Pentosaccharide lactobacillus plantarum fmbpl24020 dmx metabolizing pectin polysaccharides and applications thereof

Abstract

The present application relates to the field of biotechnology, and in particular to a pentose lactobacillus plantarum FMBL L24020DMX for metabolizing pectin polysaccharide and application, the pentose lactobacillus plantarum FMBL L24020DMX is preserved in China typical culture preservation center on August 26, 2024, and the preservation number is CCTCC NO:M 20241852;It has good ability to metabolize pectin, can effectively utilize pectin as carbon source to realize rapid proliferation, produce a large amount of short-chain fatty acid;At the same time, it has strong scavenging capacity to DPPH and ABTS free radicals;It has good inhibitory effect on alpha-glucosidase and alpha-amylase activity;It can be used for preparing antioxidant products, hypoglycemic drugs, fermented foods, health products, food additives and feed, and has wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a pentose lactobacillus FMBLL24020 DMX capable of metabolizing pectin polysaccharide and application thereof. BACKGROUND

[0002] Prebiotics refer to organic substances that are not digested and absorbed by the host, but can selectively promote the metabolism and proliferation of beneficial bacteria in the body, improve the health of the host, and include functional oligosaccharides, polysaccharides, some natural plant extracts, protein hydrolysates, polyols, etc. Pectin has two types of homopolysaccharides and heteropolysaccharides, and is mainly present in plant cell walls and cell layers, and is abundant in the peels of citrus, lemons, grapefruits, etc. Pectin has adhesion, can increase the adhesion capacity of probiotics on the intestinal wall, and improve the survival rate and physiological function of probiotics in the intestinal tract. In addition, pectin can promote intestinal peristalsis and reduce the PH value of the intestinal tract to inhibit the growth of some harmful bacteria. The prebiotic activity of pectin can bring many benefits, including enhancing intestinal health, improving digestive function, enhancing immunity, antioxidant, reducing inflammation, etc. In addition, pectin can also be widely used in food, medicine and other fields by regulating sugar metabolism, blood lipid level, improving insulin resistance, repairing organ damage, and improving type II diabetes.

[0003] However, pectin is a complex carbohydrate, and 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, 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. Existing studies have shown that intestinal flora can utilize 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 capable of utilizing pectin.

[0004] Lactobacillus pentosus is currently recognized as a safe bacterium, and plays an important role in maintaining the balance of 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 proteins in food and produce lactic acid, and is widely used in fermented foods, meat product production, and feed additives. However, there is no research to date that discloses that Lactobacillus pentosus can ferment pectin and grow on pectin as a carbon source.

[0005] The inventors isolate a strain of Lactiplantibacillus pentosus FMBL L24020 DMX from human intestinal tract, the strain can rapidly ferment pectin polysaccharide, can rapidly proliferate in a culture medium taking pectin polysaccharide as the only carbon source, and a large amount of short-chain fatty acids are fermented; the extracellular supernatant has a clearance rate of 85.51% and 83.34% on ABTS and DPPH free radicals respectively; the extracellular supernatant has an inhibition rate of 17.68% and 67.10% on alpha-glucosidase activity and alpha-amylase respectively; 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

[0006] In view of the above technical problems, the primary object of the present application is to provide a Lactiplantibacillus pentosus FMBL L24020 DMX capable of metabolizing pectin polysaccharide, which was preserved in China Center for Type Culture Collection on August 26, 2024, and the preservation number is CCTCC NO: M 20241852.

[0007] The second object of the present application is to provide a bacterial agent containing the Lactiplantibacillus pentosus FMBL L24020 DMX.

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

[0009] (1) has the ability to metabolize pectin;

[0010] (2) produces short-chain fatty acids by utilizing pectin;

[0011] (3) has antioxidant activity;

[0012] (4) has the activity of inhibiting alpha-glucosidase;

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

[0014] Preferably, the functional product is one or more of food, food additive, dietary supplement, health product, drug, feed or feed additive.

