Bifidobacterium breve BD003 and application of compound bacterial powder thereof in digestion promotion, intestine moistening and defecation

Abstract

This invention provides the application of Bifidobacterium breve BD003 and its compound powder in aiding digestion and promoting bowel movements, belonging to the field of microbial and functional development technology. This invention provides Bifidobacterium breve BD003, with accession number CGMCC No. 30721. This invention further provides a freeze-dried powder of Bifidobacterium breve BD003, and a compound powder containing Bifidobacterium breve BD003. The freeze-dried powder of Bifidobacterium breve BD003 and its compound powder can promote the production of digestive enzymes, increase fiber degradation rate and short-chain fatty acid production, and have significant bowel-regulating effects, possessing broad market potential and application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbial and functional development technology, specifically relating to the application of Bifidobacterium breve BD003 and its compound bacterial powder in aiding digestion and promoting bowel movement. Background Technology

[0002] Constipation is a common functional digestive disorder in clinical practice, characterized by reduced bowel movement frequency, hard stools, straining during defecation, and a feeling of incomplete evacuation. It can be broadly classified into organic and functional constipation. With the increasing sophistication of modern diets, the prevalence of sedentary lifestyles, increased work pressure, and an aging population, the prevalence of constipation is rising year by year, showing a trend towards affecting younger people, seriously impacting public health and quality of life. Furthermore, long-term chronic constipation not only causes bloating, abdominal pain, and intestinal dysfunction, but also induces anorectal diseases such as hemorrhoids, anal fissures, and intestinal obstruction. Simultaneously, the accumulation of toxins and the proliferation of harmful bacteria in the intestines further interfere with metabolism and immune function, increasing the risk of colitis, intestinal polyps, and even intestinal cancer, placing a heavy burden on the public healthcare system.

[0003] Currently, there are many intervention methods for relieving constipation in clinical practice and on the market, with drug intervention and dietary regulation being the mainstream approaches. Chemical drugs, including osmotic laxatives, stimulant laxatives, and prokinetic agents, while offering rapid relief and short-term improvement in bowel symptoms, have significant technical drawbacks and limitations: long-term use of stimulant laxatives can easily lead to drug dependence, causing intestinal motility deterioration and resulting in intractable constipation; osmotic laxatives can easily cause adverse reactions such as bloating, diarrhea, and electrolyte imbalance, making them unsuitable for long-term use by the elderly, children, and those with weak constitutions; most laxatives only provide symptomatic relief and cannot fundamentally improve the intestinal microecological environment, leading to a very high relapse rate after discontinuation. Traditional dietary regulation, which mainly involves supplementing dietary fiber, drinking more water, and adjusting lifestyle, has a slow onset of action and a long treatment cycle, offering limited improvement for intractable constipation caused by intestinal flora imbalance, and failing to meet the modern demand for efficient, safe, and long-lasting constipation relief.

[0004] With the deepening research on gut microbiota, gut microbiota imbalance has been confirmed as one of the core causes of functional constipation. Constipated patients commonly exhibit a decrease in the abundance of beneficial bacteria and an overgrowth of harmful bacteria in their intestines. This disrupts the gut microbiota homeostasis, leading to insufficient secretion of beneficial metabolites such as short-chain fatty acids, impaired intestinal barrier function, and reduced colonic motility, ultimately resulting in abnormal bowel function. Probiotics, as core functional strains regulating gut microbiota, can repair gut microbiota balance, promote intestinal motility, and increase stool water content by colonizing the gut, competitively inhibiting the reproduction of harmful bacteria, and secreting active substances such as organic acids. With their advantages of safety, non-toxicity, non-addictiveness, and long-term consumption, they have gradually replaced traditional drugs, becoming the preferred solution for relieving constipation and regulating gut health. However, current probiotic laxative products mostly use single strains or conventional compound strain formulations, lacking targeted functional strain screening and scientific ratios. The synergistic effect of strains is weak, only slightly improving mild constipation symptoms, resulting in poor overall conditioning effects. Therefore, screening for probiotics with high activity, high colonization, and strong laxative function, and preparing compound probiotic powder through scientific compounding, remains a key problem that urgently needs to be solved in this field. Summary of the Invention

[0005] To address the problems existing in the prior art, the primary objective of this invention is to provide a *Bifidobacterium breve* (Bifidobacterium breve) Bifidobacterium breve BD003, deposited by China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, accession number: CGMCC No. 30721, deposited on May 22, 2024.

[0006] A second objective of this invention is to provide the above-mentioned lyophilized Bifidobacterium breve BD003 powder and its compound powder.

[0007] A third objective of this invention is to provide applications of the above-mentioned Bifidobacterium breve BD003, or the above-mentioned lyophilized Bifidobacterium breve BD003 powder, or the above-mentioned compound bacterial powder.

