Bifidobacterium longum subsp. Infantis, probiotic preparation and application of probiotic preparation in hypoglycemic and hypolipidemic products

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to bifidobacterium longum subsp. Infantis, a probiotic preparation and application of the probiotic preparation in products for reducing blood sugar and blood lipid. The bifidobacterium longum subsp. Infantis provided by the invention is bifidobacterium longum subsp. Infantis A32-03, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.32811. The strain does not generate hemolysin, does not have nitrate reductase activity, does not generate any harmful metabolites such as biogenic amines and indoles, is sensitive to various antibiotics, and is good in biological safety. Particularly, the probiotic preparation obtained by fermenting the bifidobacterium longum subsp. Infantis A32-03 in a culture medium containing the auricularia auricula polysaccharide has a prominent inhibition effect on alpha-amylase, alpha-glucosidase and lipase, has an excellent degradation capability on cholesterol, has a good blood sugar and blood fat regulation effect, and can be used for preparing the probiotic preparation. The method has good popularization value in oral administration of medicines for reducing blood sugar and blood fat or health-care food for regulating blood sugar and blood fat.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and in particular relates to Bifidobacterium longum subspecies infantis, a probiotic preparation and application thereof in blood sugar and lipid lowering products. Background Art

[0002] Obesity is a metabolic disease, and its incidence has been steadily increasing over the years, becoming a global public health concern. Obesity is caused by an imbalance between energy intake and energy expenditure and is linked to multiple factors, including genetics, diet, and exercise. Furthermore, the rapid development of society and the fast-paced lifestyle have inadvertently placed increasing pressure on people, leading to irregular diets and excessive consumption of processed foods. These foods are often high in sugar and fat, causing excessive lipid accumulation in the body and leading to hyperlipidemia. Long-term hyperlipidemia can lead to various complications, including cardiovascular disease, kidney disease, and retinopathy.

[0003] Furthermore, the number of people with diabetes worldwide is growing at an unprecedented rate. Diabetes is a chronic metabolic disease closely linked to the evolution of human eating habits and lifestyles. It is characterized by persistent hyperglycemia due to decreased insulin secretion or impaired insulin activity. Long-term hyperglycemia can also lead to various complications, including cardiovascular disease and kidney disease.

[0004] Currently, widely used drugs for treating obesity and diabetes achieve weight loss by inhibiting lipase activity and stimulating the brain's feeding center to reduce appetite. They also inhibit carbohydrate hydrolase activity to delay glucose release and absorption, thereby lowering postprandial blood sugar levels, slowing carbohydrate metabolism, and preventing excessive glucose absorption. However, these drugs are often expensive and often cause adverse reactions such as abdominal distension, diarrhea, gastrointestinal cramps, and nausea during clinical use, which limits their clinical application.

[0005] Probiotics are active microorganisms that reside in the human body and play a vital role in promoting human health and enhancing immune function. Furthermore, probiotics are relatively safe and have relatively low production costs, offering promising prospects for industrial application and widespread adoption. Existing research indicates that some dominant strains of probiotics can achieve beneficial effects by modulating the composition of the intestinal microbiome, regulating digestive enzyme activity, and regulating hormone release, thereby lowering blood sugar or lipids.

[0006] However, research on probiotics with hypoglycemic and lipid-lowering properties remains limited, particularly for related probiotic products. Furthermore, existing probiotics are relatively ineffective in lowering blood sugar and lipids. Further screening for more effective probiotic strains and developing probiotic products that synergistically enhance these effects are crucial for the prevention and treatment of hyperglycemia and hyperlipidemia in patients. Summary of the Invention

[0007] In response to the deficiencies in the prior art, the present invention aims to provide a Bifidobacterium longum subsp. infantis, a probiotic preparation, and its use in blood sugar and lipid-lowering products. The Bifidobacterium longum subsp. infantis provided by the present invention does not produce harmful metabolites, has an inhibitory effect on multiple pathogens, and is sensitive to multiple antibiotics. In particular, the fermentation product of this strain in a culture medium containing black fungus polysaccharide has excellent blood sugar and lipid-lowering capabilities, making it suitable for development and application as a blood sugar and lipid-lowering product.

[0008] To achieve the above object, the first aspect of the present invention provides a Bifidobacterium longum subsp. infantis, which is named Bifidobacterium longum subsp. infantis A32-03 and is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC NO.32811.

[0009] The second aspect of the present invention provides the use of the above-mentioned Bifidobacterium longum subsp. infantis in the preparation of a probiotic preparation for lowering blood sugar and lipids.

[0010] The third aspect of the present invention provides a probiotic preparation for lowering blood sugar and lipids, which comprises a fermentation product of the above-mentioned Bifidobacterium longum subsp. infantis; the fermentation product is obtained by inoculating Bifidobacterium longum subsp. infantis in a modified culture medium and fermenting it; the modified culture medium is a basic culture medium supplemented with black fungus polysaccharide.

[0011] Black fungus is one of the common fungal resources with extremely high nutritional value, rich content of active substances, and a long history of being used as a medicine and food. Black fungus polysaccharide is a natural plant polysaccharide isolated from black fungus and is one of the key bioactive substances in black fungus. As a new prebiotic, black fungus polysaccharide has great application potential and value in related fields such as food, health care, and medicine. However, there is currently no research on the synergistic effect of Bifidobacterium longum subsp. infantis and black fungus polysaccharide on hypoglycemic and lipid-lowering effects. The present invention confirms through experiments that the fermentation product of Bifidobacterium longum subsp. infantis A32-03 in a culture medium containing black fungus polysaccharide has an outstanding synergistic inhibitory effect on α-amylase, α-glucosidase, and lipase, and has excellent cholesterol clearance ability. Therefore, it has a good blood sugar and blood lipid regulation effect. After use, it is beneficial to prevent or alleviate the complications caused by elevated blood sugar and blood lipids, and is suitable for development and utilization as a blood sugar and lipid-lowering product.

[0012] In a preferred embodiment of the present invention, the basic culture medium is MRS culture medium. The inoculation amount of Bifidobacterium longum subsp. infantis in the modified culture medium is 2 v / v% to 5 v / v%, more preferably 3 v / v%.

[0013] In a preferred embodiment of the present invention, the inoculation is to first activate and culture Bifidobacterium longum subsp. infantis to obtain a seed solution, and then adjust the concentration of the seed solution and inoculate it into the modified culture medium for fermentation. The effective bacterial concentration of the adjusted seed solution is 10 8 ~10 9 CFU / mL.

[0014] In a preferred embodiment of the present invention, the fermentation culture process conditions are: anaerobic culture at a temperature of 36-38° C. for 20-50 h; and the added concentration of black fungus polysaccharide in the improved culture medium is 5-20 mg / mL.

[0015] In a further preferred embodiment of the present invention, the fermentation culture process conditions are: anaerobic culture at 37°C for 24 hours; and the added concentration of black fungus polysaccharide in the improved culture medium is 10 mg / mL.

[0016] For the fermentation process of the strain, the fermentation broth components of the strain include bacterial cells, residual fermentation medium components, strain metabolites, etc. The bacterial cells can be obtained by centrifugation of the fermentation broth, and the precipitate is enriched with bacterial cells; while the residual fermentation medium components, strain metabolites, etc. can be obtained by centrifugation of the fermentation broth, and the fermentation broth supernatant component is enriched with fermentation medium components and strain metabolites. The strain metabolites generally include organic acids, amino acids, exopolysaccharides, etc. Among them, the organic acids (especially short-chain fatty acids, etc.) and exopolysaccharides produced by strain metabolism are the main active ingredients for regulating blood sugar and blood lipid metabolism. In addition, since the strain fermentation broth contains bacterial cells and strain metabolites, the probiotic preparation of the present invention can also be directly prepared using the fermentation products obtained by strain fermentation culture without centrifugation. In addition, after the strain fermentation, some of its metabolites and exopolysaccharides will enter the fermentation broth, while some of the metabolites and exopolysaccharides will remain in the bacterial cells. Therefore, the bacterial cells also have the activity of strain metabolites and exopolysaccharides, and can therefore also be used in the preparation of blood sugar and lipid-lowering products.

[0017] In a preferred embodiment of the present invention, based on the consideration of enhancing the blood sugar and lipid-lowering effects, the fermentation product is the supernatant fraction and / or bacterial precipitate of the fermentation broth obtained after fermentation and cultivation; the dosage form of the blood sugar and lipid-lowering probiotic preparation is a solid preparation or a liquid preparation; the solid preparation is obtained by centrifuging the fermentation broth obtained after fermentation and cultivation, and then collecting the supernatant fraction and / or bacterial precipitate and freeze-drying it; the liquid preparation is obtained by reconstituted the solid preparation with sterile water. The present invention utilizes the fermentation product in solid or liquid form, which can enrich product dosage forms and meet different application requirements.

[0018] A fourth aspect of the present invention provides a blood sugar and / or lipid lowering product, comprising an active ingredient and excipients; the active ingredient is the above-mentioned blood sugar and lipid lowering probiotic preparation.

[0019] In a preferred embodiment of the present invention, the blood sugar lowering and / or lipid lowering product is a medicine and / or a health food.