[0015] The fourth object of the present application is to provide the use of the Lactiplantibacillus pentosus FMBL L24020 DMX or the fermentation broth thereof or the sterile filtrate thereof or the bacterial agent as described in antioxidant or in the preparation of antioxidant products.

[0016] A fifth object of the present application is to provide the application of the Lactiplantibacillus pentosus FMBL L24020 DMX or its strain fermentation broth or its fermentation sterile supernatant or the microbial agent in inhibiting alpha-glucosidase or in preparing alpha-glucosidase inhibitors.

[0017] A sixth object of the present application is to provide the application of the Lactiplantibacillus pentosus FMBL L24020 DMX or its strain fermentation broth or its fermentation sterile supernatant or the microbial agent in inhibiting alpha-amylase activity or in preparing alpha-amylase inhibitors.

[0018] A seventh object of the present application is to provide the application of the Lactiplantibacillus pentosus FMBL L24020 DMX or its strain fermentation broth or its fermentation sterile supernatant or the microbial agent in preparing hypoglycemic drugs and health products.

[0019] The present application provides a Lactiplantibacillus pentosus FMBL L24020 DMX, which was deposited with the China Center for Type Culture Collection on August 26, 2024, and has the accession number CCTCC NO: M 20241852. The Lactiplantibacillus pentosus FMBL L24020 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 85.51% and 83.34%, respectively. The extracellular supernatant has an alpha-glucosidase activity and alpha-amylase inhibition rate of 17.68% and 67.10%, respectively. The Lactiplantibacillus pentosus FMBL L24020 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

[0020] Figure 1 Colony characteristics and microscope characteristics of Lactiplantibacillus pentosus FMBL L24020 DMX

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

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

[0023] Figure 4Changes in total sugar and galacturonic acid content during fermentation of pentose lactobacillus plantarum FMBL L24020 DMX

[0024] Figure 5 Alpha-amylase inhibition rate of pentose lactobacillus plantarum FMBL L24020 DMX

[0025] Figure 6 DPPH and ABTS free radical scavenging rate of pentose lactobacillus plantarum FMBL L24020 DMX DETAILED DESCRIPTION

[0026] The following examples are provided to further illustrate the present application, but should not be construed as limiting 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 were performed in triplicate, and the results were averaged. The seed pumpkin pectin used in the following examples was extracted in the laboratory.

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

[0028] Preparation of pumpkin pectin: The pumpkin pectin extracted in this experiment was prepared according to the previous method with slight modification. Fresh pumpkin was washed, deseeded, and cut into thin slices. The thin slices were dried in a constant temperature drying oven at 60°C, and then ground into powder with a pulverizer. The powder was sieved through an 80-mesh sieve and stored in a bag. 15 g of pumpkin powder was accurately weighed, and 450 mL of hydrochloric acid with a pH of 1.5 was added at a solid-liquid ratio of 1:30. The mixture was placed in a water bath at 80°C for 40 min, and then cooled to room temperature. Ultrasonic treatment was 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 was filtered, and the filtrate was concentrated to 1 / 4 of the original volume. Four times the volume of anhydrous ethanol was added for alcohol precipitation overnight. The mixture was filtered, and the same volume of anhydrous ethanol was added for secondary alcohol precipitation. After 4 h, the mixture was filtered, and the filtrate was dissolved in distilled water. The ethanol was removed by rotary evaporation, and the mixture was centrifuged at 8000 rpm for 15 min. The supernatant was dialyzed for 72 h, and then freeze-dried to obtain pumpkin pectin (PP).

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

[0030] MRS liquid medium with pectin as the only carbon source: 10.0 g of protein peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 20.0 g of pectin, 1.0 mL of Tween 80, 2.0 g of K2HPO4, 5.0 g of sodium acetate, 2.0 g of diammonium hydrogen citrate, 0.58 g of MgSO4·7H2O, 0.25 g of MnSO4·4H2O, 1000 mL of deionized water, 0.5 g of L-cysteine hydrochloride, and 20 mg of vancomycin hydrochloride were sterilized at 115°C for 20 min.

