Lactobacillus mucus fermentation FMBL L24016 DMX capable of efficiently metabolizing pectic polysaccharides and application of lactobacillus mucus fermentation FMBL L24016 DMX

Abstract

The invention relates to the technical field of biology, in particular to lactobacillus mucus fermentation FMBL L24016DMX capable of efficiently metabolizing pectic polysaccharides and application of the lactobacillus mucus fermentation FMBL L24016DMX, the lactobacillus mucus fermentation FMBL L24016DMX is preserved in China Center for Type Culture Collection on August 26, 2024, the preservation number is CCTCC NO: M 20241853, the lactobacillus mucus fermentation FMBL L24016DMX has good pectin metabolism capacity, pectin can be effectively used as a carbon source to achieve rapid proliferation, and the lactobacillus mucus fermentation FMBL L24016DMX can be used for efficiently metabolizing the pectin is fermented to generate a large amount of short-chain fatty acid, and meanwhile, the pectin has relatively strong scavenging capacity on DPPH and ABTS free radicals; the compound has a relatively good inhibition effect on the activity of alpha-glucosidase and alpha-amylase; the compound can be used for preparing antioxidant products, hypoglycemic drugs, fermented foods, health-care products, food additives and feeds, and has wide application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a fermented mucus lactobacillus FMBL L24016 DMX capable of efficiently metabolizing pectin polysaccharide and an application thereof. Background Art

[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 bacteria in the intestine and improve the health of the host. Pectin is a new type of soluble prebiotic with a complex structure. It is widely present in the primary cell walls and middle cells of fruits and vegetables. Pectin has adhesive properties, which can increase the adhesion ability of probiotics on the intestinal wall, improve their survival rate and physiological functions in the intestine, regulate the pH value and water content in the intestine, and create a microecological environment that is conducive to the growth of probiotics. Pectin is a complex carbohydrate, but the human body lacks the corresponding enzyme system to fully decompose pectin. Therefore, 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 the intestinal microbial community ferments and produces 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 studies have shown that intestinal flora can utilize pectin and adjust intestinal health, but these are the results of the combined action of complex intestinal flora, and there are few reports of single strains that can utilize pectin. Invention patent CN104498372B discloses that Fusarium oxysporum BM201 can produce polygalacturonase, pectin lyase, pectinesterase, cellulase, hemicellulase and other pectinases to form composite pectinases, and then utilize pectin; invention patent CN115806892B discloses a genetically engineered bacterium that utilizes pectin, but the above strains are obtained by genetic engineering culture. Fermented mucus lactobacillus, as an important probiotic, plays an important role in maintaining the balance of human intestinal flora, regulating immunity, and promoting digestion. However, there is currently no research to prove that fermented mucus lactobacillus has the effect of utilizing pectin.

[0004] The inventor isolated a fermentative mucus lactobacillus FMBL L24016 DMX from the human intestine. The strain of the invention can quickly ferment pectin polysaccharides, can rapidly proliferate in a culture medium with pectin polysaccharides as the sole carbon source, and ferments to produce a large amount of short-chain fatty acids; the scavenging rates of the extracellular supernatant on ABTS and DPPH free radicals reach 85.84% and 74.04% respectively; the inhibition rates of the extracellular supernatant on α-glucosidase and α-amylase activities are 49.85% and 11%; it has good antioxidant and hypoglycemic abilities. It can be used to prepare antioxidant products, hypoglycemic drugs, fermented foods, health products, food additives and feeds, and has broad application prospects. Summary of the invention

[0005] In view of the above technical problems, the primary object of the present invention is to provide a fermented mucus lactobacillus (Limosilactobacillusfermentum) FMBL L24016 DMX that efficiently metabolizes pectin polysaccharides. The fermented mucus lactobacillus FMBL L24016DMX was deposited in the China Center for Type Culture Collection on August 26, 2024, with a preservation number of CCTCC NO: M20241853.

