A high-efficiency metabolized pectin polysaccharide fermentation lactobacillus mucus FMBL L24016 DMX and application

Abstract

The present application relates to the field of biotechnology, and in particular to a high-efficiency metabolized pectin polysaccharide fermentation Lactobacillus mukaggaricus FMBL L24016DMX and application, which was preserved in China Center for Type Culture Collection on August 26, 2024, with a preservation number of CCTCC NO: M 20241853, has a good pectin metabolism capacity, can effectively utilize pectin as a carbon source to realize rapid proliferation, and can ferment pectin to produce a large amount of short-chain fatty acids, has a strong scavenging capacity for DPPH and ABTS free radicals, has a good inhibitory effect on alpha-glucosidase and alpha-amylase activity, can be used for preparing antioxidant products, hypoglycemic drugs, fermented foods, health products, food additives and feed, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a fermentation Lactobacillus mucosus FMBL L24016 DMX with high pectin polysaccharide metabolism and application. BACKGROUND

[0002] Prebiotics refer to some organic substances that are not digested and absorbed by the host, but can promote the metabolism and proliferation of beneficial flora in the intestinal tract and improve the health of the host. Pectin is a new type of soluble prebiotic, and has a complex structure and is widely present in the primary cell wall and middle layer of fruits and vegetables. Pectin has adhesion, can increase the adhesion capacity of probiotics on the intestinal wall, improve the survival rate and physiological function of probiotics in the intestinal tract, regulate the pH value and water content in the intestinal tract, and create a microecological environment conducive to the growth of probiotics. Pectin is a complex carbohydrate, but the human body lacks the corresponding enzyme system to fully decompose pectin, so pectin cannot be effectively digested and absorbed in the small intestine; after entering the large intestine, pectin becomes an important substrate for intestinal flora, and intestinal microbial communities carry out fermentation to produce a variety of beneficial metabolites, such as short-chain fatty acids (SCFAs), which play a positive role in maintaining intestinal health, enhancing immune function, improving blood lipid levels, preventing constipation, and possibly reducing the risk of certain diseases.

[0003] Existing research shows that intestinal flora can use pectin to adjust intestinal health, but these are the results of the joint action of complex intestinal flora, and there are few reports on single strains that can use pectin. Invention patent CN104498372B discloses that Fusarium oxysporum BM201 can produce polygalacturonase, pectin lyase, pectin esterase, cellulase, hemicellulase and other pectin enzymes, form a complex pectinase, and then use pectin; invention patent CN115806892B discloses a gene engineering bacterium using pectin, but the above-mentioned strains are obtained by gene engineering culture. Fermented Lactobacillus mucosus plays an important role in maintaining the balance of intestinal flora, regulating immunity, promoting digestion and the like of the human body. However, there is no research to prove that fermented Lactobacillus mucosus has the function of using pectin.

[0004] The inventors isolate a strain of Lactobacillus muciadegradus FMBL L24016 DMX from human intestinal tract, the strain can quickly ferment pectin polysaccharide, can quickly proliferate in a culture medium taking pectin polysaccharide as the only carbon source, and ferment to produce a large amount of short-chain fatty acids; the extracellular supernatant has a free radical scavenging rate of 85.84% and 74.04% on ABTS and DPPH respectively; the extracellular supernatant has an inhibition rate of 49.85% and 11% on the activities of alpha-glucosidase and alpha-amylase; and has good antioxidant and hypoglycemic abilities. The strain can be used for preparing antioxidant products, hypoglycemic drugs, fermented foods, health products, food additives and feed, and has a wide application prospect. SUMMARY

[0005] In view of the above technical problems, a primary object of the present application is to provide a Lactobacillus muciadegradus FMBL L24016 DMX capable of efficiently metabolizing pectin polysaccharide, which was deposited at the China Center for Type Culture Collection on August 26, 2024, and has a deposit number of CCTCC NO: M20241853.