[0008] A fourth objective of this invention is to provide a health food or medicine that helps to promote bowel movements.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a Bifidobacterium breve BD003, wherein the Bifidobacterium breve ( Bifidobacterium breve The accession number of BD003 is CGMCC No.30721.

[0010] This invention provides a method for preparing lyophilized Bifidobacterium breve BD003 powder, comprising the following steps: inoculating the above-mentioned Bifidobacterium breve BD003 into a fermentation medium for fermentation, centrifuging the fermentation broth to obtain bacterial sludge, mixing the bacterial sludge with a lyophilization protectant, and freeze-drying to obtain lyophilized Bifidobacterium breve BD003 powder.

[0011] Preferably, the fermentation medium comprises: lactose 8-12 g / L, glucose 8-12 g / L, sucrose 4-6 g / L, soybean peptone 8-12 g / L, bovine bone peptone 8-12 g / L, beef extract 4-6 g / L, yeast extract 8-12 g / L, anhydrous sodium acetate 4-6 g / L, diammonium citrate 1-3 g / L, dipotassium hydrogen phosphate 1-3 g / L, manganese sulfate 0.2-0.3 g / L, and Tween 0.5-1.5 g / L.

[0012] The present invention also provides lyophilized Bifidobacterium breve BD003 powder prepared by the above preparation method.

[0013] The present invention also provides a compound microbial powder, which includes freeze-dried powder of *Lactobacillus rhamnosus* GS066, freeze-dried powder of *Lactobacillus paracasei* GF027, freeze-dried powder of *Lactobacillus plantarum* LZ015, and freeze-dried powder of the above-mentioned *Bifidobacterium breve* BD003; the preservation number of *Lactobacillus rhamnosus* GS066 is CGMCC No. 29393; the preservation number of *Lactobacillus paracasei* GF027 is CGMCC No. 22831; and the preservation number of *Lactobacillus plantarum* LZ015 is CGMCC No. 29395.

[0014] Preferably, the ratio of viable bacteria in the compound bacterial powder is (15~25):(15~25):(15~25):(25~50).

[0015] This invention also provides the application of the above-mentioned compound microbial powder in the production of digestive enzymes and / or short-chain fatty acids.

[0016] Preferably, the digestive enzyme includes lipase, protease, and / or amylase.

[0017] The present invention also provides the application of the above-mentioned Bifidobacterium breve BD003 or Bifidobacterium breve BD003 lyophilized powder or compound powder in any of the following: (1) Prepare health foods or medicines that help with bowel movements; (2) Prepare health foods or medicines that help regulate the intestinal flora; (3) Prepare health foods or medicines that aid digestion; (4) Prepare drugs that improve the level of gastrointestinal regulatory peptides.

[0018] The present invention also provides a product that helps to lubricate the intestines and promote bowel movements, the product comprising the above-mentioned Bifidobacterium breve BD003 or lyophilized powder or compound powder of Bifidobacterium breve BD003.

[0019] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: This invention yields a strain of Bifidobacterium breve BD003 for the first time. When combined with freeze-dried powders of Lactobacillus rhamnosus GS066, Lactobacillus paracasei GF027, and Lactobacillus plantarum LZ015, it can promote the production of digestive enzymes, increase the fiber degradation rate and the yield of short-chain fatty acids, and has significant laxative effects. It also has high safety and broad market potential and application prospects. Attached Figure Description

[0020] Figure 1 Colony morphology of Bifidobacterium breve BD003.

[0021] Figure 2 Microscopic morphology of Bifidobacterium breve BD003.

[0022] Figure 3 Viable cell counts of strains co-cultured under different ratios.

[0023] Figure 4 Synergistic coefficient between the digestive enzyme production capacity and dietary fiber degradation rate of strains co-cultured under different ratios.

[0024] Figure 5 Effects of experimental bacterial powder on gastrointestinal regulatory peptides in mouse serum.

[0025] Biological Preservation Instructions Bifidobacterium breve BD003, classified and named Bifidobacterium breve ( Bifidobacterium breve (), deposited at: China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, accession number: CGMCC No. 30721, deposit date: May 22, 2024. Detailed Implementation

[0026] This invention provides a Bifidobacterium breve BD003, wherein the Bifidobacterium breve ( Bifidobacterium breve The accession number for BD003 is CGMCC No. 30721. The *Bifidobacterium breve* BD003 described in this invention was isolated and purified from a breast milk sample, and identification confirmed that it belongs to *Bifidobacterium breve* (…). Bifidobacterium breveIt was deposited on May 22, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0027] This invention provides a method for preparing lyophilized Bifidobacterium breve BD003 powder, comprising the following steps: inoculating the above-mentioned Bifidobacterium breve BD003 into a fermentation medium for fermentation, centrifuging the fermentation broth to obtain bacterial sludge, mixing the bacterial sludge with a lyophilization protectant, and freeze-drying to obtain lyophilized Bifidobacterium breve BD003 powder.