[0031] Pectin as the sole carbon source of Bacteroides sugar metabolism medium: 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): Bacteroides bile glycyrrhizin solid 61.6 g was weighed and dissolved in 1000 mL of deionized water; 0.01% hematin 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% hematin 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; hematin 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] In the mixed bacterial population fermentation broth with pumpkin pectin as the carbon source, the strains were isolated according to the conventional method. Pumpkin pectin (PP group) was used as the experimental group, and the fermentation broth sampled from the in vitro batch simulation fecal fermentation experiment was subjected to bacterial separation. The fermentation broth was diluted to 10 -1 , 10 -2 , 10 -3 , and spread on MRS solid medium, and cultured at 37°C for 24-48 hours. The suspected lactic acid bacteria colonies were picked and purified for 3 times, and the isolated strains were stored in MRS liquid medium with 30% glycerol and stored at -20°C. A strain was isolated and named FMBL L24020 DMX.

[0038] 2. Strain identification

[0039] DNA was extracted from the strain using a kit, and the groEL gene 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 molecular size, and suitable PCR products were selected and sent to the company for sequencing. The returned sequencing results were uploaded to the NCBI database for BLAST alignment. After alignment, the sequences of the corresponding genus were obtained from the database, and a phylogenetic tree was constructed using MEGA11.0.

[0040] Table 1. PCR amplification of the groEL gene

[0041]

[0042]

[0043] The colony characteristics and microscopic features of strain FMBL L24020 DMX are as follows: Figure 1 As shown, the phylogenetic tree is as follows: Figure 2 As shown. After identification, the strain FMBL L24020 DMX was determined to be *Lactiplantibacillus pentosus*, and was named *Lactiplantibacillus pentosus* FMBL L24020 DMX. It was deposited on August 26, 2024, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC M20241852, located at Wuhan University, Wuhan, China, telephone number 027-68754052.

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

[0045] Example 2: Metabolic effects of Lactobacillus pentosaceus FMBLL24020 DMX on pectin.

[0046] (1) The ability of isolated strains to degrade pectin and the screening of superior strains

[0047] The experimental strains: Bacteroides fragilis FMBL Ba24004 DMX, Bacteroides fragilis FMBL Ba24001 DMX, Lactobacillus fermentum FMBL L24009 DMX, Lactobacillus fermentum FMBL L24008 DMX, Lactobacillus rhamnosus FMBL L24019 DMX were isolated from human feces by the laboratory staff and preserved in the Center for Food Microbiology and Biotechnology Research of the College of Food Science of Shihezi University; Lactobacillus rhamnosus LGG was purchased from the China Industrial Microbial Culture Collection Center.

[0048] The pentose Lactobacillus plantarum FMBL L24020 DMX and several control strains were activated for two generations and cultured to the logarithmic phase. After centrifugation (10000 rpm, 5 min, 4°C) of the fermentation broth, the supernatant was discarded, the bacterial cells were washed twice with sterile physiological saline, and then resuspended in 1 mL of physiological saline (OD600=1.0±0.05). The bacterial suspension was inoculated into MRS medium with pectin polysaccharide as the sole carbon source and Bacteroides sugar metabolism medium at an inoculation amount of 2%, and the OD 600 value at 0h was OD1. The Lactobacillus was cultured at 37°C for 24h, and the OD 600 value was OD2. The final OD 600 =OD2-OD1. The final OD 600 was used as the basis to judge the pectin metabolism of the strains. The experiment was repeated three times and the average value was taken.