[0020] In a preferred embodiment of the present invention, the blood sugar lowering and / or lipid lowering product is an oral solid preparation or an oral liquid preparation.

[0021] In a preferred embodiment of the present invention, among the blood sugar lowering and / or lipid lowering products, the blood sugar lowering products exert their blood sugar lowering effect at least by inhibiting α-amylase and α-glucosidase; and the lipid lowering products exert their blood lipid lowering effect at least by inhibiting lipase and clearing cholesterol.

[0022] The present invention does not specifically limit the types of excipients, and technicians can select common excipient types based on the product type and preparation requirements. In a further preferred embodiment of the present invention, due to the adverse effects of digestive tract pH and other factors on probiotics, technicians can prepare the probiotic preparation of the present invention into enteric-coated capsules or microcapsules for use. The use of enteric-coated capsules or microcapsules can protect the probiotics, reduce the killing effect of adverse environments such as gastric acid on the probiotics, and improve the colonization of probiotics in the intestines, thereby helping the probiotics better exert their health-regulating effects after colonization in the intestines.

[0023] The technical solution of the present invention has the following advantages and beneficial effects:

[0024] The present invention provides a Bifidobacterium longum subspecies infantis, with a deposit number of CGMCC No. 32811, screened from infant feces, and having the ability to resist intestinal pathogens and promote the growth of probiotics. The strain does not produce hemolysin, does not dissolve blood cells, has no nitrate reductase activity, does not produce any biogenic amines (putrescine, cadaverine, and spermine), and does not produce harmful metabolites such as indoles. It is sensitive to various antibiotics and has good biosafety.

[0025] The probiotic preparation provided by the present invention comprises a fermentation product of Bifidobacterium longum subsp. infantis. The addition of black fungus polysaccharide during the fermentation process improves bacterial viability and tolerance to the gastrointestinal environment during freeze-drying, protects Bifidobacterium longum subsp. infantis with high activity, and promotes its proliferation, enabling the fermentation product to rapidly lower blood sugar and lipids and regulate intestinal flora homeostasis.

[0026] Comprehensive tests have confirmed that the probiotic preparation provided by the present invention is a fermentation product obtained by inoculating Bifidobacterium longum subsp. infantis A32-03 in a culture medium containing black fungus polysaccharide. The bacterial suspension and fermentation supernatant have good scavenging ability for both DPPH free radicals and hydroxyl free radicals, and have good antioxidant effects. In particular, the probiotic preparation has a prominent inhibitory effect on α-amylase, α-glucosidase, and lipase, has excellent cholesterol degradation ability, and can play a good role in regulating blood sugar and blood lipids. Used alone or in combination with other blood sugar and blood lipid lowering active substances or active bacteria, it helps to prevent or alleviate complications caused by elevated blood sugar and blood lipids.

[0027] Therefore, the Bifidobacterium longum subspecies infantis A32-03 and its fermentation products in the culture medium of black fungus polysaccharide provided by the present invention have good blood sugar and blood lipid regulation effects, can be used as oral hypoglycemic and lipid-lowering drugs or applied to health foods or drinks for improving the intestinal environment and regulating blood sugar and blood lipids, and have good promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Figure 3 is the colony morphology and microscopic morphology of Bifidobacterium longum subspecies infantis A32-03 of the present invention;

[0029] Figure 2 This is the hemolytic test result of Bifidobacterium longum subspecies infantis A32-03 in the present invention;

[0030] Figure 3 The production of harmful metabolites of Bifidobacterium longum subspecies infantis A32-03 in the present invention;

[0031] Figure 4 The effect of the black fungus polysaccharide of the present invention on the growth of Bifidobacterium longum subsp. infantis A32-03;

[0032] Figure 5 The effect of the black fungus polysaccharide of the present invention on the fermentation metabolites of Bifidobacterium longum subsp. infantis A32-03;

[0033] Figure 6 The antibacterial effect of the fermentation product of Bifidobacterium longum subspecies infantis A32-03 and black fungus polysaccharide on pathogenic bacteria in the present invention;

[0034] Figure 7 This is a schematic diagram of the digestion of the microbial ecosystem model in the present invention;

[0035] Figure 8 This is the change of intestinal microorganisms at the phylum level during in vitro simulated digestion in the present invention;

[0036] Figure 9 This represents the changes in intestinal microorganisms at the genus level during in vitro simulated digestion in the present invention. DETAILED DESCRIPTION

[0037] In order to more clearly understand the above-mentioned purposes, features and advantages of the present application, the present application will be further described in detail below in conjunction with specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict, and the protection scope of the present application is not limited by the specific embodiments disclosed below.

[0038] In the following embodiments of the present application, the writing order of the nucleotide sequences such as the primer sequences in the present application is from 5' end to 3' end without special instructions. In the present application, the inoculation amount is volume ratio of v / v% without special instructions. The raw Auricularia auricular material used in the present application is a commercially available product in Harbin, Heilongjiang Province. Other raw materials and the like are materials commonly used in the art which can be obtained through commercial channels without special instructions.

[0039] In the present application, the Auricularia auricular polysaccharide used can be prepared by itself or obtained from a commercial channel, and the present application does not make any limitation thereon. Specifically, in the following embodiments of the present application, the Auricularia auricular polysaccharide is prepared by the following method: after crushing the dried Auricularia auricular, it is passed through an 80-mesh sieve, distilled water is added at a solid-liquid ratio of 1:30 g / mL, it is extracted at 80°C for 2 h, then the mixed system is placed in an ultrasonic crusher, ultrasonic treatment is carried out at 100 W power for 10 min, then cellulase (50 u / mg, Shanghai Yuan Ye) is added at an enzyme addition amount of 1.2% of the mass of the Auricularia auricular raw material, and enzymatic treatment is carried out at 50°C and pH 5.0 for 60 min to obtain an enzymatic hydrolysate, the filtrate is collected by filtration, 3 times the volume of 98 v / v% ethanol is added to the obtained filtrate, then centrifugation is carried out, and the precipitate is collected to obtain the Auricularia auricular polysaccharide.

[0040] The MRS solid medium or MRS liquid medium used in the following embodiments of the present application is the conventional composition of such medium, which can be obtained from a commercial channel or prepared by oneself according to the formula. The present application does not make any special limitation thereon.

[0041] Specifically, in the specific embodiments of the present application, the MRS liquid medium is prepared by the following method: 10 g of proteose peptone, 10 g of beef extract, 5 g of yeast extract, 2 g of dipotassium hydrogen phosphate, 2 g of trisodium citrate, 2 g of sodium acetate, 20 g of glucose, 1 mL of Tween-80, 0.58 g of magnesium sulfate heptahydrate, and 0.25 g of manganese sulfate tetrahydrate are taken, then distilled water is added to make up to 1000 mL, after mixing, the pH is adjusted to 6.8-7.0, 121°C sterilization is carried out for 20 min, and it is ready for use. When the MRS solid medium is prepared, 1.5wt%-2wt% of agar is additionally added, and the rest of the raw materials and the preparation method are the same as those of the MRS liquid medium.

[0042] In a specific embodiment of the present invention, the modified MRS liquid culture medium used is prepared by replacing 10g of glucose in the MRS liquid culture medium with 10g of black fungus polysaccharide, while the other raw materials remain unchanged. Specifically, the modified MRS liquid culture medium is prepared as follows: 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 2g of dipotassium hydrogen phosphate, 2g of trisodium citrate, 2g of sodium acetate, 10g of glucose, 10g of black fungus polysaccharide, 1mL of Tween-80, 0.58g of magnesium sulfate heptahydrate, and 0.25g of manganese sulfate tetrahydrate are added, and then distilled water is added to make up the volume to 1000mL, mixed, and the pH is adjusted to 6.8-7.0, sterilized at 121°C for 20min, and set aside. When preparing the modified MRS solid culture medium, 1.5wt%-2wt% of agar is additionally added. The remaining raw materials and preparation method are the same as those of the modified MRS liquid culture medium.

[0043] In a specific embodiment of the present invention, the LB liquid culture medium used is prepared as follows: 10 g of tryptone, 5 g of yeast extract powder, and 10 g of sodium chloride are added, distilled water is added to make up the volume to 1000 mL, and the mixture is mixed and sterilized at 121°C and 0.1 MPa for 15 min.

[0044] In the following embodiments of the present invention, the separation, purification and identification process of Bifidobacterium longum subspecies infantis A32-03 is as follows:

[0045] (1) Sample collection

[0046] Feces were collected from healthy infants aged 1 to 3 years in Harbin, Heilongjiang Province, who were breastfed and had not taken antibiotics for the past 3 months. The fecal samples were placed in sampling tubes filled with sterile saline, then placed in anaerobic culture bags with anaerobic gas production bags, and the culture bags were placed in foam boxes filled with ice packs and quickly sent to the laboratory for subsequent processing.

