Lactobacillus pentosus FMBL L24020 DMX capable of metabolizing pectic polysaccharides and application of lactobacillus pentosus FMBL L24020 DMX

Abstract

The invention relates to the technical field of biology, in particular to lactobacillus pentosus FMBL L24020DMX for metabolism of pectic polysaccharides and application of the lactobacillus pentosus FMBL L24020DMX, the lactobacillus pentosus FMBL L24020DMX is preserved in China Center for Type Culture Collection on August 26, 2024, and the preservation number is CCTCC NO: M 20241852; the pectin can be effectively utilized as a carbon source to realize rapid proliferation, and a large amount of short-chain fatty acid is generated; meanwhile, the cleaning agent has a relatively strong capability of removing 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 in particular relates to a lactobacillus pentosus FMBLL24020 DMX capable of 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 selectively promote the metabolism and proliferation of beneficial bacteria in the body and improve the health of the host, including functional oligosaccharides, polysaccharides, some natural plant extracts, protein hydrolysates, polyols, etc. Pectin is composed of two types: homogeneous polysaccharides and heteropolysaccharides. It is mostly found in plant cell walls and cell layers, and is found in large quantities in the peels of citrus, lemons, grapefruits, etc. Pectin has adhesive properties, which can increase the adhesion of probiotics to the intestinal wall and improve their survival rate and physiological functions in the intestine. In addition, pectin can also promote intestinal peristalsis, reduce intestinal pH, and inhibit the growth of some harmful bacteria. The prebiotic activity of pectin can bring a variety of benefits, including enhancing intestinal health, improving digestive function, enhancing immunity, anti-oxidation and reducing inflammatory response. In addition, pectin can also improve type 2 diabetes by regulating sugar metabolism and blood lipid levels, improving insulin resistance, and repairing organ damage. It is widely used in food, medicine and other fields.

[0003] However, pectin is a complex carbohydrate. The human body lacks the corresponding enzyme system to fully decompose pectin. Pectin cannot be effectively digested and absorbed in the small intestine. After entering the large intestine, pectin becomes an important substrate for intestinal flora. The intestinal microbial community ferments it 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. 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.

[0004] Lactobacillus pentosus is currently recognized as a safe bacterium, and plays an important role in maintaining the balance of human intestinal flora, promoting digestion, inhibiting the growth of harmful bacteria, regulating the host immune system, and improving diabetes. Lactobacillus pentosus has the ability to decompose complex carbohydrates and protein utilization in food and produce lactic acid, and is widely used in fermented foods, meat products, and feed additives. However, there is currently no research that Lactobacillus pentosus can ferment pectin and grow with pectin as a carbon source.

[0005] The inventor isolated a strain of Lactobacillus pentosaceus FMBL L24020 DMX from the human intestine. The strain 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 rate of the extracellular supernatant to ABTS and DPPH free radicals reaches 85.51% and 83.34% respectively; the inhibition rate of the extracellular supernatant to α-glucosidase activity and α-amylase is 17.68% and 67.10% respectively; 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

[0006] In view of the above technical problems, the primary purpose of the present invention is to provide a Lactiplantibacillus pentosus FMBL L24020 DMX that metabolizes pectin polysaccharides, which was deposited in the China Center for Type Culture Collection on August 26, 2024, with a preservation number of CCTCC NO: M 20241852.

[0007] The second object of the present invention is to provide a bacterial agent containing the lactobacillus pentosus FMBL L24020DMX.

[0008] The third object of the present invention is to provide a functional product, which comprises the lactobacillus pentosus FMBL L24020 DMX or the bacterial agent as described or is obtained by fermenting the lactobacillus pentosus FMBL L24020 DMX or the bacterial agent as described, and has one or more of the following effects:

[0009] (1) Ability to metabolize pectin;

[0010] (2) Using pectin to produce short-chain fatty acids;

[0011] (3) Has antioxidant activity;

[0012] (4) Has the activity of inhibiting α-glucosidase;

[0013] (5) It has the activity of inhibiting α-amylase.

[0014] Preferably, the functional product is one or more of food, food additives, dietary supplements, health products, medicines, feed or feed additives.

[0015] The fourth object of the present invention is to provide the use of the lactobacillus pentosus FMBL L24020 DMX or its fermentation liquid or its fermentation sterile filtrate or the bacterial agent in anti-oxidation or in the preparation of anti-oxidation products.