[0006] A second object of the present application is to provide a bacterial agent containing the Lactobacillus muciadegradus FMBL L24016 DMX.

[0007] A third object of the present application is to provide a functional product containing the Lactobacillus muciadegradus FMBL L24016 DMX or the bacterial agent, which has one or more of the following effects:

[0008] (1) has the ability to metabolize pectin;

[0009] (2) produces short-chain fatty acids by utilizing pectin;

[0010] (3) has antioxidant activity;

[0011] (4) has the activity of inhibiting alpha-glucosidase;

[0012] (5) has the activity of inhibiting alpha-amylase.

[0013] Preferably, the functional product is one or more of food, food additive, dietary supplement, health product, drug, feed or feed additive.

[0014] A fourth object of the present application is to provide the use of the Lactobacillus muciadegradus FMBL L24016 DMX or its fermentation liquor or its fermentation sterile filtrate or the bacterial agent in antioxidant or in the preparation of antioxidant products.

[0015] A fifth object of the present application is to provide the application of the fermented Limosilactobacillus fermentum FMBL L24016 DMX or its strain fermentation broth or its fermented sterile supernatant or the bacterial agent in inhibiting alpha-glucosidase or in preparing alpha-glucosidase inhibitors.

[0016] A sixth object of the present application is to provide the application of the fermented Limosilactobacillus fermentum FMBL L24016 DMX or its strain fermentation broth or its fermented sterile supernatant or the bacterial agent in inhibiting alpha-amylase activity or in preparing alpha-amylase inhibitors.

[0017] A seventh object of the present application is to provide the application of the fermented Limosilactobacillus fermentum FMBL L24016 DMX or its strain fermentation broth or its fermented sterile supernatant or the bacterial agent in preparing hypoglycemic drugs and health products.

[0018] The present application provides a fermented Limosilactobacillus fermentum FMBL L24016 DMX, which was deposited with the China Center for Type Culture Collection on August 26, 2024, and has the accession number CCTCC NO: M 20241853. The fermented Limosilactobacillus fermentum FMBL L24016 DMX can rapidly ferment pectin polysaccharide, proliferate rapidly in MRS medium with pectin polysaccharide as the sole carbon source, and produce a large amount of short-chain fatty acids. The extracellular supernatant has an ABTS and DPPH free radical scavenging rate of 85.84% and 74.04%, respectively. The extracellular supernatant has an alpha-glucosidase activity and alpha-amylase inhibition rate of 11.1% and 49.85%, respectively. The fermented Limosilactobacillus fermentum FMBL L24016 DMX has good antioxidant and hypoglycemic ability, and can be used for preparing antioxidant products, hypoglycemic drugs, fermented foods, health products, food additives, and feed, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Colony characteristics and microscope characteristics of the fermented Limosilactobacillus fermentum FMBL L24016 DMX

[0020] Figure 2 Phylogenetic tree constructed based on the GroEL gene sequence of the fermented Limosilactobacillus fermentum FMBL L24016 DMX

[0021] Figure 3 Growth curve of the fermented Limosilactobacillus fermentum FMBL L24016 DMX fermenting pectin

[0022] Figure 4 Changes in total sugar and galacturonic acid content during fermentation of the fermented Limosilactobacillus fermentum FMBL L24016 DMX

[0023] Figure 5 Inhibition rate of Lactobacillus mucosae FMBL L24016 DMX on α-glucosidase and α-amylase

[0024] Figure 6 Scavenging rate of Lactobacillus mucosae FMBL L24016 DMX on DPPH and ABTS free radicals DETAILED DESCRIPTION

[0025] The following examples are provided to further illustrate the present application, but should not be construed to limit the scope of the claims. The experimental methods used in the following examples are routine laboratory methods unless otherwise specified. The experimental materials used in the following examples are routine biochemical reagents unless otherwise specified. The quantitative tests in the following examples are triplicate, and the results are averaged. The seed pumpkin pectin in the following examples is extracted in the laboratory.