[0047] (2) Strain isolation and purification

[0048] Spread culture: The collected fecal sample is shaken and diluted with sterile saline at a mass concentration of 0.9% to prepare suspensions with different concentration gradients. 100 μL of the appropriate gradient suspension is spread on a plate of MRS solid culture medium containing mupirocin lithium salt, and then placed in an anaerobic jar for aeration and anaerobic culture for 48 hours. Isolation and purification: After the spread culture is completed, single colonies with different morphologies on the plate are selected, streaked on the plate, and anaerobically cultured at 37°C for 48 hours. Following the above steps, the plate is streaked and purified for three generations to obtain pure bacteria. Microscopic examination of bacteria: The purified three-generation single colonies are photographed to record the colony morphology, and half of the single colony is selected for Gram staining to observe its microscopic morphology. Then, use a sterilized inoculation loop to pick up half of the remaining single colony for microscopic examination and inoculate it into MRS liquid culture medium. Incubate it anaerobically at 37°C for 16 hours. Take 600 μL of the pure cultured bacterial liquid and add it to an EP tube that has been pre-added with 400 μL of glycerol. Seal the tube with sealing film and store it in a -80°C ultra-low temperature refrigerator.

[0049] The colony morphology and microscopic examination results of Bifidobacterium longum subsp. infantis A32-03 strain are as follows: Figure 1 As shown (A is the colony morphology, B is the microscopic morphology). Figure 1 The colonies of Bifidobacterium longum subsp. infantis A32-03 were translucent, milky white, and had a convex, smooth, and moist surface. Furthermore, Gram staining of Bifidobacterium longum subsp. infantis A32-03 was positive, and microscopic observation revealed Y- and V-shaped rods with blunt ends at one or both ends, consistent with the characteristics of Bifidobacterium longum.

[0050] (3) Molecular biological identification

[0051] After activating and culturing the cells of Bifidobacterium longum subsp. infantis A32-03 isolated and purified in step (2), genomic DNA was extracted using a bacterial genomic DNA extraction kit (Nanjing Novozymes Biotechnology Co., Ltd.). The 16S rDNA sequence was amplified using the upstream primer 27F: AGTTTGATCMTGGCTCAG and the downstream primer 1492R: GGTTACCTTGTTACGACTT to obtain a PCR product. The PCR reaction system included 25 μL of 2xRapidTaq Master Mix, 2 μL of each upstream and downstream primers, 3 μL of DNA template, and 18 μL of ddH2O. PCR reaction conditions included 95°C for 5 min, 35 cycles of 95°C for 30 s, 51°C for 30 s, and 72°C for 1 min, and 72°C for 10 min.

[0052] The PCR reaction product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained gene sequence was submitted to the NCBI database for BLAST analysis and comparison, with 16S rDNA gene sequence homology ≥99% as the identification standard. The obtained 16S rDNA gene sequence of the strain is shown in SEQ ID NO. 1.

[0053] Specifically, the 16S rDNA gene sequence of Bifidobacterium longum subsp. infantis A32-03 is (SEQ ID NO.1):

[0054]

[0055] Molecular biological identification revealed that the 16S rDNA gene sequence of the strain was over 99% homologous to that of Bifidobacterium longum subsp. infantis. The present invention named the strain Bifidobacterium longum subsp. infantis A32-03 and simultaneously deposited it as a biological deposit. The deposit information is as follows:

[0056] Deposit name: Bifidobacterium longum subsp. infantis A32-03,

[0057] Taxonomic nomenclature: Bifidobacterium longum subsp. infantis;

[0058] Deposit number: CGMCC NO.32811;

[0059] Depository: General Microbiology Center, China Culture Collection Administration;

[0060] Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0061] Date of preservation: November 26, 2024.

[0062] Example 1

[0063] This example provides a strain of Bifidobacterium longum subsp. infantis, named Bifidobacterium longum subsp. infantis A32-03, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC NO.32811.

[0064] Example 2

[0065] This embodiment provides a blood sugar and lipid lowering probiotic preparation, comprising a fermentation product of Bifidobacterium longum subsp. infantis of Example 1; the fermentation product is obtained by inoculating Bifidobacterium longum subsp. infantis and fermenting it in a modified culture medium; the modified culture medium is an MRS liquid culture medium supplemented with black fungus polysaccharide.

[0066] The inoculation amount of Bifidobacterium longum subsp. infantis in the modified culture medium is 3% (v / v), and Bifidobacterium longum subsp. infantis is activated before inoculation. The activation culture is to inoculate Bifidobacterium longum subsp. infantis in MRS liquid culture medium, subculture once at 37°C under anaerobic conditions to obtain seed liquid; then adjust the effective bacterial concentration of the seed liquid to 10 8 ~10 9CFU / mL, the seed liquid was inoculated into the modified culture medium at an inoculum size of 3% (v / v) for fermentation. The fermentation process conditions were: anaerobic culture at 37°C for 24 hours; the concentration of black fungus polysaccharide added to the modified culture medium was 10 mg / mL.

[0067] Test Example 1: Safety of Bifidobacterium longum subsp. infantis A32-03

[0068] 1.1 Hemolytic test

[0069] The experimental method is as follows: (1) Preparation of inoculum: The frozen Bifidobacterium longum subsp. infantis A32-03 strain was streaked and inoculated into MRS solid medium, cultured anaerobically at 37°C for 24-48 h, and then subcultured once in MRS liquid medium to obtain the seed solution of Bifidobacterium longum subsp. infantis A32-03. The seed solution (effective bacterial concentration 10 8 ~10 9 CFU / mL) is inoculated into fresh MRS liquid culture medium at an inoculation rate of 3%, and cultured at 37°C for 24 to 48 hours to obtain fresh bacterial liquid, which is used as inoculation liquid for standby use. (2) Preparation of blood cell culture medium: TBS basal culture medium is prepared according to the standard formula, and sterilized under high pressure at 121°C for 15 minutes. When the culture medium is cooled to 50°C, 5% v / v sterile defibrinated sheep blood is added, mixed, and poured into a plate to obtain a blood cell plate for standby use. (3) Streak culture: The inoculation liquid of the test strain obtained in step (1) is streaked and inoculated onto the blood cell plate prepared in step (2), and cultured in an incubator at 37°C for 24 to 48 hours to observe whether the test strain has hemolysis, and Staphylococcus aureus ATCC 13565 is used as a positive control. The hemolytic test results of Bifidobacterium longum subspecies infantis A32-03 and the positive control strain are shown as follows: Figure 2 shown. Figure 2 In the figure, A is Bifidobacterium longum subsp. infantis A32-03; B is the positive control strain Staphylococcus aureus ATCC 13565.

[0070] like Figure 2 As shown, after Bifidobacterium longum subsp. infantis A32-03 was streaked onto the blood cell culture medium, no hemolytic zone appeared around the colonies, proving that Bifidobacterium longum subsp. infantis A32-03 did not produce hemolysin, would not lyse blood cells, and would not cause hemolytic hazards.

[0071] 1.2. Detection of harmful metabolites

[0072] Bifidobacterium longum subsp. infantis A32-03 and a positive control strain (Escherichia coli IQCC 10126) were inoculated into broth containing ornithine decarboxylase, lysine decarboxylase, or arginine decarboxylase, respectively. The broth was incubated at 37°C for 24 hours and tested for biogenic amines. Following the manufacturer's instructions, a purple color change in the broth indicated positive biogenic amine production. The Kovacs indole assay was used to assess the strain's ability to produce indole; a red ring indicated a positive result. Finally, nitroreductase activity was measured using a nitrate reductase assay kit; a positive result was indicated by an orange-red color change in the broth.

[0073] Figure 3 The production of harmful metabolites by Bifidobacterium longum subsp. infantis A32-03 and the positive control strain Escherichia coli IQCC 10126. Figure 3 In the figure, A is the result of ornithine conversion to putrescine; B is the result of lysine conversion to cadaverine; C is the result of arginine conversion to spermine; D is the result of indole test; E is the result of nitrate reductase activity test. Figure 3 In Figures A to E of , the left side shows the production of harmful metabolites by Bifidobacterium longum subsp. infantis A32-03, and the right side shows the production of harmful metabolites by the positive control strain Escherichia coli IQCC 10126.

[0074] like Figure 3 As shown, compared with Escherichia coli, Bifidobacterium longum subspecies infantis A32-03 does not have nitrate reductase activity, does not produce any biogenic amines (putrescine, cadaverine and spermine), and does not produce harmful metabolites such as indoles, indicating that it has high biosafety.

[0075] 1.3 Antibiotic tolerance test

[0076] After the test bacteria (Bifidobacterium longum subsp. infantis A32-03) were streaked on an MRS solid plate for activation, 3 to 5 colonies were picked and added to 5 mL of normal saline to prepare a bacterial suspension, and the concentration of the bacterial suspension was adjusted to 10 8 CFU / mL, aspirate 100 μL of bacterial suspension and add it to the plate of MRS solid culture medium, evenly spread the bacterial liquid on the plate with a sterile cotton swab, stick an antibiotic sensitivity piece (biocin, clindamycin, oxacillin, penicillin, ceftriaxone, cephalothin, vancomycin, ampicillin, erythromycin, azithromycin, cefixime, amoxicillin, ciprofloxacin, rifampicin), let it stand for 15 minutes, replace the air with oxygen-free air, and then culture it upside down at 37°C. After 24 hours, use a vernier caliper to measure the antibiotic sensitivity diameter of the strain.