[0016] The fifth object of the present invention is to provide the use of the Lactobacillus pentosus FMBL L24020 DMX or its strain fermentation broth or its fermentation sterile supernatant or the bacterial agent in inhibiting α-glucosidase or preparing an α-glucosidase inhibitor.

[0017] The sixth object of the present invention is to provide the use of the Lactobacillus pentosus FMBL L24020 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.

[0018] The seventh object of the present invention is to provide the use of the Lactobacillus pentosus FMBL L24020 DMX or its strain fermentation liquid or its fermentation sterile supernatant or the bacterial agent according to claim 2 in the preparation of hypoglycemic drugs and health products.

[0019] The beneficial effects of the present invention are as follows: the present invention provides a Lactiplantibacillus pentosus FMBL L24020 DMX, which was deposited in the China Center for Type Culture Collection on August 26, 2024, with a deposit number of CCTCC NO: M 20241852. The Lactiplantibacillus pentosus FMBL L24020 DMX can quickly ferment pectin polysaccharides, can rapidly proliferate in an MRS 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 for ABTS and DPPH free radicals are 85.51% and 83.34% respectively; the inhibition rates of the extracellular supernatant for α-glucosidase activity and α-amylase are 17.68% and 67.10% respectively; it has good antioxidant and hypoglycemic abilities, can be used to prepare antioxidant products, hypoglycemic drugs, fermented foods, health products, food additives and feeds, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Colony characteristics and microscopic features of Lactobacillus pentosus FMBL L24020 DMX

[0021] Figure 2 Phylogenetic tree based on the DMX GroEL gene sequence of Lactobacillus pentosus FMBL L24020

[0022] Figure 3 Growth curve of Lactobacillus pentosus FMBL L24020 DMX

[0023] Figure 4Changes of total sugar and galacturonic acid contents during DMX fermentation of Lactobacillus pentosus FMBL L24020

[0024] Figure 5 Inhibition rate of α-amylase and α-amylase inhibition rate of Lactobacillus pentosus FMBL L24020 DMX

[0025] Figure 6 Scavenging rate of DPPH and ABTS free radicals by DMX of Lactobacillus pentosus FMBL L24020 DETAILED DESCRIPTION

[0026] 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.

[0027] It should be noted that in the following examples, the method for extracting pectin and the medium formula used are as follows:

[0028] 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). MRS solid 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; 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 24-48 hours. After picking the suspected lactic acid bacteria colonies and purifying them three times, the isolates were stored in MRS liquid medium supplemented with 30% glycerol and stored at -20℃. A strain was isolated from it and named FMBL L24020 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]

[0043] The bacterial strain FMBL L24020 DMX has the following colony characteristics and microscopic characteristics: Figure 1 The phylogenetic tree is shown in Figure 2 After identification, the strain FMBL L24020 DMX was obtained as Lactobacillus pentosus, with a Latin name of Lactiplantibacillus pentosus, and was named Lactiplantibacillus pentosus FMBL L24020 DMX, which was deposited in the China Center for Type Culture Collection on August 26, 2024, with a deposit number of CCTCC M20241852, and a deposit address of Wuhan University, Wuhan, China, with a telephone number of 027-68754052.

[0044] In the following examples, Lactiplantibacillus pentosus FMBL L24020DMX is abbreviated as Lactiplantibacillus pentosus FMBL L24020 DMX.

[0045] Example 2: Metabolic effect of Lactobacillus pentosus FMBLL24020 DMX on pectin

[0046] (1) Pectin degradation ability of isolated strains and screening of excellent strains

[0047] Experimental strains: Bacteroides fragilis FMBL Ba24004 DMX, Bacteroides fragilis FMBLBa24001 DMX, Lactobacillus fermentative mucus FMBL L24009 DMX, Lactobacillus fermentative mucus FMBL L24008 DMX, and Lactobacillus rhamnosus FMBLL24019 DMX were all isolated from human feces by our laboratory staff 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.

[0048] Lactobacillus pentosus FMBL L24020 DMX and several control strains were activated and cultured for two generations until the logarithmic phase. The fermentation broth was centrifuged (10000rpm, 5min, 4℃) and the supernatant was discarded. The bacteria were washed twice with sterile saline and resuspended in 1mL 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 the OD at 0h 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.