[0026] It should be noted that the method used to extract pectin and the medium formula used in the following examples are as follows:

[0027] Preparation of seed pumpkin pectin: The pumpkin pectin extracted in this experiment is prepared according to the previous method in the laboratory with slight modifications. Fresh pumpkin is washed, seeded, and cut into thin slices. The thin slices are dried in a constant temperature drying oven at 60°C. After drying, the slices are pulverized into powder with a pulverizer, sieved through an 80-mesh sieve, and stored in a bag. 15 g of pumpkin powder is accurately weighed, and 450 mL of pH 1.5 hydrochloric acid is added at a solid-liquid ratio of 1:30. The mixture is placed in a water bath at 80°C for 40 min, and then cooled to room temperature. Ultrasonic treatment is performed under the following conditions: ultrasonic power of 600 w, ultrasonic pulse duty cycle of 50% (10 s on and 10 s off), and ultrasonic time of 15 min. After ultrasonic treatment, the mixture is filtered, and the filtrate is concentrated to 1 / 4 of the original volume. Four times the volume of anhydrous ethanol is added for alcohol precipitation overnight. The mixture is filtered, and the same volume of anhydrous ethanol is added for secondary alcohol precipitation. After 4 h, the mixture is filtered, and the filtrate is dissolved in distilled water. The ethanol is removed by rotary evaporation, and the mixture is centrifuged at 8000 rpm for 15 min. The supernatant is dialyzed for 72 h, and then freeze-dried to obtain pumpkin pectin (PP).

[0028] MRS solid medium: proteose peptone 10.0 g; beef extract 10.0 g; yeast extract 5.0 g; glucose 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; L-cysteine hydrochloride 0.5 g; vancomycin hydrochloride 20 mg; sterilized at 115°C for 20 min.

[0029] MRS liquid medium: Proteose peptone 10.0 g; beef extract 10.0 g; yeast extract 5.0 g; glucose 20.0 g; Tween 80 1.0 mL; K2HPO4 2.0 g; sodium acetate 5.0 g; ammonium citrate 2.0 g; MgSO4·7H2O 0.58 g; MnSO4·4H2O 0.25 g; deionized water 1000 mL, L-cysteine hydrochloride 0.5 g; vancomycin hydrochloride 20 mg; sterilized at 115°C for 20 min.

[0030] MRS liquid medium with pectin as the sole carbon source: Proteose peptone 10.0 g; beef extract 10.0 g; yeast extract 5.0 g; pectin 20.0 g; Tween 80 1.0 mL; K2HPO4 2.0 g; sodium acetate 5.0 g; ammonium citrate 2.0 g; MgSO4·7H2O 0.58 g; MnSO4·4H2O 0.25 g; deionized water 1000 mL, L-cysteine hydrochloride 0.5 g; vancomycin hydrochloride 20 mg (added when culturing lactobacillus); sterilized at 115°C for 20 min.

[0031] Bacteroides sugar metabolism medium with pectin as the sole carbon source: Proteose peptone 20.0 g; yeast extract 5.0 g; sodium chloride 5.0 g; potassium phosphate dibasic 0.05 g; potassium phosphate monobasic 0.05 g; cysteine hydrochloride 1 g; pumpkin pectin 5 g / L; deionized water 1000 mL.

[0032] Bacteroides solid medium (BBE): 61.6 g of Bacteroides bile seven glycosides solid was weighed into 1000 mL of deionized water; 0.01% hemin chloride and 0.01% vitamin K1 were added; sterilized at 115°C for 20 min.

[0033] Bacteroides liquid medium (BHI): 38.5 g of brain heart infusion medium powder was weighed; dissolved in 1000 mL of deionized water; 0.01% hemin chloride and 0.01% vitamin K1 were added; sterilized at 115°C for 20 min.