[0077] The results showed that the sensitivity diameters (mm) of Bifidobacterium longum subsp. infantis A32-03 to novobiocin, clindamycin, oxacillin, penicillin, ceftriaxone, cephalothin, vancomycin, ampicillin, erythromycin, azithromycin, cefixime, amoxicillin, ciprofloxacin, and rifampicin were 23, 40, 23, 38, 36, 35, 42, 33, 41, 35, 13, 35, 16, and 24 mm, respectively. This indicates that Bifidobacterium longum subsp. infantis A32-03 is sensitive to all 14 common antibiotics on the market, proving that Bifidobacterium longum subsp. infantis A32-03 is safe for production and application.

[0078] Experimental Example 2: Effect of black fungus polysaccharide on the growth of Bifidobacterium longum subsp. infantis A32-03

[0079] 2.1 Effects on strain growth

[0080] The seed solution of activated cultured Bifidobacterium longum subsp. infantis A32-03 (effective bacterial concentration 10 8 ~10 9 CFU / mL, activation culture conditions see Example 2, the same below) were inoculated with 3% (v / v) in MRS liquid medium and improved MRS liquid medium (containing black fungus polysaccharide, 10 mg / mL), and cultured anaerobically at 37°C for 30 h. During the culture process, the OD of the culture medium was measured every 2 h. 600 and pH values, and make a growth curve. The results are as follows Figure 4 shown.

[0081] like Figure 4 As shown, the growth rate of Bifidobacterium longum subsp. infantis A32-03 cultured in the modified MRS medium (containing black fungus polysaccharide) was faster in the logarithmic phase, and it had the maximum growth (OD600 was 1.12±0.03) and acid production (pH was 4.06±0.01). This shows that the modified MRS medium (containing black fungus polysaccharide) can better promote the proliferation of Bifidobacterium longum subsp. infantis A32-03 than MRS medium.

[0082] 2.2 Effects on fermentation metabolites (short-chain fatty acids) of the strain

[0083] Bifidobacterium can produce short-chain fatty acids by fermenting prebiotics, common short-chain fatty acids are acetic acid, butyric acid, isobutyric acid, propionic acid and valeric acid, this test evaluates the influence of strains on fermentation metabolism by testing the content of short-chain fatty acids. In the specific experiment, 5 mL of the fermentation broth after culture in section 2.1 was taken as the sample, the sample was centrifuged under the condition of 13000g for 5 min. The supernatant of the sample was taken and filtered with a 0.45 μm filter membrane, and then HPLC analysis was used to determine the short-chain fatty acids in the sample. The HPLC detection conditions are as follows: an HPX-87H column (300x7.8mm, 5 μm, Bio-Rad) was used, 20 μL aliquot of the sample was injected into the column, 5 mM H2SO4 was used as the mobile phase, the column temperature was 50℃, the flow rate was 0.35 mL / min, and the detection wavelength was 210 nm. The content of short-chain fatty acids produced by B. longum subsp. infantis A32-03 after fermentation is shown in Table 2. Figure 5

[0084] As can be seen from Figure 5 , compared with the fermentation product after culture in the MRS medium, the total content of short-chain fatty acids (729.67 μg / mL) in the fermentation broth is the highest when the modified MRS medium with added black fungus polysaccharide is used, especially the content of acetic acid (425.36 μg / mL) is 1.24 times that after fermentation in the MRS medium.

[0085] Test Example Three, Protective Effect of Black Fungus Polysaccharide on Strains in the Process of Freeze-drying and Gastrointestinal Fluid Stress

[0086] 3.1, Protective Effect on Strains in Freeze-drying Environment

[0087] The activated seed liquid of B. longum subsp. infantis A32-03 (effective bacterial concentration 10 8 ~ 10 9 CFU / mL) was inoculated in the MRS liquid medium and the modified MRS liquid medium (containing black fungus polysaccharide, 10 mg / mL) at an inoculation amount of 3% (v / v) at 37℃ under anaerobic conditions for 24 h, the fermentation product (precipitated part) was collected by centrifugal separation, and was mixed with reconstituted skim milk with a milk powder concentration of 14% (100 mL of the precipitate after centrifugation of the fermentation broth was mixed with 10 mL of skim milk), then freeze-dried to obtain bacterial powder. The bacterial powder obtained after freeze-drying was suspended in sterile physiological saline, and the activities of β-galactosidase and lactate dehydrogenase in it were determined, and the results are shown in Table 1.

[0088] Table 1, Key enzyme activities of B. longum subsp. infantis A32-03 after freeze-drying

[0089]

[0090] ​As shown in Table 1, compared with Bifidobacterium longum subsp. infantis A32-03 grown in MRS liquid medium, the key enzyme activity of glucose metabolism was significantly increased after growth in modified MRS liquid medium (containing Auricularia auricular polysaccharide). This indicates that Auricularia auricular polysaccharide improves the tolerance of Bifidobacterium longum subsp. infantis A32-03 in freeze-drying environment by protecting the activity of key metabolic enzymes.

[0091] 3.2, Protective effect of strain on simulated artificial gastrointestinal fluid stress

[0092] The seed liquid (10 8 ~ 10 9 CFU / mL) of Bifidobacterium longum subsp. infantis A32-03 activated after cultivation was inoculated into MRS liquid medium and modified MRS liquid medium (containing Auricularia auricular polysaccharide, 10 mg / mL) at a ratio of 3%, respectively. After 3 generations of activation, 1 mL was taken and placed in 9 mL of filtered and sterilized artificial gastric juice with a pH of 2.5, and was shaken uniformly and cultured anaerobically at 37°C. Samples were taken at the beginning and 2 h after the start of the culture, and the viable cell count was determined. Then, 1 mL of the culture after 2 h of digestion in artificial gastric juice with a pH of 2.5 was inoculated into 9 mL of filtered and sterilized artificial intestinal juice with a pH of 8.0, and was continued to be cultured anaerobically at 37°C. Each group had 3 replicates, and the viable cell count was determined at 0 and 4 h, respectively, and the survival rate was calculated. The artificial gastric juice formula was as follows: physiological saline with a mass concentration of 0.85% was prepared, then 0.3 wt% pepsin was added, dilute hydrochloric acid was used to adjust the pH to 2.5, and the solution was filtered with a 0.22 μm microporous filter to sterilize and prepare for use. The artificial intestinal juice formula was as follows: physiological saline with a mass concentration of 0.85% was prepared, then 0.1 wt% trypsin was added, sodium hydroxide was used to adjust the pH to 8.0, and the solution was filtered with a 0.22 μm microporous filter to sterilize and prepare for use. The artificial gastric juice and artificial intestinal juice were prepared and used immediately, and were stored in a refrigerator at 4°C for a short time. The survival rate calculation formula was as follows: The results of the gastrointestinal fluid tolerance evaluation of Bifidobacterium longum subsp. infantis A32-03 are shown in Table 2.

[0093] Table 2, gastrointestinal fluid tolerance evaluation of Bifidobacterium longum subsp. infantis A32-03

[0094]

[0095]

[0096] As shown in Table 2, the number of viable bacteria of Bifidobacterium longum subsp. infantis A32-03 was significantly reduced after treatment with gastric and intestinal fluids. The number of viable bacteria of Bifidobacterium longum subsp. infantis A32-03 in modified MRS medium supplemented with black fungus polysaccharide was significantly higher than that of Bifidobacterium longum subsp. infantis A32-03 grown in MRS medium, indicating that black fungus polysaccharide can improve the tolerance of Bifidobacterium longum subsp. infantis A32-03 to gastrointestinal fluids and has certain intestinal colonization potential.

[0097] Experimental Example 4: Determination of the Antioxidant Function of Fermentation Products of Bifidobacterium longum subsp. infantis A32-03 and Auricularia auriculariae Polysaccharide

[0098] This study evaluated the antioxidant function of the fermentation products of Bifidobacterium longum subsp. infantis A32-03 and black fungus polysaccharide by measuring the strains' ability to scavenge DPPH and hydroxyl radicals.

[0099] The experimental methods for scavenging DPPH and hydroxyl free radicals are as follows:

[0100] (1) Preparation of bacterial suspension: The seed solution of the test bacteria (Bifidobacterium longum subsp. infantis A32-03, effective bacterial concentration 10 8 ~10 9 CFU / mL) were inoculated into MRS liquid medium and modified MRS liquid medium (containing black fungus polysaccharide, 10 mg / mL) at a ratio of 3%, and incubated anaerobically at 37°C for 24 hours to obtain culture fluids of the strains. 1 mL of the culture fluid was aspirated and centrifuged at 8000 rpm for 8 minutes to collect the cells, which were then washed twice with 1 mL of PBS buffer and resuspended in 2 mL of PBS solution to obtain a bacterial suspension for later use.

[0101] (2) Preparation of fermentation supernatant of strains: The seed solution of the test bacteria (Bifidobacterium longum subsp. infantis A32-03, effective bacterial concentration 10 8 ~10 9 CFU / mL) were inoculated into MRS liquid medium and modified MRS liquid medium at a ratio of 3%, cultured anaerobically at 37°C for 24 h, and then centrifuged at 8000 rpm for 8 min. The supernatant was collected and filtered through a 0.22 μm microporous filter to obtain the fermentation supernatant for use.