[0006] The second object of the present invention is to provide a bacterial agent containing the fermented mucus lactobacillus FMBL L24016DMX.

[0007] The third object of the present invention is to provide a functional product, wherein the functional product comprises the fermented mucus lactobacillus FMBL L24016 DMX or the bacterial agent, and the functional product has one or more of the following effects:

[0008] (1) Ability to metabolize pectin;

[0009] (2) Using pectin to produce short-chain fatty acids;

[0010] (3) Has antioxidant activity;

[0011] (4) Has the activity of inhibiting α-glucosidase;

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

[0013] Preferably, the functional product is one or more of food, food additives, dietary supplements, health products, medicines, feed or feed additives.

[0014] The fourth object of the present invention is to provide the use of the fermented Lactobacillus mucilaginosus FMBL L24016 DMX or its fermentation liquid or its fermentation sterile filtrate or the bacterial agent in anti-oxidation or in the preparation of antioxidant products.

[0015] The fifth object of the present invention is to provide the use of the fermented Lactobacillus mucilaginosus FMBL L24016 DMX or its strain fermentation broth or its fermentation sterile supernatant or the bacterial agent in inhibiting α-glucosidase or preparing α-glucosidase inhibitors.

[0016] The sixth object of the present invention is to provide the use of the fermented Lactobacillus mucilaginosus FMBL L24016 DMX or its strain fermentation broth or its fermentation sterile supernatant or the bacterial agent in inhibiting α-amylase activity or preparing α-amylase inhibitors.

[0017] The seventh object of the present invention is to provide the use of the fermented Lactobacillus mucilaginosus FMBL L24016 DMX or its strain fermentation liquid or its fermentation sterile supernatant or the bacterial agent in the preparation of hypoglycemic drugs and health products.

[0018] The beneficial effects of the present invention are as follows: the present invention provides a fermented mucus lactobacillus (Limosilactobacillusfermentum) FMBL L24016 DMX, which was deposited in the China Center for Type Culture Collection on August 26, 2024, with a deposit number of CCTCC NO: M 20241853; the fermented mucus lactobacillus FMBL L24016 DMX can quickly ferment pectin polysaccharides, can rapidly proliferate in an MRS culture medium with pectin polysaccharides as the sole carbon source, and ferments to produce a large amount of short-chain fatty acids; the ABTS and DPPH free radical scavenging rates of the extracellular supernatant are 85.84% and 74.04%, respectively; the inhibition rates of the extracellular supernatant on α-glucosidase activity and α-amylase are 11.1% and 49.85%, respectively; it has good antioxidant and blood sugar lowering abilities, can be used to prepare antioxidant products, blood sugar lowering drugs, fermented foods, health products, food additives and feed, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Colony characteristics and microscopic features of Lactobacillus mucilaginosus FMBL L24016 DMX

[0020] Figure 2 Phylogenetic tree based on the DMX GroEL gene sequence of Lactobacillus mucilaginosus FMBL L24016

[0021] Figure 3 Growth curve of pectin fermentation by Lactobacillus mucilaginosus FMBL L24016 DMX

[0022] Figure 4 Changes of total sugar and galacturonic acid contents during pectin fermentation by Lactobacillus mucilaginosus FMBL L24016 DMX

[0023] Figure 5 Inhibition rate of α-glucosidase and α-amylase by fermented Lactobacillus mucilaginosus FMBL L24016 DMX

[0024] Figure 6 Scavenging rate of DPPH and ABTS free radicals by fermented Lactobacillus mucilaginosus FMBL L24016 DMX DETAILED DESCRIPTION

[0025] The following examples are used to describe the specific implementation methods of the present invention in detail, thereby further illustrating the present invention, but the claims of the present invention are not limited in any way. The experimental methods in the following examples, unless otherwise specified, are all routine laboratory methods. 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. The pumpkin pectin for seeds in the following implementation cases is extracted in the laboratory.