[0049] Table 2 Evaluation of the pectin metabolism ability of the strains

[0050]

[0051] Note: -: indicates that the OD value change is <0.2, not metabolized; +: indicates that the OD value change is between 0.2-0.5, poor metabolism; ++: indicates that the OD value change is between 0.5-0.8, general metabolism ability; +++: indicates that the OD value change is between 0.8-1.0, good metabolism ability; ++++: indicates that the OD value change is >1.0, strong metabolism ability

[0052] From Table 2, the maximum OD value of pentose lactobacillus FMBL L24020 DMX before and after change, other control strains of fermentation mucilaginous lactobacillus FMBL L24009 DMX, fermentation mucilaginous lactobacillus FMBL L24008 DMX grow worse than pentose lactobacillus FMBL L24020 DMX on pectin medium, OD value change between 0.2-0.8, wherein rhamnose lactobacillus FMBL L24019 DMX, fragile Bacteroides FMBL Ba24004 DMX, fragile Bacteroides FMBL Ba24001 DMX, rhamnose lactobacillus FMBL L24019 DMX OD value change between 0.2-0.5, poor metabolism; Further illustrates the pentose lactobacillus FMBL L24020 DMX has the potential to metabolize pectin.

[0053] (2) Growth curve of pentose lactobacillus FMBL L24020 DMX in pectin as the only carbon source medium

[0054] Pentose lactobacillus FMBL L24020 DMX and control strains of rhamnose lactobacillus LGG were activated, and after 2-3 generations, the bacterial liquid was centrifuged to remove the supernatant, the bacterial slurry was washed twice with sterile physiological saline and resuspended, 2% inoculum was added to the MRS liquid medium with pectin as the only carbon source, and the inoculated medium was placed in a 37℃ 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.

[0055] From Figure 3 It can be seen that pentose lactobacillus FMBL L24020 DMX can effectively utilize pectin as a carbon source to grow rapidly, and enter the logarithmic growth phase after 8-10h of fermentation, and enter the stationary phase after 12h, with an OD value of about 1.24. Rhamnose lactobacillus LGG has an OD value of less than 0.34 after 24h of fermentation. Therefore, pentose lactobacillus 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 fermentation of pentose lactobacillus FMBL L24020 DMX with pectin

[0057] The total sugar content was determined by anthrone-sulfuric acid method. Total sugar content determination: take glucose standard solution 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2mL into 10mL test tube, add water to 2mL, add 6mL of sulfuric acid anthrone solution, boil in water bath for 15min, take out, cool quickly for 15min, measure OD 620The pectin fermentation liquid of Lactobacillus pentosus FMBL L24020 DMX was diluted 20 times with distilled water, and the absorbance of the pectin fermentation liquid of Lactobacillus pentosus FMBL L24020 DMX was determined according to the above method, and the total sugar content was calculated by substituting the standard curve.

[0058] The galacturonic acid content was determined by the m-hydroxydiphenyl method: 100 μL of the fermentation liquid of Lactobacillus pentosus FMBL L24020 DMX was taken into a 1.5 mL EP tube, 500 μL of sodium tetraborate / sulfuric acid solution was added in an ice water bath, and the mixture was mixed well with a vortex instrument, heated in a boiling water bath for 10 min, cooled in an ice water bath, and then 100 μL of 1.5 mg / mL m-hydroxydiphenyl solution was added, mixed well, and shaken for 5 min. The absorbance was measured at a wavelength of 524 nm.

[0059] The experimental results are shown in Table 1. Figure 4 As shown in Table 1, the total sugar consumption of pectin fermentation by Lactobacillus pentosus FMBL L24020 DMX was 3.39 mg / mL, the galacturonic acid content decreased by 2.57 mg / mL, and the change was significant; while the total sugar content of Lactobacillus rhamnosus LGG fermentation process decreased by 1.16 mg / mL, and the galacturonic acid content decreased by 1.27 mg / mL. Pectin was rapidly utilized by Lactobacillus pentosus FMBL L24020 DMX within 0-12 h, and the growth of Lactobacillus pentosus FMBL L24020 DMX entered the stable phase after 12 h. The total sugar consumption rate reached 51.36% after 24 h of fermentation, while the total sugar consumption rate of Lactobacillus rhamnosus LGG was 17.52%, which further indicated that Lactobacillus pentosus FMBL L24020 DMX could better metabolize pectin.