[0028] The fermentation medium of the present invention preferably comprises: lactose 8-12 g / L, glucose 8-12 g / L, sucrose 4-6 g / L, soybean peptone 8-12 g / L, bovine bone peptone 8-12 g / L, beef extract 4-6 g / L, yeast extract 8-12 g / L, anhydrous sodium acetate 4-6 g / L, diammonium citrate 1-3 g / L, dipotassium hydrogen phosphate 1-3 g / L, manganese sulfate 0.2-0.3 g / L, and Tween 0.5-1.5 g / L. More preferably, the mixture includes: 10 g / L lactose, 10 g / L glucose, 5 g / L sucrose, 10 g / L soybean peptone, 10 g / L bovine bone peptone, 5 g / L beef extract, 10 g / L yeast extract, 5 g / L anhydrous sodium acetate, 2 g / L diammonium citrate, 2 g / L dipotassium hydrogen phosphate, 0.25 g / L manganese sulfate, and 1 g / L Tween; the fermentation medium is preferably prepared with water. The fermentation temperature is preferably 36-38℃, the alkali content is controlled at 5.0, and the fermentation time is preferably 13-15 h. The centrifugation conditions are preferably 8000 rpm for 90 min. The freeze-drying protectant is preferably composed of 200 g / L skim milk, 200 g / L trehalose, and 60 g / L sucrose, and is prepared with distilled water. The bacterial sludge and freeze-drying protectant are mixed at a mass-to-volume ratio of 1 g:1 mL. The freeze-drying process of this invention includes pre-freezing, primary drying, and desorption drying; the pre-freezing condition is preferably -40℃ for 4 hours; the primary drying involves controlling the temperature sequentially at -40℃, -35℃, -30℃, -25℃, -20℃, -15℃, -10℃, -5℃, and 0℃, with the preferred conditions being -40℃ for 1 hour, -35℃ for 2 hours, -30℃ for 4 hours, -25℃ for 4 hours, -20℃ for 4 hours, -15℃ for 4 hours, -10℃ for 4 hours, -5℃ for 4 hours, and 0℃ for 3 hours; the desorption drying involves controlling the temperature sequentially at 6℃, 12℃, and 24℃, with the preferred conditions being 6℃ for 4 hours, 12℃ for 3 hours, and 24℃ for 12 hours.

[0029] This invention also provides a lyophilized powder of *Bifidobacterium breve* BD003 prepared by the above method, wherein the viable count of the lyophilized powder is 1 × 10⁻⁶. 11cfu / g ~1.2×10 11 cfu / g.

[0030] The present invention also provides a compound bacterial powder, which includes freeze-dried powders of *Lactobacillus rhamnosus* GS066, *Lactobacillus paracasei* GF027, *Lactobacillus plantarum* LZ015, and *Bifidobacterium breve* BD003; wherein the ratio of viable bacteria counts of *Lactobacillus rhamnosus* GS066, *Lactobacillus paracasei* GF027, *Lactobacillus plantarum* LZ015, and *Bifidobacterium breve* BD003 is (15~25):(15~25):(15~25):(25~50), preferably 1:1:1:1.

[0031] The compound bacterial powder of this invention contains Lactobacillus rhamnosus GS066, with accession number CGMCC No. 29393, disclosed in patent CN202411154693.X; Lactobacillus paracasei GF027, with accession number CGMCC No. 22831, disclosed in patent CN202311142064.0; and Lactobacillus plantarum LZ015, with accession number CGMCC No. 29395, disclosed in patent CN202411336531.8.

[0032] As an optional implementation, the method for preparing Lactobacillus rhamnosus GS066 freeze-dried powder in this invention includes the following steps: inoculating the above-mentioned Lactobacillus rhamnosus GS066 into a fermentation medium for fermentation, centrifuging the fermentation broth to obtain bacterial sludge, mixing the bacterial sludge with a freeze-drying protectant, and freeze-drying to obtain Lactobacillus rhamnosus GS066 freeze-dried powder.