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

[0027] Preparation of pumpkin pectin for seeds: The pumpkin pectin extracted in this experiment was extracted according to the previous laboratory method with slight modifications. The fresh pumpkin was washed, seeded and pulped, and then cut into thin slices. The slices were placed in a constant temperature drying oven at 60°C and dried. After drying, they were crushed into powder with a grinder, sieved through an 80-mesh sieve, and stored in bags. Accurately weigh 15g pumpkin powder, add 450mL of pH1.5 hydrochloric acid at a solid-liquid ratio of 1:30, incubate in a water bath at 80℃ for 40min, cool to room temperature, and then perform ultrasound. The ultrasound conditions are as follows: the ultrasound power is 600w, the ultrasound pulse duty cycle is set to 50% (on 10s, off 10s), and the ultrasound time is 15min. After the ultrasound is completed, filter and collect the filtrate. After concentrating the filtrate to 1 / 4 of the original volume, add 4 times the volume of anhydrous ethanol for alcohol precipitation overnight, filter, collect the filter residue, add the same volume of anhydrous ethanol for secondary alcohol precipitation, filter after 4h, collect the filter residue, dissolve it in distilled water, remove the ethanol by rotary evaporation, centrifuge at 8000rpm for 15min, take the supernatant and dialyze it for 72h, and freeze-dry it to finally obtain pumpkin pectin (PP).

[0028] MRS solid culture medium: 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 culture medium: 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; deionized water 1000 mL, L-cysteine ​​hydrochloride 0.5 g; vancomycin hydrochloride 20 mg; sterilize at 115°C for 20 min.

[0030] MRS liquid culture medium with pectin as the sole carbon source: 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; diammonium hydrogen 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 lactobacilli); sterilize at 115°C for 20 min.

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

[0032] Bacteroides solid medium (BBE): weigh 61.6 g of Bacteroides bile esculin solid and dissolve it in 1000 mL of deionized water; add 0.01% hemin chloride and 0.01% vitamin K1; sterilize at 115° C. for 20 min.

[0033] Bacteroides liquid medium (BHI): weigh 38.5 g of brain heart infusion medium powder; dissolve in 1000 mL of deionized water; add 0.01% hemin chloride and 0.01% vitamin K1; sterilize at 115°C for 20 min.

[0034] Basic nutrient medium: yeast extract 2.0g; peptone 2.0g; sodium bicarbonate 2.0g; bile salt 0.5g; cysteine ​​hydrochloride 0.5g; NaCl 0.1g; dipotassium hydrogen phosphate 0.04g; potassium dihydrogen phosphate 0.04g; heme 50mg; MgSO4·7H2O 0.01g; CaCl2·6H2O 0.01g; vitamin K110μL; Tween 80 2.0mL and distilled water 1.0L; adjust the pH of the basic nutrient medium to 7.0; pectin 10g.

[0035] Example 1: Isolation and identification of strains

[0036] 1. Strain Isolation

[0037] The strains were isolated from the mixed bacterial fermentation broth with pumpkin pectin as the carbon source according to the conventional method. The pumpkin pectin (PP group) was used as the experimental group to perform in vitro batch simulation of feces fermentation experiment and the fermentation broth was isolated. The fermentation broth was diluted to 10 -1 , 10 -2 , 10 -3 , and spread on MRS solid medium and cultured at 37℃ for 24-48h. 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℃. A strain was isolated and purified from it and 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 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.

[0040] Table 1 PCR amplification of groEL gene

[0041]

[0042] The colony characteristics and microscopic characteristics of FMBL L24016 DMX are as follows Figure 1 The phylogenetic tree is shown in Figure 2 After identification, the strain FMBL L24016 DMX is fermented mucus lactobacillus, with a Latin name of Limosilactobacillus fermentum, and is named as fermented mucus lactobacillus (Limosilactobacillusfermentum) FMBL L24016 DMX, which was deposited in the China Center for Type Culture Collection on August 26, 2024, with a deposit number of CCTCC NO: M 20241853, and the deposit address is: Wuhan University, Wuhan, China, and the telephone number is 027-68754052.