[0060] Example Three, Pectin Fermentation by Lactobacillus pentosus FMBL L24020 DMX to Produce Short-Chain Fatty Acids

[0061] The short-chain fatty acids (SCFA) in the fermentation liquid of Lactobacillus pentosus FMBL L24020 DMX were analyzed by gas chromatography. The fermentation liquid was centrifuged at 10000 r / min for 15 min to remove cells and impurities, and then filtered through a 0.22 μm filter membrane for loading. The content of SCFA produced by Lactobacillus pentosus FMBL L24020 DMX during fermentation was analyzed by 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, at 160 °C for 1 min, then increase by 20 °C / min, at 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 pentose Lactiplantibacillus FMBL L24020 DMX fermentation pectin

[0064]

[0065] Short-chain fatty acids are the main postbiotics produced by probiotic fermentation of dietary fiber, which play an important role in the body. In addition to providing energy, they can enhance the barrier function of the intestinal tract, maintain the integrity of the intestinal barrier, have anti-inflammatory, anti-tumor and immune regulation effects, and can regulate glucose and fat metabolism, and have an important role in intestinal function and the immune system. As can be seen from Table 3, a large amount of SCFA was produced during the fermentation of pectin by pentose Lactiplantibacillus FMBL L24020 DMX, and the total short-chain fatty acid content reached 265.83 pg / mL, of which the acetic acid content was the highest, reaching 134.27 pg / mL, followed by valeric acid, with a content of 86.23 pg / mL.

[0066] Example Four, Inhibition Effect of Pentose Lactiplantibacillus FMBL L24020 DMX on a-Glucosidase and a-Amylase

[0067] Activated pentose Lactiplantibacillus FMBL L24020 DMX bacterial suspension (OD 600 : 1.0 ± 0.05) was added to MRS liquid medium with pectin as the only carbon source at an inoculation amount of 2% (v / v), and cultured at 37 °C for 24 h under aerobic conditions, then centrifuged at 8000 rpm for 5 min, and the supernatant was filtered through a 0.22 pm water system microfiltration membrane, and the cell-free supernatant was harvested for use.

[0068] 2. Inhibition activity of pentose Lactiplantibacillus on a-glucosidase

[0069] The sample solution was mixed with 100 μL of α-glucosidase (0.2 U / mL) and 50 μL of p-nitrophenyl-α-D-glucopyranoside (PNPG) solution, and then incubated at 37 °C for 20 min. Finally, 50 μL of Na2CO3 (0.2 mol / L) was added to terminate the reaction. The absorbance of the sample was measured at 405 nm. The α-glucosidase inhibition rate was calculated using the following formula:

[0070]

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

[0072] 3. Pentosanase inhibition rate determination

[0073] The sample solution was mixed with 125 μL of α-amylase solution, and then incubated at 37 °C for 10 min. Then, 250 μL of 1 mg / mL soluble starch was added, and the mixture was incubated at 37 °C for 15 min. Finally, 500 μL of 3,5-dinitrosalicylic acid (DNS) reagent was added, and the mixture was boiled in a water bath for 5 min. The absorbance of the sample was measured 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, in which 125 μL of PBS (0.1 mol / L, pH 6.8) solution was used instead of the α-amylase solution; A D is the blank, in which 125 μL of PBS solution was used instead of the sample solution; and A B is the blank control, in which 125 μL of PBS solution was used instead of the sample solution, and 125 μL of PBS solution was used 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. Human blood glucose is mainly derived from diet, among which the main carbohydrate is large-chain starch 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 (alpha-amylase and alpha-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. As shown in the results Figure 5 As shown in the results, the inhibition activity of pentose Lactiplantibacillus FMBL L24020 DMX on alpha-glucosidase was 17.86%, and the inhibition activity on alpha-amylase reached 67.10%. Therefore, the pectin fermentation broth of Lactiplantibacillus FMBL L24016 DMX can control the increase of blood glucose and insulin level by inhibiting the activity of alpha-glucosidase and alpha-amylase in the intestine. Therefore, the above results also indicate the application potential of pentose Lactiplantibacillus FMBL L24020 DMX and its pectin fermentation broth in the preparation of blood glucose-lowering drugs, health products, synbiotics, and metabiotics.

