180 CLL can better metabolize pectin.

[0051] Example Three, Lactobacillus plantarum FMBL L23180 CLL Fermentation of Pectin Produces Short-Chain Fatty Acids

[0052] The short-chain fatty acids (SCFA) in the lactic acid bacteria fermentation broth were analyzed by gas chromatograph. The fermentation broth was centrifuged at 10000 r / min for 15 min to remove cells and impurities, filtered through a 0.22 μm filter membrane, and then analyzed for the content of SCFA produced by Lactobacillus plantarum FMBL L23180 CLL during fermentation using an Agilent 7890B gas chromatograph.

[0053] Chromatographic column uses DB-WAX analysis column (60 m x 250 pm x 0.25 pm), injection port temperature: 250 °C; column oven conditions: initial temperature 100 °C for 30 s, 8 °C per minute, 160 °C for 1 min, 20 °C / min, 200 °C for 5 min; injection volume 1 pL; carrier gas conditions: N2, flow rate 1.2 mL / min, split ratio 2:1; FID detector conditions: temperature 250 °C, tail gas: N2(25 mL / min), H2(30 mL / min), air (400 mL / min).

[0054] Table 2. The content of SCFA produced by Lactiplantibacillus plantarum FMBL L23180 CLL fermentation pectin

[0055]

[0056] Short-chain fatty acids are the main postbiotics produced by probiotic fermentation of dietary fiber, which plays an important role in the body. In addition to providing energy, it can enhance the barrier function of the intestinal tract, maintain the integrity of the intestinal barrier, and has anti-inflammatory, anti-tumor and immune regulation effects, and can regulate glucose and fat metabolism, and has an important role in intestinal function and the immune system. As can be seen from Table 2, a large amount of SCFA is produced during the fermentation of pectin by Lactiplantibacillus plantarum FMBL L23180 CLL, among which the content of valeric acid is the highest, reaching 542.23 pg / mL, followed by acetic acid, with a content of 109.96 pg / mL.

[0057] Example Four, Acid and Cholosalt Tolerance of Lactiplantibacillus plantarum FMBL L23180 CLL

[0058] Activated Lactiplantibacillus plantarum FMBL L23180 CLL (OD 600 : 1.0 ± 0.05) was inoculated in MRS liquid medium at an inoculation amount of 2%, and cultured at 37 °C for 24 h. The supernatant was discarded by centrifuging at 8000 rpm for 10 min, and the bacterial precipitate was collected. Then, different pH values (2.5, 3, 3.5, 4) and different concentrations of cholate (0.05%, 0.1%, 0.5%) were added to the MRS liquid medium, and it was placed at 37 °C for 4 h. The bacterial liquid at 0 h and 4 h was respectively plated and counted, and the survival rate under different pH conditions was calculated.

[0059] Table 3. Cholosalt Tolerance Survival Rate

[0060]

[0061] Table 4. Acid Tolerance Survival Rate

[0062]

[0063] As shown in Table 3, the survival rate of the plantaricin FMBL L23180 CLL reached 27.11% and 17.38% after 4h treatment at 0.05% and 0.1% bile salt concentration, respectively, and the plantaricin FMBL L23180 CLL still survived at 0.1% bile salt concentration. As shown in Table 4, the survival rate of the plantaricin FMBL L23180 CLL was still 6.46% at pH 2.5. The above results show that the plantaricin 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 exerting the probiotic effect in the gastrointestinal tract.

[0064] Example Five, Carbohydrate Metabolism Experiment

[0065] The activated plantaricin FMBL L23180 CLL was inoculated into modified MRS liquid medium with prebiotic: resistant starch, chitosan, D-trehalose, soybean oligosaccharide, galactooligosaccharide, raffinose, maltodextrin, fructooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, stachyose, inulin as the only carbon source at an inoculation amount of 2% (v / v) (OD 600 : 1.0 ± 0.05), with glucose as the positive control, and no carbon source as the negative control. The absorbance of OD 600 at 0h was OD1, and the absorbance of OD 600 at 37℃ after 24h culture was OD2, and the final OD 600 = OD1- OD2. The experiment was repeated three times, and the average value was taken.

[0066] Prebiotics are not 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 show that the plantaricin FMBL L23180 CLL strain can effectively utilize 11 kinds of carbon sources, including resistant starch, D-trehalose, soybean oligosaccharide, galactooligosaccharide, raffinose, maltodextrin, fructooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, stachyose and inulin, but cannot effectively utilize chitosan.

[0067] Table 5 Carbohydrate utilization ability of plantaricin FMBL L23180 CLL

[0068] soybean oligosaccharides 1.460 resistant starch 0.308 maltodextrin 1.376 raffinose 1.441 xylooligosaccharides 0.502 galactooligosaccharides 1.220 fructooligosaccharides 1.592 stachyose 0.843 isomaltooligosaccharides 1.406 inulin 0.486 chitooligosaccharides 0.084 D-fucosylated oligosaccharides 1.490 positive control 1.610 negative control 0.200

[0069] In summary, the present application provides a plant lactobacillus FMBL L23180 CLL capable of metabolizing pectin polysaccharide, a microbial inoculant and application thereof. 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, can ferment pectin to produce a large amount of short-chain fatty acids, has good carbon source utilization ability and acid and bile salt tolerance, and can be used for preparing fermented food, health products, food additives and feed, and has a broad application prospect.

Claims

1. A plant lactobacillus that metabolizes pectin polysaccharides ( Lactiplantibacillus plantarum FMBLL23180 CLL, characterized in that, The *Lactobacillus plantarum* FMBL L23180 CLL was deposited at the China Center for Type Culture Collection on August 15, 2024, with accession number CCTCC NO: M 20241793.

2. A microbial agent, characterized in that, Contains *Lactobacillus plantarum* FMBL L23180 CLL as described in claim 1.

3. A lyophilizing agent, characterized in that, Contains *Lactobacillus plantarum* FMBL L23180 CLL as described in claim 1.

4. The use of *Lactobacillus plantarum* FMBL L23180 CLL as described in claim 1, or the microbial agent as described in claim 2, or the freeze-drying agent as described in claim 3, in the preparation of food, food additives, dietary supplements, or feed additives.

5. The application of *Lactobacillus plantarum* FMBL L23180 CLL as described in claim 1, or the microbial agent as described in claim 2, or the freeze-drying agent as described in claim 3, in the preparation of fermented dairy products, soy products, or fruit and vegetable products.

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