[0049] Table 2 Evaluation of strains' ability to metabolize pectin

[0050]

[0051] 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

[0052] It can be seen from Table 2 that the OD value of Lactobacillus pentosus FMBL L24020 DMX changed the most before and after. The other control strains, Lactobacillus mucilaginosus FMBL L24009 DMX and Lactobacillus mucilaginosus FMBL L24008 DMX, grew worse than Lactobacillus pentosus FMBL L24020 DMX on pectin medium, and the OD values ​​varied between 0.2 and 0.8. Among them, the OD values ​​of Lactobacillus rhamnosus LGG, Bacteroides fragilis FMBL Ba24004 DMX, Bacteroides fragilis FMBL Ba24001 DMX, and Lactobacillus rhamnosus FMBLL24019 DMX varied between 0.2 and 0.5, indicating poor metabolism. This further illustrates that Lactobacillus pentosus FMBL L24020 DMX has the potential to metabolize pectin.

[0053] (2) Growth curve of Lactobacillus pentosus FMBLL24020 DMX in medium with pectin as the sole carbon source

[0054] Lactobacillus pentosus FMBL L24020 DMX and control strain Lactobacillus rhamnosus LGG were activated, and after 2-3 generations of activation, the bacterial solution was centrifuged to remove the supernatant, and the bacterial mud was washed twice with sterile physiological saline and resuspended, and 2% of the seed was added to the MRS liquid culture medium with pectin as the sole carbon source, and the inoculated culture medium was placed in a 37° C. incubator for fermentation. The ultraviolet absorbance value (OD value) at 600 nm was measured every 2 hours, and the growth curve of the strain was drawn.

[0055] Depend on Figure 3 It can be seen that Lactobacillus pentosus FMBL L24020 DMX can effectively utilize pectin as a carbon source to grow rapidly, enter the logarithmic growth phase after 8-10 hours of fermentation, and enter the stable phase after 12 hours, with an OD value of about 1.24. However, the OD value of Lactobacillus rhamnosus LGG was less than 0.34 when fermented for 24 hours. Therefore, Lactobacillus pentosus FMBL L24020 DMX has the ability to metabolize pectin and can better utilize pectin to achieve rapid proliferation.

[0056] (3) Changes in total sugar and galacturonic acid content during pectin fermentation by Lactobacillus pentosus FMBLL24020 DMX

[0057] 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 OD 620Dilute the fermentation liquid of Lactobacillus pentosus FMBL L24020 DMX with distilled water 20 times, determine the absorbance value of the fermentation liquid of Lactobacillus pentosus FMBL L24020 DMX according to the above method, and substitute it into the standard curve to calculate the content of total sugar.

[0058] The galacturonic acid content was determined by the m-hydroxybiphenyl method: 100 μL of the fermentation broth of Lactobacillus pentosus FMBL L24020DMX 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 a 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.

[0059] The experimental results are as follows Figure 4 As shown, it can be seen that the total sugar consumed by Lactobacillus pentosus FMBL L24020 DMX fermented pectin, and the total sugar decreased by 3.39 mg / mL, and the galacturonic acid content decreased by 2.57 mg / mL, which was a significant change; while the total sugar content decreased by 1.16 mg / mL and the galacturonic acid content decreased by 1.27 mg / mL during the fermentation process of Lactobacillus rhamnosus LGG. Pectin was rapidly utilized by Lactobacillus pentosus FMBL L24020 DMX within 0-12 hours, and the growth of Lactobacillus pentosus FMBL L24020 DMX entered a stable period after 12 hours. The consumption rate of total sugar reached 51.36% at 24 hours of fermentation, while the consumption rate of total sugar by Lactobacillus rhamnosus LGG was 17.52%, which further shows that Lactobacillus pentosus FMBL L24020 DMX can metabolize pectin well.

[0060] Example 3: Production of short-chain fatty acids by fermentation of pectin by Lactobacillus pentosus FMBLL24020 DMX

[0061] The short-chain fatty acids (SCFA) in the fermentation broth of Lactobacillus pentosus FMBL L24020 DMX were analyzed by gas chromatography. The fermentation broth was centrifuged at 10000r / min for 15min to remove cells and impurities, and then filtered through a 0.22μm filter membrane before loading. The content of SCFA produced by Lactobacillus pentosus FMBL L24020 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 pentosus FMBL L24020 DMX

[0064]

[0065] Short-chain fatty acids are the main postbiotic substances produced by probiotic fermentation of dietary fiber. They play an important role in the body. In addition to providing energy to the body, they can enhance the barrier function of the intestine and maintain the integrity of the intestinal barrier. They also have anti-inflammatory, anti-tumor and immunomodulatory effects, and can regulate glucose and fat metabolism, which plays an important role in intestinal function and the immune system. As shown in Table 8, a large amount of SCFA is produced during the fermentation of pectin by Lactobacillus pentosus FMBL L24020 DMX, and the total short-chain fatty acid content reaches 265.83 μg / mL, of which acetic acid content is the highest, reaching 134.27 μg / mL, followed by valeric acid, with a content of 86.23 μg / mL.