[0102] (3) Determination of the ability of strains to scavenge DPPH free radicals: Take 400 μL of the bacterial suspension and fermentation supernatant of the test strain, add 600 μL of 0.4 mM freshly prepared DPPH free radical solution, mix well, and place in the dark at room temperature for 30 minutes. Then measure the absorbance A of the sample at a wavelength of 517 nm. The sample was tested in parallel three times. The control group sample was 400 μL of PBS bacterial suspension and 600 μL of 80% methanol solution, and the blank group sample was 400 μL of 80% methanol solution and 600 μL of DPPH free radical solution. The DPPH scavenging rate was calculated according to the following formula:

[0103] (4) Determination of the ability of the strain to scavenge hydroxyl radicals: 1 mL each of 1,10-phenanthroline (2.5 mmol / L), PBS (0.02 mmol / L, pH 7.4) and distilled water were mixed, and then 1 mL each of the bacterial suspension or fermentation supernatant of Bifidobacterium longum subsp. infantis A32-03 prepared above, FeSO4 (2.5 mmol / L) and H2O2 (20 mmol / L) were added and incubated at 37°C for 1.5 h. The absorbance of the mixture was measured at 536 nm. b ; Replace distilled water with an equal volume of sample to measure absorbance As; Replace H2O2 with an equal volume of distilled water to measure absorbance Ac. The hydroxyl radical scavenging rate is calculated as follows:

[0104] The DPPH free radical test results are shown in Table 3. The hydroxyl free radical test results are shown in Table 4.

[0105] Table 3. DPPH radical scavenging ability of Bifidobacterium longum subsp. infantis A32-03 and black fungus polysaccharide fermentation products

[0106]

[0107] Table 4. Hydroxyl radical scavenging ability of Bifidobacterium longum subsp. infantis A32-03 and black fungus polysaccharide fermentation products

[0108]

[0109] As shown in Table 3, the DPPH radical scavenging rates of the bacterial suspension and fermentation supernatant of Bifidobacterium longum subsp. infantis A32-03 after culture in MRS medium were 21.54 ± 0.41% and 68.46 ± 0.18%, respectively. The DPPH radical scavenging rates of the bacterial suspension and fermentation supernatant after culture in modified MRS medium (containing black fungus polysaccharide) were 24.43 ± 1.12% and 72.13 ± 0.31%, respectively, indicating that black fungus polysaccharide can significantly improve the ability of Bifidobacterium longum subsp. infantis A32-03 to scavenge DPPH radicals, which is beneficial to the improvement of the antioxidant capacity of the strain. In addition, the DPPH radical scavenging ability of the fermentation supernatant was significantly better than that of the bacterial suspension.

[0110] As shown in Table 4, the hydroxyl radical scavenging rates of the bacterial suspension and fermentation broth of Bifidobacterium longum subsp. infantis A32-03 after culture in MRS medium were 86.35 ± 0.22% and 24.36 ± 0.32%, respectively. The hydroxyl radical scavenging rates of the bacterial suspension and fermentation supernatant after culture in modified MRS medium (containing black fungus polysaccharide) were 90.01 ± 0.19% and 29.11 ± 0.24%, respectively. This indicates that black fungus polysaccharide can significantly improve the antioxidant capacity of Bifidobacterium longum subsp. infantis A32-03 and can effectively scavenge hydroxyl radicals. In addition, the hydroxyl radical scavenging ability of the bacterial suspension is significantly better than that of the fermentation supernatant.

[0111] Experimental Example 5: Evaluation of the antibacterial activity of the fermentation product of Bifidobacterium longum subsp. infantis A32-03 and black fungus polysaccharide

[0112] The experimental steps of antibacterial activity are as follows: (1) Bifidobacterium longum subsp. infantis A32-03 was activated and cultured in MRS liquid medium to obtain seed solution, and the concentration of seed solution was adjusted to 10 8 ~10 9 CFU / mL, inoculated into the modified MRS liquid culture medium (containing black fungus polysaccharide, 10 mg / mL) at an inoculum volume of 3% (v / v), and cultured anaerobically at 37°C for 24 hours to obtain the culture fluid of the strain. (2) Escherichia coli IQCC 10126 and Staphylococcus aureus ATCC 13565 were used as indicator bacteria, and the indicator bacteria liquid was inoculated into 10 mL of LB liquid culture medium at an inoculum volume of 3% respectively, and cultured at a constant temperature of 37°C in an incubator for 24 hours. (3) First pour 10 mL of plain agar culture medium on the lower layer of the plate, wait for it to cool and solidify, and then put it into the Oxford cup. Take 20 μL of each pathogen suspension and add it to 20 mL of LB solid culture medium at an appropriate temperature (about 50°C) and mix well (the number of viable indicator bacteria is 10 6 CFU / mL), pour it into the plain agar plate prepared in advance, shake it gently to mix it, and after it solidifies, use tweezers to remove the Oxford cup, and add 160 μL of the culture solution of the strain obtained in step (1) into the hole left after the Oxford cup is removed (the number of viable bacteria is 108 CFU / mL), wherein the control was replaced with modified MRS liquid medium. The plates were placed in an anaerobic culture bag with an anaerobic gas generator bag, the air was replaced with anaerobic air, and the culture bag was carefully moved into the incubator and incubated horizontally at 37°C for 18 h. After the incubation, the diameters of the inhibition zones were measured with a vernier caliper.

[0113] The results of the antibacterial effect test are shown in Table 5 and Figure 6 . Figure 6 Table 5, wherein A is Staphylococcus aureus and B is Escherichia coli, and the uppermost side of each plate is the test group.

[0114] Table 5, wherein A is Staphylococcus aureus and B is Escherichia coli, and the uppermost side of each plate is the test group.

[0115]

[0116] As can be seen from Figure 6 and Table 5, Bifidobacterium longum subsp. infantis A32-03 and the fermentation product of Auricularia auricular polysaccharide have excellent inhibitory effects on common intestinal pathogenic bacteria, such as Escherichia coli and Staphylococcus aureus.

[0117] Test Example Six, in Vitro Hypoglycemic and Hypolipidemic Effects of Bifidobacterium longum subsp. infantis A32-03 and the Fermentation Product of Auricularia auricular Polysaccharide

[0118] 6.1, Preparation of Fermentation Supernatant of Strains

[0119] After Bifidobacterium longum subsp. infantis A32-03 was activated and cultured in MRS liquid medium, a seed liquid was obtained, the concentration of the bacterial liquid of the seed liquid was adjusted to 10 8 ~ 10 9 CFU / mL, 3% (v / v) of the inoculation amount was inoculated into modified MRS liquid medium (containing Auricularia auricular polysaccharide, 10 mg / mL), and anaerobic culture was performed at 37°C for 24 h. Subsequently, centrifugation was performed at 8000 r / min for 8 min, the supernatant was collected, and filtration was performed through a 0.22 μm microporous filter to obtain the fermentation supernatant, which was used as the test group of the present application.

[0120] Meanwhile, the modified MRS liquid medium (containing Auricularia auricular polysaccharide, 10 mg / mL) without inoculation of strains was used as a blank control group; and the following comparative groups of fermentation supernatants were set:

[0121] Comparative Group 1: the modified MRS liquid medium in the test group was replaced with MRS liquid medium (without Auricularia auricular polysaccharide), and the remaining process parameters of the fermentation supernatant were the same as those of the test group;

[0122] Comparative Group 2: the Auricularia auricular polysaccharide in the modified MRS liquid medium in the test group was replaced with Lycium barbarum polysaccharide (the amount was kept consistent), and the remaining process parameters of the fermentation supernatant were the same as those of the test group;

[0123] Comparative group 3: The Bifidobacterium longum subsp. infantis A32-03 in the experimental group was replaced with Bifidobacterium lactis YQWS2301, and the rest of the process parameters of the fermentation supernatant were the same as those of the experimental group;

[0124] Comparative group 4: The Bifidobacterium longum subsp. infantis A32-03 in the experimental group was replaced with Bifidobacterium lactis Bb-12, and the rest of the process parameters of the fermentation supernatant were the same as those of the experimental group;

[0125] Comparative group 5: Bifidobacterium longum subsp. infantis A32-03 in the experimental group was replaced by Bifidobacterium longum subsp. infantis A32-02, and the remaining process parameters of the fermentation supernatant were the same as those of the experimental group.

[0126] Bifidobacterium lactis YQWS2301 is an existing strain reported in prior art CN118956656A and deposited by the applicant. Bifidobacterium longum subsp. infantis A32-02 was isolated from the intestines of healthy infants in Harbin, Heilongjiang Province. Its isolation, purification, and identification procedures were similar to those for Bifidobacterium longum subsp. infantis A32-03. Bifidobacterium lactis Bb-12 was purchased from Chr. Hansen.