[0077] Example Five, Antioxidant Capacity of Pentose Lactiplantibacillus FMBL L24020 DMX

[0078] Activated Lactiplantibacillus FMBL L24016 DMX bacterial suspension (OD 600 : 1.0 ± 0.05) was added to MRS liquid medium with pectin as the only carbon source at an inoculation amount of 2% (v / v), and after 24 h of aerobic culture at 37°C, centrifugation was performed at 8000 rpm for 5 min, 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 and 4°C for 10 min, the supernatant was filtered through a 0.22 μm water system filter membrane, and the filtrate was prepared for determination of 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 sample was dissolved in 0.25 mL of DPPH mixture, and placed at room temperature in the dark for 30 min. The absorbance was measured at 517 nm and recorded as A i ; 1 mL of sample was dissolved in 0.25 mL of 75% methanol solution, and placed at room temperature in the dark for 30 min. The absorbance was measured at 517 nm and recorded as A j; Take 1 mL of 75% methanol solution, dissolve in 0.25 mL DPPH mixed solution, room temperature, light protection for 30 min, measured at 517 nm absorbance, accounting for A0. Using the following formula to calculate the DPPH free radical scavenging activity:

[0081]

[0082] 2. Fermentation broth ABTS free radical scavenging activity

[0083] The fermentation broth was centrifuged at 8000 rpm, 4°C, for 10 min, and the supernatant was filtered through a 0.22 μm water filter membrane. 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 and 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. Using the following formula to calculate the ABTS free radical scavenging activity, 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 shown in Figure 6 , the highest DPPH free radical scavenging rate of Pentosus lactis FMBL L24020 DMX is 83.44%, and the highest ABTS free radical scavenging rate is 85.51%. 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 components 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, the above results also further indicate the application potential of Pentosus lactis FMBL L24020 DMX and its pectin fermentation broth in the preparation of antioxidant drugs, health products, synbiotics and metabiotics.

[0086] In summary, the present application provides a pentose lactobacillus FMBL L24020 DMX, which was preserved in China Center for Type Culture Collection on August 26, 2024, and the preservation number is CCTCC NO: M 20241852. The strain can effectively utilize pectin as a carbon source to realize rapid proliferation, and a large amount of short-chain fatty acids can be produced by fermenting pectin. Meanwhile, the pentose lactobacillus FMBL L24020 DMX has strong scavenging ability on DPPH and ABTS free radicals, and has good inhibitory effect on alpha-glucosidase and alpha-amylase activity. It can be used for preparing antioxidant products, hypoglycemic drugs, fermented foods, health products, food additives and feed, and has a wide application prospect.

Claims

1. A Pentosaccharide-based Lactobacillus that metabolizes pectin polysaccharides ( Lactiplantibacillus pentosus The FMBLL24020 DMX is characterized by: The pentosus lactis FMBL L24020 DMX described above was preserved in the China Center for Type Culture Collection on August 26, 2024, and the preservation number is CCTCC NO: M 20241852.

2. An inoculant characterized in that, The pentosus lactis FMBL L24020 DMX described above was preserved in the China Center for Type Culture Collection on August 26, 2024, and the preservation number is CCTCC NO: M 20241852.

3. Use of the pentosus lactis FMBL L24020 DMX or the fermentation liquor thereof of claim 1 or the microbial agent of claim 2 in the preparation of an antioxidant product.

4. Use of the pentosus lactis FMBL L24020 DMX or the fermentation liquor of a strain thereof of claim 1 or the microbial agent of claim 2 in the preparation of an alpha-glucosidase inhibitor.

5. Use of the pentosus lactis FMBL L24020 DMX or the fermentation liquor of a strain thereof of claim 1 or the microbial agent of claim 2 in the preparation of an alpha-amylase inhibitor.

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