[0034] Basic nutrient medium: yeast extract 2.0 g; proteose peptone 2.0 g; sodium bicarbonate 2.0 g; bile salt 0.5 g; cysteine hydrochloride 0.5 g; NaCl 0.1 g; potassium phosphate dibasic 0.04 g; potassium phosphate monobasic 0.04 g; hemin 50 mg; MgSO4·7H2O 0.01 g; CaCl2·6H2O 0.01 g; vitamin K1 10 μL; Tween 80 2.0 mL and distilled water 1.0 L; the pH of the basic nutrient medium was adjusted to 7.0; pectin 10 g.

[0035] Example 1, Strain isolation and identification

[0036] 1. Strain isolation

[0037] The strains were isolated from the fermentation broth of the mixed bacteria group using pumpkin pectin as the carbon source. The fermentation broth sampled from the in vitro batch simulation of fecal fermentation experiment was separated according to the conventional method. Pumpkin pectin (PP group) was used as the experimental group. The fermentation broth was diluted to 10 -1 , 10 -2 , 10 -3 , and spread on MRS solid medium, and incubated at 37°C for 24-48h. The suspected lactic acid bacteria colonies were picked and purified for 3 times. The isolated strains were stored in MRS liquid medium with 30% glycerol and stored at -20°C. A strain was isolated and purified, named FMBL L24016 DMX.

[0038] 2. Strain identification

[0039] 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 products were subjected to 1.2% agarose gel electrophoresis to observe the molecular size, and the appropriate PCR products were sent to the company for sequencing. The returned sequencing results were uploaded to the NCBI database for BLAST comparison. After comparison, the corresponding genus sequence was obtained from the database, and a phylogenetic tree was established using MEGA11.0.

[0040] Table 1. PCR amplification of groEL gene

[0041]

[0042] The colony characteristics and microscopic characteristics of the FMBL L24016 DMX strain are shown in Figure 1 , and the phylogenetic tree is shown in Figure 2 . After identification, the strain FMBL L24016 DMX is a fermenting limosilactobacillus, with the Latin name: Limosilactobacillus fermentum. It is named Limosilactobacillus fermentum FMBL L24016 DMX, and was preserved in the China Center for Type Culture Collection on August 26, 2024, with the preservation number: CCTCC NO: M 20241853, and the preservation address: Wuhan University, Wuhan, China, telephone 027-68754052.

[0043] In the following examples, the Limosilactobacillus fermentum FMBL L24016 DMX is simply written as Limosilactobacillus fermentum FMBL L24016 DMX.

[0044] Example 2: Metabolic effects of fermented Lactobacillus mucilaginosus FMBLL24016 DMX on pectin.

[0045] (1) The ability of isolated strains to degrade pectin and the screening of superior strains

[0046] Experimental strains: Clostridium butyricum FMBL C24001 DMX, Clostridium butyricum FMBL C24005 DMX, Bacteroides fragilis FMBLBa24008 DMX, Bacteroides fragilis FMBLBa24001 DMX, Lactobacillus fermentum FMBLL24017 DMX, Lactobacillus fermentum FMBL L24018 DMX, and Lactobacillus rhamnosus FMBL L24019 DMX were all isolated from human feces and deposited at the Food Microbiology and Biotechnology Research Center of the College of Food Science and Technology, Shihezi University; Lactobacillus rhamnosus LGG was purchased from the China Industrial Microbial Culture Collection Center.

[0047] After activating *Lactobacillus fermentatus* FMBL L24016 DMX and the control strains mentioned above, they were fermented in MRS liquid medium for 24 h. The fermentation broth was centrifuged (10000 rpm, 5 min, 4℃), and the supernatant was discarded. The bacterial cells were washed twice with sterile physiological saline and resuspended in 1 mL of physiological saline (OD600 = 1.0 ± 0.05). The bacterial suspension was inoculated at a 2% inoculum into MRS medium with pectin polysaccharide as the sole carbon source and into sugar metabolism media of *Bacteroides* and *Clostridium butyricum*. The OD600 at 0 h was measured. 600 The value was OD1. Lactobacillus was cultured at 37℃ for 24 hours, and the OD value was measured. 600 The value is OD2, and the final OD 600 =OD2 - OD1. The final OD 600 To determine the strain's pectin metabolism, the experiment was repeated three times, and the average value was taken.