[0127] 6.2. α-Glucosidase inhibition assay

[0128] Solution preparation: 1.5U / mL α-glucosidase test solution: Dissolve 100U of α-glucosidase in 1mL of PBS buffer to a 100U / mL stock solution. Take 300μL of the stock solution and dilute to 1.5U / mL with 20mL of PBS. 20mmol / LPNPG (p-nitrophenyl-α-D-pyranoglucopyranoside): Accurately weigh 0.0788g of PNPG in a 10mL centrifuge tube, dissolve in 5mL of PBS buffer, aliquot, and store in a -20°C refrigerator until needed. 0.2mol / LNa2CO3 terminator: Accurately weigh 2.12g of Na2CO3, dissolve in an appropriate amount of distilled water, and dilute to volume in a 100mL volumetric flask.

[0129] α-glucosidase inhibition activity test: 35 μL of the supernatant of the sample to be tested is aspirated, mixed with 165 μL of PBS buffer, and then 50 μL of α-glucosidase solution (1.5 U / mL) is added. After mixing, the mixture is incubated at 37°C for 5 minutes. Then, 75 μL of PNPG (20 mmol / L) is added and mixed evenly. The mixture is incubated at 37°C for 15 minutes. Finally, 400 μL of Na2CO3 is added to terminate the reaction. The OD value at a wavelength of 405 nm is measured in a microplate reader, and the inhibition rate is calculated. Each sample is tested in parallel 3 times, and acarbose (5 μg / mL) is used as a positive control. The inhibition rate is calculated as follows: Where: A1 is the absorbance with enzyme but not sample; A2 is the absorbance without enzyme and sample; A3 is the absorbance with enzyme and sample; A4 is the absorbance with sample but not enzyme.

[0130] 6.3. α-Amylase inhibition assay

[0131] Solution preparation process: 2.5U / mL α-amylase test solution: Take 50mg of α-amylase and dissolve it in 0.2mol / L PBS buffer to 25mL, filter, and prepare it before use. 1% soluble starch solution: Take 0.5g of soluble starch and place it in a 100mL beaker. Take 50mL of PBS buffer, first moisten the starch with a small amount of PBS buffer, boil the remaining buffer and add it to the soluble starch, place the soluble starch solution in a boiling water bath and stir for 10min to fully gelatinize and dissolve it. Take it out, cool it to room temperature, and transfer it to a 50mL volumetric flask. Wash the beaker with an appropriate amount of water, add the washing liquid to the volumetric flask, and dilute to the scale line, shake well and filter, and prepare it before use. 3,5-Dinitrosalicylic acid test solution (DNS): Dissolve 6.5 g of 3,5-dinitrosalicylic acid in 325 mL of 2 mol / L sodium hydroxide solution and 45 mL of glycerol. Add water to 1000 mL and dissolve thoroughly. Store at room temperature until ready to use. Acarbose solution: Dissolve 5 mg of acarbose in 5 mL of distilled water to prepare a 1 mg / mL solution.

[0132] α-Amylase inhibition test: 200 μL of fermentation supernatant was mixed with 200 μL of α-amylase working solution and incubated at 37°C for 10 minutes. Then, 200 μL of 10 g / L soluble starch solution was added and incubated for another 10 minutes. Then, 400 μL of DNS was added and the mixture was incubated in a boiling water bath for 5 minutes. After cooling, 4 mL of deionized water was added. After mixing, the OD value at 540 nm was measured in a microplate reader. Acarbose (1 mg / mL) was used as a positive control. The inhibition rate was calculated as follows: Where: A1 is the absorbance with enzyme but not sample; A2 is the absorbance without enzyme and sample; A3 is the absorbance with enzyme and sample; A4 is the absorbance with sample but not enzyme.

[0133] The results of the test on the inhibitory effects of the fermentation supernatants of the experimental group and the control group on α-glucosidase and α-amylase are shown in Table 6.

[0134] Table 6. Inhibitory effect on α-glucosidase and α-amylase (%)

[0135]

[0136] The results in Table 6 show that the fermentation product of Bifidobacterium longum subsp. infantis A32-03 and black fungus polysaccharide has a strong inhibitory ability against α-glucosidase and α-amylase, with an inhibition rate of 82.32±0.13% for α-glucosidase and 85.21±0.23% for α-amylase. The inhibitory effect is significantly better than the combination of the control strains Bifidobacterium lactis YQWS2301, Bifidobacterium lactis Bb-12 and Bifidobacterium longum subsp. infantis A32-02 and black fungus polysaccharide (P<0.05). In addition, the inhibitory effect of the fermentation product of Bifidobacterium longum subsp. infantis A32-03 and black fungus polysaccharide is significantly better than the control group and control group 1 using either alone, and is significantly better than the combination of Bifidobacterium longum subsp. infantis A32-03 and wolfberry polysaccharide (P<0.05). This shows that the fermentation product of Bifidobacterium longum subsp. infantis A32-03 combined with black fungus polysaccharide has excellent blood sugar lowering ability.

[0137] 6.4 Lipase inhibition assay

[0138] Preparation of polyvinyl alcohol triolein emulsion: Weigh 40g of polyvinyl alcohol and add 800mL of water. Heat in a boiling water bath while stirring continuously until completely dissolved. Cool and dilute to 1000mL. Filter through a clean double-layer gauze and set aside. Measure 150mL of the filtrate and add 50mL of triolein. Process using a high-speed homogenizer for 6 minutes (3 minutes each time, two times) to obtain the polyvinyl alcohol triolein emulsion, which is ready for use.

[0139] Lipase activity assay: Add 4 mL of polyvinyl triolein emulsion and 5 mL of PBS buffer (pH = 7.4, 0.025 mol / L) to a conical flask, preheat in a 37°C water bath for 10 min, then add 1 mL of lipase solution (2 mg / mL, prepared in PBS buffer), mix immediately, react accurately for 15 min in a 37°C water bath, and add 15 mL of ethanol solution (95%) to terminate the enzyme reaction. In the blank group, add 95% ethanol after 1 minute of reaction, and then add 1 mL of lipase solution. Add 2 drops of phenolphthalein indicator and titrate with 0.05 mol / L sodium hydroxide until it turns slightly red and remains unfaded for 30 seconds as the titration endpoint. Record the volume of sodium hydroxide solution consumed. Calculate lipase activity using the following formula: Where: U is the enzyme activity per gram of lipase, IU; Vs is the volume of NaOH solution consumed by the sample group, mL; Vc is the volume of NaOH solution consumed by the blank group, mL; c is the concentration of the NaOH standard solution, mol / L; w is the amount of lipase added, g; t is the reaction time after adding lipase, min.

[0140] Lipase inhibition rate determination: Add 4 mL of polyvinyl triolein emulsion and 5 mL of PBS to a conical flask and incubate at 37°C in a water bath for 10 min. Then, add 1 mL of the fermentation supernatant prepared in Section 6.1 and continue the reaction at 37°C for 10 min. Then, add 1 mL of lipase (2 mg / mL) and incubate at 37°C for 15 min. Immediately after the reaction, add 15 mL of 95% ethanol to terminate the reaction. Add 2 drops of phenolphthalein indicator to the reaction system and titrate with 0.05 mol / L sodium hydroxide until a slightly reddish color remains unchanged for 30 seconds, marking the endpoint. Record the volume of sodium hydroxide solution consumed. For the control group, add the same volume of fermentation supernatant, followed by 15 mL of 95% ethanol and 1 mL of lipase (2 mg / mL). Incubate at 37°C for 15 min. Titration is performed using the same method as above. Calculate the residual enzyme activity using the following formula: Where: U - enzyme activity per gram of lipase, IU; Vs - volume of NaOH solution consumed by the sample group, mL; V0 - volume of NaOH solution consumed by the control group, mL; c - concentration of NaOH standard solution, mol / L; w - amount of lipase added, g; t - reaction time after adding lipase, min. The lipase inhibition rate is calculated using the formula: The test results of lipase inhibition rate are shown in Table 7.

[0141] Table 7. Inhibition ability on lipase (%)

[0142]

[0143] The results of Table 7 show that the fermentation product of Bifidobacterium longum subspecies infantis A32-03 and black fungus polysaccharide has a strong inhibitory ability on lipase, and its inhibition rate on lipase can reach 72.96±0.09%, and the inhibitory effect is significantly better than the fermentation product of the control strains Bifidobacterium lactis YQWS2301, Bifidobacterium lactis Bb-12 and Bifidobacterium longum subspecies infantis A32-02 and black fungus polysaccharide combination (P<0.05). In addition, the inhibitory effect of the fermentation product of Bifidobacterium longum subspecies infantis A32-03 and black fungus polysaccharide is significantly better than that of the two alone, and is significantly better than the fermentation product of Bifidobacterium longum subspecies infantis A32-03 and wolfberry polysaccharide combination (P<0.05). This shows that the fermentation product of Bifidobacterium longum subspecies infantis A32-03 and black fungus polysaccharide has excellent lipid-lowering ability.

[0144] 6.5 Cholesterol Clearance Test

[0145] Preparation of MRS / modified MRS-cholesterol medium: Add 0.1 g / L cholesterol, 0.2 g / L ox bile salt, 1 mL / L Tween 80, and 5 mL / L glacial acetic acid to modified MRS or MRS liquid medium, and sterilize at 121°C for 20 min to obtain MRS / modified MRS-cholesterol medium.