[0043] In the following examples, Limosilactobacillusfermentum FMBLL24016 DMX is abbreviated as Limosilactobacillusfermentum FMBL L24016 DMX.

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

[0045] (1) Pectin degradation ability of isolated strains and screening of excellent strains

[0046] Experimental strains: Clostridium butyricum FMBL C24001 DMX, Clostridium butyricum FMBL C24005 DMX, Bacteroides fragilis FMBLBa24008DMX, Bacteroides fragilis FMBLBa24001 DMX, Lactobacillus fermentative mucus FMBLL24017 DMX, Lactobacillus fermentative mucus FMBL L24018 DMX, and Lactobacillus rhamnosus FMBL L24019 DMX were all isolated from human feces 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 Microbiology Culture Collection Management Center.

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

[0048] Table 2 Evaluation of strains' ability to metabolize pectin

[0049]

[0050] Note: -: indicates that the OD value changes <0.2, no metabolism; +: indicates that the OD value changes between 0.2-0.5, poor metabolism; ++: indicates that the OD value changes between 0.5-0.8, average metabolic capacity; +++: indicates that the OD value changes between 0.8-1.0, good metabolic capacity; ++++: indicates that the OD value changes >1.0, strong metabolic capacity

[0051] As shown in Table 2, the OD value of fermented Lactobacillus mucilaginosus FMBL L24016 DMX changed the most before and after, between 0.8-1.0, while the OD value of Clostridium butyricum FMBL C24001 DMX and Clostridium butyricum FMBL C24005 DMX changed less than 0.2, and almost no pectin was metabolized and utilized; the OD values ​​of Lactobacillus rhamnosus LGG, Bacteroides fragilis FMBL Ba24008 DMX, Bacteroides fragilis FMBL Ba24001DMX, and Lactobacillus rhamnosus FMBL L24019 DMX changed between 0.2-0.5, and the metabolism was poor; the OD values ​​of fermented Lactobacillus mucilaginosus FMBL L24017 DMX and fermented Lactobacillus mucilaginosus FMBL L24018 DMX changed between 0.5-0.8, and the metabolic capacity was average, which further demonstrated that fermented Lactobacillus mucilaginosus FMBL L24016 DMX had a good ability to metabolize pectin.

[0052] (2) Growth curve of fermentative Lactobacillus mucilaginosus FMBLL24016 DMX in medium with pectin as the sole carbon source

[0053] The fermented Lactobacillus mucilaginosus FMBL L24016 DMX and the control strain Lactobacillus rhamnosus LGG were activated in MRS liquid medium and centrifuged to remove the supernatant. The bacterial sludge was washed twice with sterile saline and prepared to a concentration of 1.0×10 7 cfu / mL of bacterial liquid was added to MRS liquid medium with pectin as the sole carbon source at an inoculation rate of 2%, and the inoculated medium was placed in a 37°C incubator for fermentation. The ultraviolet absorbance value (OD value) at 600nm was measured every 2 hours, and the growth curve of the strain was drawn.

[0054] The results are as follows Figure 3 As shown, the fermented Lactobacillus mucilaginosus FMBL L24016 DMX entered the logarithmic growth phase after 2 hours of fermentation, and entered the stable phase after 10 hours, with an OD value of about 1.2, while the OD value of Lactobacillus rhamnosus LGG was less than 0.34 at 24 hours of fermentation; the fermented Lactobacillus mucilaginosus FMBL L24016 DMX has a good ability to metabolize pectin and can effectively use pectin as a carbon source to achieve rapid proliferation.

[0055] (3) Changes in total sugar and galacturonic acid content during pectin fermentation by Lactobacillus mucilaginosus FMBLL24016 DMX

[0056] The total sugar content was determined by the anthrone-sulfuric acid method. Determination of total sugar content: 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 OD620 Dilute the fermented Lactobacillus mucilaginosus broth 20 times with distilled water, determine the absorbance of the fermented Lactobacillus mucilaginosus broth according to the above method, and substitute it into the standard curve to calculate the content of total sugar.