[0048] Table 2 Evaluation of the strain's ability to metabolize pectin

[0049]

[0050] Note: -: Indicates OD value change < 0.2, no metabolism; +: Indicates OD value change between 0.2 and 0.5, poor metabolism; ++: Indicates OD value change between 0.5 and 0.8, moderate metabolic capacity; +++: Indicates OD value change between 0.8 and 1.0, good metabolic capacity; ++++: Indicates OD value change > 1.0, strong metabolic capacity.

[0051] From Table 2, it can be seen that the OD value of the fermented L. mesci L24016 DMX changed the most before and after fermentation, between 0.8-1.0, while the OD value of C. butyricum FMBL C24001 DMX, C. butyricum FMBL C24005 DMX changed less than 0.2, and they hardly metabolized pectin; the OD value of L. rhamnosus LGG, B. fragilis FMBL Ba24008 DMX, B. fragilis FMBL Ba24001 DMX, L. rhamnosus FMBL L24019 DMX changed between 0.2-0.5, and they had poor metabolism; the OD value of L. mesci L24017 DMX and L. mesci L24018 DMX changed between 0.5-0.8, and they had general metabolism, which further proved that L. mesci L24016 DMX had good ability to metabolize pectin.

[0052] (2) Growth curve of L. mesci L24016 DMX in MRS liquid medium with pectin as the sole carbon source

[0053] After activation in MRS liquid medium, L. mesci L24016 DMX and the control strain L. rhamnosus LGG were centrifuged to remove the supernatant, and the bacterial slurry was washed twice with sterile physiological saline, and then prepared into a bacterial solution with a concentration of 1.0x10 7 cfu / mL. The inoculation amount was 2%, and the inoculated medium was placed in a 37°C incubator for fermentation. The ultraviolet absorbance value (OD value) at 600 nm was measured every 2h, and the growth curve of the strain was plotted.

[0054] The results are shown in Table 3. Figure 3 As shown in Table 3, L. mesci L24016 DMX entered the logarithmic growth phase after 2h of fermentation, and entered the stationary phase after 10h, with an OD value of about 1.2, while the OD value of L. rhamnosus LGG was less than 0.34 after 24h of fermentation; L. mesci L24016 DMX had good ability to metabolize pectin, and could effectively utilize pectin as a carbon source to achieve rapid proliferation.

[0055] (3) Changes in total sugar and galacturonic acid content during fermentation of L. mesci L24016 DMX

[0056] The total sugar content was determined by anthrone-sulfuric acid method. Total sugar content determination: 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2mL of glucose standard solution was taken into a 10mL test tube, water was added to 2mL, 6mL of sulfuric acid anthrone solution was added, and it was boiled in a water bath for 15min, then it was taken out and quickly cooled for 15min, and the OD620 A standard curve was plotted. The fermentation broth of *Lactobacillus myxoidis* was diluted 20 times with distilled water, and the absorbance of the fermentation broth was measured according to the method described above. The absorbance was then substituted into the standard curve to calculate the total sugar content.

[0057] The galacturonic acid content was determined using the m-hydroxybiphenyl method: 100 μL of fermentation broth was placed in a 1.5 mL EP tube, and 500 μL of sodium tetraborate / sulfuric acid solution was added in an ice-water bath. The mixture was vortexed and heated in a boiling water bath for 10 min. After cooling in an ice-water bath, 100 μL of 1.5 mg / mL m-hydroxybiphenyl solution was added, and the mixture was shaken for 5 min. The absorbance was measured at a wavelength of 524 nm.