[0146] The cholesterol removal ability test process is: in tool stopper test tube, respectively add 9mMRS / improved MRS-cholesterol culture medium, inoculate respectively activated Bifidobacterium longum infantis subspecies A32-03, Bifidobacterium longum infantis subspecies A32-02, Bifidobacterium lactis YQWS2301 and Bifidobacterium lactis Bb-12 in 2% ratio, uninoculated culture medium is placed in 37 ℃ of incubators and cultivated for 18h, after cultivation finishes, centrifugal (4000r / min, 4 ℃, 10min).According to the operating instructions of total cholesterol (TC) content detection kit, measure cholesterol content in the fermentation supernatant.Calculate cholesterol clearance rate with following formula: Where: Cs = cholesterol content in the sample group, μmol / L; Cc = cholesterol content in the blank control group, μmol / L. The cholesterol clearance test results are shown in Table 8.

[0147] Table 8. Cholesterol scavenging ability (%)

[0148]

[0149] The results in Table 8 show that the cholesterol scavenging ability of Bifidobacterium longum subspecies infantis A32-03 cultured in the modified MRS medium with the addition of black fungus polysaccharide is significantly higher than that of Bifidobacterium longum subspecies infantis A32-03 grown in MRS medium, with a cholesterol clearance rate of 29.27 ± 0.13%, and the cholesterol scavenging ability is significantly better than that of the control strains Bifidobacterium lactis YQWS2301, Bifidobacterium lactis Bb-12 and Bifidobacterium longum subspecies infantis A32-02 grown in the modified MRS medium with the addition of black fungus polysaccharide (P < 0.05). In addition, the cholesterol scavenging ability of Bifidobacterium longum subspecies infantis A32-03 cultured in the modified MRS medium with the addition of black fungus polysaccharide is significantly higher than that of Bifidobacterium longum subspecies infantis A32-03 grown in the modified MRS medium with the addition of wolfberry polysaccharide. This shows that the fermentation product of Bifidobacterium longum subspecies infantis A32-03 and black fungus polysaccharide has a strong lipid-lowering effect.

[0150] Experimental Example 7: Effect of in vitro simulation of Bifidobacterium longum subsp. infantis A32-03 and black fungus polysaccharide composition on colonic pathogens

[0151] 7.1. Construction and operation of SHIME in vitro digestion

[0152] This experiment refers to the SHIME in vitro model that simulates the human intestinal microbial ecosystem and has been slightly improved (see the schematic diagram Figure 7 The parameters of the human intestinal microbial ecosystem (SHIME) model components and the basal culture medium are shown in Tables 9 and 10, respectively.

[0153] Table 9. Parameters of the Human Intestinal Microbial Ecosystem (SHIME) model components

[0154]

[0155] Table 10. Basic culture medium for microbial ecosystem model

[0156] Element Mass (g) Xylan 4 pectin 2 Arabinogalactan 1 glucose 0.4 starch 1 yeast extract 3 Peptone 3 mucin 0.75 Cysteine 0.5 distilled water 1000 (mL)

[0157] The SHIME model consists of five fermentation glass tubes as reactors, namely the stomach, small intestine, ascending colon (AC), transverse colon (TC), and descending colon (DC). Each glass fermenter is placed in a water bath at a constant temperature of 37°C, and latex tubes are used for connection and food delivery. A peristaltic pump is used to simulate the peristaltic process of the human intestine, and a magnetic stirrer is used to simulate the stirring process of various parts of the human body at 37°C. In this experiment, nitrogen is introduced for 20 minutes every 4 hours to simulate the anaerobic environment of the human gastrointestinal tract. Table 9 details the retention time, pH, flow rate and fermenter volume of this device. The pH of the five reactors is adjusted by a pH controller (0.1 mol / L hydrochloric acid or 0.1 mol / L sodium hydroxide) to meet the pH environment of various parts of the human body.

[0158] Before the experiment began, fecal suspensions from healthy adult volunteers were inoculated into the ascending colon (AC), transverse colon (TC), and descending colon (DC). Subjects had to have no history of gastrointestinal disease, no antibiotic use within the past 6 months, and signed informed consent before participating in the study. Freshly donated fecal samples were stored in a collection box at 4°C along with anaerobic bacteria bags. Fifty grams of feces were weighed and added to 250 mL of inoculum (sterile PBS buffer 0.1 mol / L, pH 7, with 1 g / L sodium thioglycolate as a reducing agent). The suspension was homogenized using a homogenizer and shaker. The suspension was then centrifuged at 4°C and 1000 g for 10 minutes. Finally, the supernatant was added to 50 mL of AC, 50 mL of TC, and 60 mL of DC, respectively.

[0159] The experiment is divided into four phases: stabilization period (1 week), pathogenic period (1 day), treatment period (1 week) and washout period (1 week). The stabilization period is to allow the microbial community in the feces to better colonize and grow in the fermentation tank, accurately simulating the human intestinal microbial community. During the pathogenic period, Staphylococcus aureus and Escherichia coli were added to the ascending colon, transverse colon, and descending colon, and the peristaltic pump was stopped for natural colonization for 24 hours. The purpose of the treatment period is to test the effect of the fermentation product of Bifidobacterium longum infantis subspecies A32-03 and black fungus polysaccharide on the intestinal microbial community. Among them, the fermentation product is to activate and culture Bifidobacterium longum infantis subspecies A32-03 in MRS liquid culture medium, and then adjust the bacterial liquid concentration to 10 8 ~10 9 CFU / mL was inoculated at a 3% inoculum (v / v) into modified MRS liquid medium (containing black fungus polysaccharide, 10 mg / mL) and incubated anaerobically at 37°C for 24 hours. A washout period was performed to verify the sustained therapeutic effects of Bifidobacterium longum subsp. infantis A32-03 and black fungus polysaccharide ferment. Samples were collected on the last day of each period for intestinal microbial analysis and short-chain fatty acid determination. During the trial, 250 mL of sterile culture medium was added twice daily (Table 10). Based on the recommended intake of probiotics in existing research literature and relevant international standards and regulations, 3.0 g / day of Bifidobacterium longum subsp. infantis A32-03 and black fungus polysaccharide ferment were added in addition to the culture medium during the one-week treatment period. Following the addition of the culture medium, 50 mL of saliva, 100 mL of gastric juice, and 60 mL of intestinal fluid were added to the stomach and small intestine, respectively. The digestive fluid composition is shown in Table 11, and the electrolyte composition is shown in Table 12. After the gastric juice was prepared, the pH was adjusted to 2 with 0.1 mol / L HCl.

[0160] Table 11. Composition of various digestive juices

[0161]

[0162]

[0163] Table 12. Electrolyte stock solutions of various digestive solutions

[0164]

[0165] 7.2. Intestinal Microbial Analysis

[0166] Samples were collected from each colon and placed in cryovials, immediately placed in liquid nitrogen, and then stored in a −80°C freezer. The samples were then shipped on dry ice to Harbin Haruo Biotechnology Co., Ltd. for 16S rDNA gene amplicon sequencing. Bioinformatics analysis of the sequencing data was performed using Quantitative Insights Into Microbial Ecology (QIIME) software.

[0167] Figure 8 、 Figure 9 In Tables 13 and 14, AB, AI, AT, AW, TB, TI, TT, TW, DB, DI, DT, and DW are AB: ascending colon during the stable period, AI: ascending colon during the period of inoculation of pathogens, AT: ascending colon during the treatment period, AW: ascending colon during the washout period, TB: transverse colon during the stable period, TI: transverse colon during the period of inoculation of pathogens, TT: transverse colon during the treatment period, TW: descending colon during the washout period, DB: descending colon during the stable period, DI: descending colon during the period of inoculation of pathogens, DT: descending colon during the treatment period, and DW: descending colon during the washout period.

[0168] This study used high-throughput sequencing analysis to study the effects of Bifidobacterium longum subsp. infantis A32-03 and black fungus polysaccharide fermentation on intestinal flora. Figure 8 As shown, the relative abundance of intestinal microorganisms at the phylum level varied to varying degrees during the different treatment periods. The predominant phyla in the intestinal microbiota were Firmicutes, Proteobacteria, Actinobacteriota, and Bacteroidetes. During the stable period, Firmicutes was the dominant phylum in all three colons. However, after inoculation with pathogens, the Proteobacteria level significantly changed, with the relative abundance of Proteobacteria increasing from 0.25%, 2.15%, and 15.42% to 43.50%, 40.99%, and 43.12% in the three colons, respectively. The relative abundance of Proteobacteria decreased significantly during treatment (P < 0.05). Conversely, the relative abundance of Firmicutes increased significantly in all three colon segments during treatment (P < 0.05). The percentages of Firmicutes increased from 46.67%, 49.42%, and 48.59% to 65.11%, 58.76%, and 66.98%, respectively. There was no significant change during the elution period. Since almost all Proteobacteria species are harmful bacteria, such as Salmonella and Escherichia coli, this demonstrates the potential of Bifidobacterium longum subsp. infantis A32-03 and black fungus polysaccharides to inhibit the growth of pathogens.