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

[0058] Depend on Figure 4 It can be seen that fermentation of mucilaginous lactobacillus FMBL L24016 DMX consumed total sugar in fermentation of pectin, causing the total sugar to drop rapidly by 3.34 mg / mL, with a significant change; while the total sugar content of rhamnosus lactobacillus LGG decreased by 1.16 mg / mL during the fermentation process. Pectin was rapidly utilized by fermentation of mucilaginous lactobacillus FMBL L24016 DMX within 0-10 hours, and the growth of fermentation of mucilaginous lactobacillus FMBL L24016 DMX entered a stable period after 10 hours. The consumption rate of total sugar reached 49.4% at 24 hours of fermentation, and the content of galacturonic acid decreased by 2.86 mg / mL; while the consumption rate of total sugar of rhamnosus lactobacillus LGG was 17.52%, which further shows that fermentation of mucilaginous lactobacillus FMBL L24016 DMX can metabolize pectin well.

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

[0060] Short-chain fatty acids (SCFA) 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 intestinal barrier function and maintain the integrity of the intestinal barrier. They also have anti-inflammatory, anti-tumor and immunomodulatory effects. They can regulate glucose and fat metabolism and play an important role in intestinal function and the immune system.

[0061] In this embodiment, the content of SCFA in the fermentation broth of Lactobacillus mucilaginosus FMBL L24016 DMX with pectin as the carbon source was analyzed by gas chromatography. The fermentation broth was centrifuged at 10000r / min for 15min to remove cells and impurities, and then sampled after filtering through a 0.22μm filter membrane. The content of SCFA produced by fermentation of Lactobacillus mucilaginosus FMBL L24016 DMX during the fermentation process was analyzed by Agilent 7890B gas chromatograph.

[0062] GC measurement conditions: DB-WAX analytical column (60m×250μm×0.25μm) was used for measurement. Inlet temperature: 250℃; Column box conditions: starting temperature 100℃ for 30s, increasing by 8℃ per minute, maintaining at 160℃ for 1min, then increasing the temperature at 20℃ / min, and maintaining at 200℃ for 5min; injection volume 1μL; carrier gas conditions: N2, flow rate 1.2mL / min, split ratio 2:1; FID detector conditions: temperature 250℃, tail gas: N2 (25mL / min), H2 (30mL / min), air (400mL / min).

[0063] Table 3 SCFA content produced by fermentation of pectin by Lactobacillus mucilaginosus FMBL L24016 DMX

[0064]

[0065] As shown in Table 3, a large amount of SCFA was produced during the fermentation of pectin by Lactobacillus mucilaginosus FMBL L24016 DMX, and the total short-chain fatty acid content reached 335.32 μg / mL, among which acetic acid had the highest content, reaching 145.67 μg / mL, followed by valeric acid, which was 129.54 μg / mL.

[0066] Example 4: Inhibition rate of α-glucosidase and α-amylase by fermentation of Lactobacillus mucilaginosus FMBL L24016 DMX 1. Experimental strains

[0067] Activated fermented Lactobacillus mucilaginosus FMBL L24016 DMX bacterial suspension (OD 600 : 1.0±0.05) was added at an inoculum size of 2% (v / v) to MRS liquid culture medium with pectin polysaccharide as the sole carbon source, cultured aerobically at 37°C for 24 h, and then centrifuged at 8000 rpm for 5 min. The supernatant was filtered through a 0.22 μm water microfiltration membrane, and the cell-free supernatant was harvested for later use.