[0058] Depend on Figure 4 It was found that the fermentation of pectin by *Lactobacillus mucilaginosus* FMBL L24016 DMX consumed total sugar, causing a rapid decrease of 3.34 mg / mL in total sugar content, a significant change. In contrast, the fermentation of *Lactobacillus rhamnosus* LGG resulted in a decrease of 1.16 mg / mL in total sugar content. Within 0-10 hours, pectin was rapidly utilized by *Lactobacillus mucilaginosus* FMBL L24016 DMX. After 10 hours, the growth of *Lactobacillus mucilaginosus* FMBL L24016 DMX entered a stable phase, with a total sugar consumption rate of 49.4% and a galacturonic acid content decrease of 2.86 mg / mL after 24 hours of fermentation. In contrast, the total sugar consumption rate of *Lactobacillus rhamnosus* LGG was only 17.52%, further demonstrating that *Lactobacillus mucilaginosus* FMBL L24016 DMX can effectively metabolize pectin.

[0059] Example 3: Fermentation of Lactobacillus mucilaginosus FMBLL24016 DMX to produce short-chain fatty acids from pectin.

[0060] Short-chain fatty acids (SCFAs) are the main post-biotic substances produced by probiotic fermentation of dietary fiber. They play an important role in the body. In addition to providing energy, they can enhance the intestinal barrier function, maintain the integrity of the intestinal barrier, and also have anti-inflammatory, anti-tumor and immunomodulatory effects. They can regulate glucose and lipid metabolism and play an important role in intestinal function and the immune system.

[0061] In this embodiment, gas chromatography was used to analyze the SCFA content in the fermentation broth of *Lactobacillus myxoides* FMBL L24016 DMX using pectin as a carbon source. The fermentation broth was centrifuged at 10000 r / min for 15 min to remove cells and impurities, and then filtered through a 0.22 μm filter before loading. The SCFA content produced by *Lactobacillus myxoides* FMBL L24016 DMX during fermentation was analyzed using an Agilent 7890B gas chromatograph.

[0062] GC measurement conditions: measurement with DB-WAX analysis column (60 m x 250 pm x 0.25 pm). Inlet temperature: 250 °C; column oven conditions: initial temperature 100 °C for 30 s, increase by 8 °C per minute, 160 °C for 1 min, increase by 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).

[0063] Table 3. SCFA content produced by fermentation of L. mesenteroides FMBL L24016 DMX

[0064]

[0065] As can be seen from Table 3, a large amount of SCFA is produced during the fermentation of pectin by L. mesenteroides FMBL L24016 DMX, and the total short-chain fatty acid content reaches 335.32 pg / mL, among which the content of acetic acid is the highest, reaching 145.67 pg / mL, followed by valeric acid, with a content of 129.54 pg / mL.

[0066] Example Four, Inhibition Rate of Fermented L. mesenteroides FMBL L24016 DMX on a-Glucosidase and a-Amylase Inhibitory Effect

[0067] Activated L. mesenteroides FMBL L24016 DMX bacterial suspension (OD 600 : 1.0 ± 0.05) was added to MRS liquid medium with pectin polysaccharide as the sole carbon source at an inoculation amount of 2% (v / v), and after aerobic culture at 37 °C for 24 h, centrifugation was performed at 8000 rpm for 5 min, and the supernatant was filtered through a 0.22 pm water-based microfiltration membrane, and the cell-free supernatant was harvested for use.

[0068] 2. Inhibition activity of fermented L. mesenteroides FMBL L24016 DMX on a- glucosidase

[0069] With p-nitrophenyl-a-D-glucopyranoside solution (PNPG) as the substrate and Na2CO3 as the terminator, 50 pL of fermented L. mesenteroides FMBL L24016 DMX and 100 pL of a-glucosidase (0.2 U / mL) were thoroughly mixed, and then reacted at 37 °C for 10 min. Subsequently, 50 pL of PNPG was added, and the reaction was continued at 37 °C for 20 min. Finally, 50 pL of Na2CO3 (0.2 mol / L) was added to terminate the reaction. The absorbance value of the sample was measured at 405 nm. The inhibition rate of a-glucosidase was calculated using the following formula:

[0070]

[0071] Wherein, A S is the sample solution; A C is the sample control, 100 μL of PBS (0.1 mol / L, pH 6.8) solution instead of the α-glucosidase solution; A D is the blank, 50 μL of PBS solution instead of the sample solution; A B is the blank control, 50 μL of PBS solution instead of the sample solution, and 100 μL of PBS solution instead of the α-glucosidase solution.