[0169] In order to study the essential differences in the intestinal microbial structure, the top 20 genera with the highest relative abundance were selected for comparison, such as Figure 9 As shown. After inoculation with pathogenic bacteria, the intestinal flora of the three colons underwent significant changes. When inoculated with pathogenic bacteria, the microbial homeostasis of the intestinal flora was disrupted, among which Escherichia, Klebsiella, Salmonella, Staphylococcus, and Enterococcus increased significantly (P < 0.05). Megamonas, Lactobacillus, Bacteroides, Lachnoclostridium, Bifidobacterium, Dialister, and Phascolarctobacterium decreased significantly (P < 0.05). During treatment with the combination of Bifidobacterium longum subsp. infantis A32-03 and black fungus polysaccharide, the abundance of Escherichia, Klebsiella, Salmonella, and Staphylococcus decreased significantly (P < 0.05). The abundance of probiotics, including Lactobacillus, Bifidobacterium, Bacteroides, Lachnoclostridium, and Phascolarctobacterium, increased significantly (P < 0.05). The abundance of each genera did not fluctuate significantly during the washout period.

[0170] Based on the above experimental results, Bifidobacterium longum subspecies infantis A32-03 and black fungus polysaccharide fermentation products can significantly regulate the homeostasis of intestinal flora, promote the growth of intestinal probiotics, and inhibit certain pathogenic bacteria, thereby allowing the intestinal flora to reach a balanced state again.

[0171] 7.3 Determination of Short-Chain Fatty Acids

[0172] A 5 mL sample was centrifuged at 13,000 g for 5 min. The supernatant was filtered through a 0.45 μm filter and analyzed by HPLC to determine the short-chain fatty acids in the sample. HPLC analysis conditions: A 20 μL aliquot was injected onto an HPX-87H column (300 × 7.8 mm, 5 μm, Bio-Rad) with 5 mM H₂SO₄ as the mobile phase, a column temperature of 50°C, a flow rate of 0.35 mL / min, and a detection wavelength of 210 nm.

[0173] Short-chain fatty acids are important metabolites of microorganisms and can indirectly reflect changes in the microbial flora. The changes in short-chain fatty acids throughout the digestion process are shown in Tables 13 and 14.

[0174] Table 13. Changes in short-chain fatty acid content in SHIME at different stages (mmol / L)

[0175] lactic acid Acetic acid Propionic acid Butyric acid AB <![CDATA[27.57±0.12 c ]]> <![CDATA[24.37±0.84 b ]]> 14.28 ± 0.76 c ]] <![CDATA[3.73±0.16 c ]]> AI <![CDATA[23.60±0.43 d ]]> <![CDATA[16.28±0.41 c ]]> <![CDATA[10.34±0.18 d ]]> <![CDATA[2.85±0.15 d ]]> AT <![CDATA[68.92±0.08 a ]]> <![CDATA[35.61±0.47 a ]]> <![CDATA[19.35±0.36 a ]]> 4.47 ± 0.19 a ]] AW <![CDATA[48.92±0.08 b ]]> <![CDATA[31.07±0.68 a ]]> <![CDATA[15.18±0.23 b ]]> <![CDATA[4.09±0.10 b ]]> TB <![CDATA[33.87±0.46 c ]]> <![CDATA[29.11±0.49 c ]]> 13.43 ± 0.15 c ]] 4.66 ± 0.06 c ]] TI 30.00 ± 0.86 d ]] <![CDATA[21.96±0.74 d ]]> <![CDATA[11.79±0.08 d ]]> <![CDATA[3.87±0.31 d ]]> TT <![CDATA[50.46±0.98 a ]]> 43.30 ± 1.05 a ]] <![CDATA[16.51±0.22 a ]]> <![CDATA[6.18±0.18 a ]]> TW <![CDATA[44.46±0.98 b ]]> <![CDATA[33.67±0.71 b ]]> <![CDATA[15.18±0.73 b ]]> <![CDATA[5.07±0.21 b ]]> DB <![CDATA[47.01±1.09 c ]]> 41.22 ± 0.74 b ]] <![CDATA[17.63±0.93 b ]]> 5.04 ± 0.12 b ]] DI <![CDATA[39.92±0.60 d ]]> <![CDATA[29.62±2.74 c ]]> 11.31 ± 0.33 c ]] <![CDATA[3.79±0.21 c ]]> DT <![CDATA[61.03±0.55 a ]]> 50.69 ± 2.59 a ]] <![CDATA[21.42±1.64 a ]]> <![CDATA[6.78±0.27 a ]]> DW <![CDATA[51.03±0.55 b ]]> 51.03 ± 0.55 b ]] <![CDATA[18.61±1.17 b ]]> <![CDATA[5.62±0.52 b ]]>

[0176] Table 14. Changes in short-chain fatty acid content at different stages of SHIME (mmol / L)

[0177]

[0178]

[0179] In Tables 13 and 14, a total of six short-chain fatty acids were tested. Among them, lactic acid and acetic acid levels were relatively high. When the three colons were inoculated with pathogenic bacteria, the short-chain fatty acid levels changed significantly (P < 0.05). The levels of all six short-chain fatty acids decreased. The total short-chain fatty acid content in the ascending colon, transverse colon, and descending colon decreased from 89.45 ± 0.46 mmol / L, 110.80 ± 62 mmol / L, and 141.36 ± 1.35 mmol / L to 66.71 ± 6.34 mmol / L, 85.81 ± 0.78 mmol / L, and 106.54 ± 2.63 mmol / L, respectively. During the treatment period, the addition of Bifidobacterium longum subsp. infantis A32-03 and black fungus polysaccharide fermentation products significantly increased all six short-chain fatty acids. The sum of short-chain fatty acids in the ascending colon, transverse colon, and descending colon increased to 158.35±0.73 mmol / L, 151.98±1.49 mmol / L, and 178.63±4.79 mmol / L, respectively. Although there was a downward trend during the washout period, the total fatty acid content significantly increased compared to the stable period (P<0.05). The total fatty acid content in the ascending colon, transverse colon, and descending colon ultimately reached 121.92±1.31 mmol / L, 130.91±1.03 mmol / L, and 151.08±1.69 mmol / L, respectively. This suggests that Bifidobacterium longum subsp. infantis A32-03 and black fungus polysaccharide fermentation products can increase the content of short-chain fatty acids in the intestine.

[0180] In summary, the Bifidobacterium longum subspecies infantis A32-03 provided by the present invention and the probiotic preparation fermented with black fungus polysaccharide have good blood sugar and blood lipid regulation effects, can be used as oral hypoglycemic and lipid-lowering drugs or applied to health foods or beverages for improving the intestinal environment and regulating blood sugar and blood lipids, and have good promotion value.

[0181] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Bifidobacterium longum subsp. infantis, characterized in that The name of the Bifidobacterium longum subspecies infantis is Bifidobacterium longum subspecies infantis ( Bifidobacterium longum subsp. infantis ) A32-03, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number CGMCC NO. 32811.

2. A use of Bifidobacterium longum subsp. infantis as claimed in claim 1, characterized in that: Application in the preparation of probiotic preparations for lowering blood sugar and lipids.

3. A probiotic preparation for lowering blood sugar and lipids, characterized in that: The blood sugar and lipid lowering probiotic preparation comprises the fermentation product of Bifidobacterium longum subsp. infantis as claimed in claim 1; the fermentation product is obtained by inoculating Bifidobacterium longum subsp. infantis into a modified culture medium and fermenting it; the modified culture medium is a basic culture medium supplemented with black fungus polysaccharide.

4. The blood sugar and lipid lowering probiotic preparation according to claim 3, characterized in that The basic culture medium is MRS culture medium; the inoculation amount of Bifidobacterium longum subspecies infantis in the improved culture medium is 2v / v% to 5v / v%.

5. The blood sugar and lipid lowering probiotic preparation according to claim 3, characterized in that The fermentation culture process conditions are: anaerobic culture at a temperature of 36-38° C. for 20-50 hours; and the added concentration of black fungus polysaccharide in the improved culture medium is 5-20 mg / mL.

6. The blood sugar and lipid lowering probiotic preparation according to claim 5, characterized in that The fermentation culture process conditions are: anaerobic culture at 37° C. for 24 hours; and the added concentration of black fungus polysaccharide in the improved culture medium is 10 mg / mL.

7. The blood sugar and lipid lowering probiotic preparation according to claim 3, characterized in that The fermentation product is the supernatant component and / or bacterial precipitate of the fermentation liquid obtained after fermentation culture; the dosage form of the blood sugar and lipid lowering probiotic preparation is a solid preparation or a liquid preparation; the solid preparation is obtained by centrifuging the fermentation liquid obtained after fermentation culture, and then collecting the supernatant component and / or bacterial precipitate and freeze-drying it; the liquid preparation is obtained by reconstituted the solid preparation with sterile water.

8. A product for reducing blood sugar and / or blood lipids, characterized in that: The invention comprises active components and excipients; the active component is the blood sugar and lipid lowering probiotic preparation according to any one of claims 3 to 7.

9. The blood sugar and / or lipid lowering product according to claim 8, characterized in that The blood sugar lowering and / or lipid lowering product is a medicine and / or a health food.

10. The blood sugar and / or lipid lowering product according to claim 8, characterized in that: The blood sugar lowering and / or lipid lowering product is an oral solid preparation or an oral liquid preparation.

Images