[0066] Example 4. Inhibitory effect of Lactobacillus pentosus FMBLL24020 DMX on α-glucosidase and α-amylase 1. Experimental strains

[0067] Activated Lactobacillus pentosus FMBL L24020 DMX bacterial suspension (OD 600 : 1.0±0.05) was added to MRS liquid culture medium with pectin as the sole carbon source at an inoculum size of 2% (v / v), 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 Lactobacillus pentosus against α-glucosidase

[0069] Using p-nitrophenyl-α-D-glucopyranoside solution (PNPG) as substrate and Na2CO3 as terminator, 50 μL sample and 100 μL α-glucosidase (0.2 U / mL) were fully mixed and reacted at 37°C for 10 min. Subsequently, 50 μL PNPG was added and the reaction continued at 37°C for 20 min. Finally, 50 μL Na2CO3 (0.2 mol / L) was added to terminate the reaction. The sample absorbance 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 Lactobacillus pentosus

[0073] Mix 125 μL of sample 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. Use the following formula to calculate the α-amylase inhibition rate:

[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 5 As shown, the inhibitory activity of Lactobacillus pentosus FMBLL24020 DMX on α-glucosidase was 17.86%, and the inhibitory activity on α-amylase reached 67.10%. Therefore, the fermented Lactobacillus mucilaginosus FMBL L24016 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. Therefore, the above results also show the application potential of Lactobacillus pentosus FMBLL24020 DMX and its pectin fermentation broth in the preparation of hypoglycemic drugs, health products, synbiotics, and postbiotic preparations.

[0077] Example 5. Antioxidant capacity of Lactobacillus pentosus FMBLL24020 DMX

[0078] Activated Lactobacillus pentosus FMBL L24016 DMX bacterial suspension (OD 600 : 1.0±0.05) was added to MRS liquid culture medium with pectin as the sole carbon source at an inoculum size of 2% (v / v), 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 scavenging rate of DPPH free radicals by Lactobacillus pentosus FMBL L24020 DMX was as high as 83.44%, and the scavenging rate of ABTS free radicals was as high as 85.51%. 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, the above results also further demonstrate the application potential of Lactobacillus pentosus FMBLL24020 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 plant bacillus pentosus lactobacillus FMBL L24020 DMX, which was deposited in the China Center for Type Culture Collection on August 26, 2024, with a deposit number of CCTCC NO: M 20241852. The strain of the present invention can effectively utilize pectin as a carbon source to achieve rapid proliferation, and ferment pectin to produce a large amount of short-chain fatty acids; at the same time, the plant bacillus pentosus lactobacillus FMBL L24020 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 Lactobacillus pentosus FMBLL24020 DMX that metabolizes pectin polysaccharides, characterized in that: The lactobacillus pentosus FMBL L24020 DMX was deposited in the China Center for Type Culture Collection on August 26, 2024, with a deposit number of CCTCC NO: M 20241852.

2. A bacterial agent, characterized in that Contains the Lactobacillus pentosus FMBL L24020 DMX according to claim 1.

3. A functional product, characterized in that: The functional product comprises the lactobacillus pentosus FMBL L24020 DMX according to claim 1 or the bacterial agent according to claim 2, or is obtained by fermenting the lactobacillus pentosus FMBL L24020 DMX according to claim 1 or the bacterial agent according to claim 2, 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 lactobacillus pentosus FMBL L24020 DMX or its fermentation liquid or its fermentation sterile filtrate as claimed in claim 1 or the bacterial agent as claimed in claim 2 in anti-oxidation or in the preparation of antioxidant products.

6. Use of the Lactobacillus pentosus FMBL L24020 DMX or its fermentation broth or its sterile fermentation 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 Lactobacillus pentosus FMBL L24020 DMX or its fermentation broth or its sterile fermentation 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 Lactobacillus pentosus FMBL L24020 DMX or its 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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