[0068] 2. Inhibitory activity of DMX against α-glucosidase by fermentation of Lactobacillus mucilaginosus FMBL L24016

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

[0070]

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

[0072] 3. Determination of the inhibition rate of α-amylase by fermented Lactobacillus mucilaginosus FMBL L24016 DMX

[0073] Mix 125 μL of fermented Lactobacillus mucilaginosus FMBL L24016 DMX with 125 μL of α-amylase solution and react at 37°C for 10 min. Then add 250 μL of 1 mg / mL soluble starch and react at 37°C for 15 min. Finally, add 500 μL of 3,5-dinitrosalicylic acid (DNS) reagent and react in a boiling water bath for 5 min. Measure the sample absorbance at 540 nm. Calculate the α-amylase inhibition rate using the following formula:

[0074]

[0075] Among them, A S is the sample solution; A C For sample control: 125 μL PBS (0.1 mol / L, pH 6.8) solution was used instead of the α-amylase solution; A D For blank: replace the sample solution with 125 μL PBS solution; A B As blank control, 125 μL PBS solution was used to replace the sample solution, and 125 μL PBS solution was used to replace the α-amylase solution.

[0076] Diabetes is a metabolic disease characterized by elevated blood sugar, and its pathogenesis is related to elevated blood sugar. Human blood sugar mainly comes from diet, in which the main carbohydrates are starch with larger chain segments and complex polysaccharide molecules, which cannot be directly absorbed into the blood circulation and need to be hydrolyzed into glucose monomers by key digestive enzymes in the intestine (α-amylase and α-glucosidase) before they can be absorbed into the blood and increase blood sugar. Pectin is a non-starch polysaccharide that can delay the intestinal absorption of fat and sugar, lower cholesterol and blood sugar. In addition, pectin, as a prebiotic, can promote the proliferation of probiotics, regulate immune responses, and enable insulin to better play its blood sugar-lowering role. The results are as follows: Figure 5As shown, the inhibitory activity of Lactobacillus mucilaginosus FMBLL24016 DMX pectin fermentation broth on α-glucosidase and α-amylase was 11.1% and 49.85%, respectively. Therefore, Lactobacillus mucilaginosus FMBLL24016 DMX pectin fermentation broth can control the increase of blood sugar and insulin levels by inhibiting the activity of α-glucosidase and α-amylase in the intestine. This result also shows the application potential of Lactobacillus mucilaginosus FMBLL24016 DMX and its pectin fermentation broth in the preparation of hypoglycemic drugs, health products, synbiotics, and postbiotic preparations.

[0077] Example 5. Antioxidant capacity of fermented Lactobacillus mucilaginosus FMBLL24016 DMX

[0078] Activated fermented Lactobacillus mucilaginosus FMBL L24016 DMX bacterial suspension (OD 600 : 1.0±0.05) was added at an inoculum size of 2% (v / v) to MRS liquid culture medium with pectin polysaccharide as the sole carbon source, cultured aerobically at 37°C for 24 h, and then centrifuged at 8000 rpm for 5 min. The supernatant was filtered through a 0.22 μm water microfiltration membrane, and the cell-free supernatant was harvested for later use.

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

[0080] Centrifuge the fermentation broth at 8000rpm, 4℃ for 10min, and filter the supernatant through a 0.22μm water filter membrane. The filtrate is used to determine the DPPH free radical scavenging rate. Weigh 39.4mg DPPH, dissolve it in 75% methanol and dilute it to 100mL to obtain a 1mmol / L DPPH solution. Store it at 0-4℃ away from light for later use. Take 1mL of sample, dissolve it in 0.25mL DPPH mixed solution, place it at room temperature away from light for 30min, and measure the absorbance at 517nm, which is calculated as A i Take 1 mL of sample solution, dissolve it in 0.25 mL of 75% methanol solution, place it at room temperature and away from light for 30 minutes, and measure the absorbance at 517 nm, which is calculated as A j ; Take 1mL of 75% methanol solution, dissolve it in 0.25mL of DPPH mixed solution, place it at room temperature and away from light for 30min, measure the absorbance at 517nm, and calculate it as A0. Use the following formula to calculate the DPPH free radical scavenging activity:

[0081]

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

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

[0084]

[0085] The results are as follows Figure 6 As shown in the results, the fermented Lactobacillus mucilaginosus FMBL L24016 DMX had the highest scavenging rate of 74.03% for DPPH free radicals and the highest scavenging rate of 85.84% for ABTS free radicals. Probiotics can effectively stimulate the host's antioxidant system and produce antioxidant metabolites such as short-chain fatty acids. Pectin is rich in antioxidant components, which can neutralize free radicals in the body, reduce oxidative stress, protect cells from injury, and delay the aging process. At the same time, pectin can provide nutrients for probiotics, promote the growth of probiotics, and maintain intestinal microecology. Therefore, this result also shows the application potential of fermented Lactobacillus mucilaginosus FMBL L24016 DMX and its pectin fermentation broth in the preparation of antioxidant drugs, health products, synbiotics and postbiotic preparations.

[0086] In summary, the present invention provides a fermented mucus lactobacillus FMBL L24016 DMX, which was deposited in the China Center for Type Culture Collection on August 26, 2024, with a deposit number of CCTCC NO: M 20241853. The fermented mucus lactobacillus FMBL L24016 DMX can quickly ferment pectin polysaccharides, and can rapidly proliferate in a culture medium with pectin polysaccharides as the sole carbon source to produce a large amount of short-chain fatty acids; at the same time, the fermented mucus lactobacillus FMBL L24016 DMX has a strong scavenging ability for DPPH and ABTS free radicals; it has a good inhibitory effect on the activity of α-glucosidase and α-amylase; it can be used to prepare antioxidant products, hypoglycemic drugs, fermented foods, health products, food additives and feed, and has broad application prospects.

Claims

1. A fermentative mucus lactobacillus (Limosilactobacillusfermentum) FMBLL24016DMX that efficiently metabolizes pectin polysaccharides, characterized in that: The fermented mucus lactobacillus FMBL L24016 DMX was deposited in the China Center for Type Culture Collection on August 26, 2024, with a deposit number of CCTCC NO: M 20241853.

2. A bacterial agent, characterized in that Contains the fermented mucus lactobacillus FMBL L24016 DMX according to claim 1.

3. A functional product, characterized in that: The functional product comprises the fermented mucus lactobacillus FMBL L24016 DMX according to claim 1 or the bacterial agent according to claim 2 or is obtained by fermentation, and the functional product has one or more of the following effects: (1) Ability to metabolize pectin; (2) Using pectin to produce short-chain fatty acids; (3) Has antioxidant activity; (4) Has the activity of inhibiting α-glucosidase; (5) It has the activity of inhibiting α-amylase.

4. The functional product according to claim 3, characterized in that: The functional product is one or more of food, food additives, dietary supplements, health products, medicines, feed or feed additives.

5. Use of the fermented mucilaginous lactobacillus FMBL L24016 DMX as claimed in claim 1 or its fermentation liquid or its fermentation sterile filtrate or the bacterial agent as claimed in claim 2 in anti-oxidation or in preparing antioxidant products.

6. Use of the fermented Lactobacillus mucilaginosus FMBL L24016 DMX or its strain fermentation broth or its fermentation sterile supernatant as claimed in claim 1 or the bacterial agent as claimed in claim 2 in inhibiting α-glucosidase or preparing an α-glucosidase inhibitor.

7. Use of the fermented Lactobacillus mucilaginosus FMBL L24016 DMX or its strain fermentation broth or its fermentation sterile supernatant as claimed in claim 1 or the bacterial agent as claimed in claim 2 in inhibiting α-amylase activity or preparing an α-amylase inhibitor.

8. Use of the fermented Lactobacillus mucilaginosus FMBL L24016 DMX or its strain fermentation liquid or its fermentation sterile supernatant as claimed in claim 1 or the bacterial agent as claimed in claim 2 in the preparation of hypoglycemic drugs and health products.

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