[0072] 3. Fermented L. mesci LMBL L24016 DMX α-amylase inhibition rate determination

[0073] 125 μL of fermented L. mesci LMBL L24016 DMX was mixed with 125 μL of α-amylase solution, and reacted at 37 °C for 10 min. Then 250 μL of 1 mg / mL soluble starch was added, and reacted at 37 °C for 15 min. Finally, 500 μL of 3,5-dinitrosalicylic acid (DNS) reagent was added, and reacted in a boiling water bath for 5 min. The sample absorbance value was determined at 540 nm. The α-amylase inhibition rate was calculated using the following formula:

[0074]

[0075] Wherein, A S is the sample solution; A C is the sample control: 125 μL of PBS (0.1 mol / L, pH 6.8) solution instead of the α-amylase solution; A D is the blank: 125 μL of PBS solution instead of the sample solution; A B is the blank control, 125 μL of PBS solution instead of the sample solution, and 125 μL of PBS solution instead of the α-amylase solution.

[0076] Diabetes is a metabolic disease characterized by elevated blood glucose, and its pathogenesis is related to elevated blood glucose. The main source of blood glucose in the human body is diet, among which the main carbohydrate is starch with a larger chain segment and complex polysaccharide molecules, which cannot be directly absorbed into the blood circulation and needs to be hydrolyzed into glucose monomers by key digestive enzymes (α-amylase and α-glucosidase) in the intestine to be absorbed into the blood and then increase blood glucose. Pectin is a non-starch polysaccharide, which can delay the absorption of fat and sugar in the intestine, reduce cholesterol and blood glucose. In addition, pectin as a prebiotic can promote the proliferation of probiotics, regulate immune response, and make insulin better play a role in reducing blood sugar. The results are as follows Figure 5As shown, the inhibitory activity of the fermented L. muclebredecis FMBL L24016 DMX pectin fermentation broth on a-glucosidase and a-amylase was 11.1% and 49.85%, respectively. Therefore, the fermented L. muclebredecis FMBL L24016 DMX pectin fermentation broth can control the increase of blood glucose and insulin levels by inhibiting the activity of a-glucosidase and a-amylase in the intestine. This result also indicates the application potential of fermented L. muclebredecis FMBL L24016 DMX and its pectin fermentation broth in the preparation of hypoglycemic drugs, health products, synbiotics, and metabiotics.

[0077] Example Five, Antioxidant Capacity of Fermented L. muclebredecis FMBL L24016 DMX

[0078] Activated L. muclebredecis FMBL L24016 DMX bacterial suspension (OD 600 : 1.0 ± 0.05) was added to MRS liquid medium with pectin polysaccharide as the only carbon source at an inoculation amount of 2% (v / v), and after aerobic culture at 37°C for 24 h, centrifugation was performed at 8000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm water system microfiltration membrane, and the cell-free supernatant was harvested for use.

[0079] 1. DPPH free radical scavenging activity of fermentation broth

[0080] The fermentation broth was centrifuged at 8000 rpm at 4°C for 10 min, and the supernatant was filtered through a 0.22 μm water system filter membrane, and the filtrate was used to determine the DPPH free radical scavenging rate. 39.4 mg of DPPH was weighed, dissolved in 75% methanol and diluted to 100 mL to obtain a 1 mmol / L DPPH solution, which was stored at 0-4°C in the dark and used as needed. 1 mL of the sample was dissolved in 0.25 mL of the DPPH mixture, and the absorbance value at 517 nm was measured after 30 min of incubation at room temperature in the dark, and was recorded as A i ; 1 mL of the sample was dissolved in 0.25 mL of 75% methanol solution, and the absorbance value at 517 nm was measured after 30 min of incubation at room temperature in the dark, and was recorded as A j ; 1 mL of 75% methanol solution was dissolved in 0.25 mL of the DPPH mixture, and the absorbance value at 517 nm was measured after 30 min of incubation at room temperature in the dark, and was recorded as A0. The DPPH free radical scavenging activity was calculated using the following formula:

[0081]

[0082] 2. ABTS free radical scavenging activity of fermentation broth

[0083] The fermentation broth was centrifuged at 8000 rpm, 4 DEG C for 10 min, and the supernatant was filtered through a 0.22 mu m water filter membrane, and the filtrate was used to determine the ABTS free radical scavenging rate. 7 mmol of ABTS solution was mixed with 2.45 mmol of potassium sulfate (1:1, v / v) and reacted in the dark for 12-16 h to generate ABTS free radical cations. Dilute with distilled water, adjust the absorbance to 0.700+ / -0.02 at 734 nm. Add 0.8 mL of ABTS solution (1-5 mg / mL) to 0.2 mL of sample, and react in the dark for 6 min. Then measure the absorbance of each sample at 734 nm. The ABTS free radical scavenging activity is calculated using the following formula, where A0 is the absorbance value of the blank group, and A s is the absorbance value of the fermentation broth sample. The formula is as follows:

[0084]

[0085] The results are shown in Figure 6 , the highest DPPH free radical scavenging rate of the fermented Lactobacillus mui L24016 DMX is 74.03%, and the highest ABTS free radical scavenging rate is 85.84%. Probiotics can effectively stimulate the host's antioxidant system and produce antioxidant metabolites such as short-chain fatty acids. Pectin contains a wealth of antioxidant ingredients that can neutralize free radicals in the body, reduce oxidative stress, protect cells from injury, and slow down the aging process. At the same time, pectin can provide nutrients for probiotics, promote the growth of probiotics, and maintain the intestinal microecology. Therefore, this result also shows that the fermented Lactobacillus mui L24016 DMX and its pectin fermentation broth have potential applications in the preparation of antioxidant drugs, health products, synbiotics and metabiotics.

[0086] In summary, the present application provides a fermented Lactobacillus mui L24016 DMX, which was deposited with the China Center for Type Culture Collection on August 26, 2024, and has the accession number CCTCC NO:M 20241853. It can rapidly ferment pectin polysaccharide, proliferate rapidly in a medium with pectin polysaccharide as the sole carbon source, and produce a large amount of short-chain fatty acids. It also has strong scavenging ability for DPPH and ABTS free radicals, and good inhibitory effect on alpha-glucosidase and alpha-amylase activity. It can be used to prepare antioxidant products, hypoglycemic drugs, fermented foods, health products, food additives and feed, and has a wide application prospect.

Claims

1. A fermented Lactobacillus mucilaginosus that efficiently metabolizes pectin polysaccharides ( Limosilactobacillus fermentum FMBL L24016 DMX, characterized in that, The fermenting Lactobacillus mucinus FMBL L24016 DMX was deposited at the China Center for Type Culture Collection on August 26, 2024, with accession number CCTCC NO: M 20241853.

2. A microbial agent, characterized in that, Contains the fermenting Lactobacillus mucinus FMBL L24016 DMX as described in claim 1.

3. The application of the fermented Lactobacillus mucinus FMBL L24016 DMX or its fermentation broth as described in claim 1, or the bacterial agent as described in claim 2, in the preparation of antioxidant products.

4. The use of the fermentation broth of *Lactobacillus mucinus* FMBL L24016 DMX or its strain as described in claim 1, or the bacterial agent as described in claim 2, in the preparation of α-glucosidase inhibitors.

5. The use of the fermentation broth of *Lactobacillus mucilaginosus* FMBL L24016 DMX or its strain as described in claim 1, or the bacterial agent as described in claim 2, in the preparation of α-amylase inhibitors.

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