[0033] The fermentation medium for Lactobacillus rhamnosus GS066 described in this invention preferably comprises: 28-32 g / L glucose, 14-16 g / L beef extract, 8-12 g / L tryptone, 8-12 g / L yeast extract, 4-6 g / L anhydrous sodium acetate, 1-3 g / L diammonium citrate, 1-3 g / L dipotassium hydrogen phosphate, 0.2-0.3 g / L manganese sulfate, and 0.5-1.5 g / L Tween; more preferably, it comprises 30 g / L glucose, 15 g / L beef extract, 10 g / L tryptone, 10 g / L yeast extract, 5 g / L anhydrous sodium acetate, 2 g / L diammonium citrate, 2 g / L dipotassium hydrogen phosphate, 0.25 g / L manganese sulfate, and 1 g / L Tween; the fermentation medium is preferably prepared with water. The fermentation temperature of this invention is preferably 36-38℃, the alkali content is controlled at 5.0, and the fermentation time is preferably 10-12 hours. The centrifugation conditions of this invention are preferably 8000 rpm for 90 minutes. The freeze-drying protectant of this invention preferably comprises 200 g / L skim milk, 200 g / L trehalose, and 60 g / L sucrose, and the freeze-drying protectant is prepared with distilled water. The bacterial sludge and freeze-drying protectant of this invention are mixed at a mass-to-volume ratio of 1 g:1 mL. The freeze-drying process of this invention includes pre-freezing, primary drying, and desorption drying; the pre-freezing condition is preferably -40℃ for 4 hours; the primary drying involves controlling the temperature sequentially at -40℃, -35℃, -30℃, -25℃, -20℃, -15℃, -10℃, -5℃, and 0℃, with the preferred conditions being -40℃ for 1 hour, -35℃ for 2 hours, -30℃ for 4 hours, -25℃ for 4 hours, -20℃ for 4 hours, -15℃ for 4 hours, -10℃ for 4 hours, -5℃ for 4 hours, and 0℃ for 3 hours; the desorption drying involves controlling the temperature sequentially at 6℃, 12℃, and 24℃, with the preferred conditions being 6℃ for 4 hours, 12℃ for 3 hours, and 24℃ for 12 hours.

[0034] As an optional implementation, the method for preparing Lactobacillus paracasei GF027 lyophilized powder in this invention includes the following steps: inoculating the above-mentioned Lactobacillus paracasei GF027 into a fermentation medium for fermentation, centrifuging the fermentation broth to obtain bacterial sludge, mixing the bacterial sludge with a lyophilization protectant, and freeze-drying to obtain Lactobacillus paracasei GF027 lyophilized powder.

[0035] The fermentation medium for Lactobacillus paracasei GF027 described in this invention preferably comprises: 28-32 g / L glucose, 8-12 g / L soybean peptone, 14-16 g / L tryptone, 8-12 g / L yeast extract, 4-6 g / L anhydrous sodium acetate, 1-3 g / L diammonium citrate, 1-3 g / L dipotassium hydrogen phosphate, 0.2-0.3 g / L manganese sulfate, and 0.5-1.5 g / L Tween; more preferably, it comprises 30 g / L glucose, 10 g / L soybean peptone, 15 g / L tryptone, 10 g / L yeast extract, 5 g / L anhydrous sodium acetate, 2 g / L diammonium citrate, 2 g / L dipotassium hydrogen phosphate, 0.25 g / L manganese sulfate, and 1 g / L Tween; the fermentation medium is preferably prepared with water. The fermentation temperature of this invention is preferably 36-38℃, the alkali content is controlled at 5.0, and the fermentation time is preferably 10-12 hours. The centrifugation conditions of this invention are preferably 8000 rpm for 90 minutes. The freeze-drying protectant of this invention preferably comprises 200 g / L skim milk, 200 g / L trehalose, and 60 g / L sucrose, and the freeze-drying protectant is prepared with distilled water. The bacterial sludge and freeze-drying protectant of this invention are mixed at a mass-to-volume ratio of 1 g:1 mL. The freeze-drying process of this invention includes pre-freezing, primary drying, and desorption drying; the pre-freezing condition is preferably -40℃ for 4 hours; the primary drying involves controlling the temperature sequentially at -40℃, -35℃, -30℃, -25℃, -20℃, -15℃, -10℃, -5℃, and 0℃, with the preferred conditions being -40℃ for 1 hour, -35℃ for 2 hours, -30℃ for 4 hours, -25℃ for 4 hours, -20℃ for 4 hours, -15℃ for 4 hours, -10℃ for 4 hours, -5℃ for 4 hours, and 0℃ for 3 hours; the desorption drying involves controlling the temperature sequentially at 6℃, 12℃, and 24℃, with the preferred conditions being 6℃ for 4 hours, 12℃ for 3 hours, and 24℃ for 12 hours.

[0036] As an optional implementation, the method for preparing freeze-dried LZ015 powder of *Lactobacillus plantarum* in this invention includes the following steps: inoculating the above-mentioned *Lactobacillus plantarum* LZ015 into a fermentation medium for fermentation, centrifuging the fermentation broth to obtain bacterial sludge, mixing the bacterial sludge with a freeze-drying protectant, and freeze-drying to obtain freeze-dried LZ015 powder.

[0037] The fermentation medium for *Lactobacillus plantarum* LZ015 described in this invention preferably comprises: 28-32 g / L glucose, 8-12 g / L soybean peptone, 14-16 g / L bovine bone peptone, 8-12 g / L yeast extract, 4-6 g / L anhydrous sodium acetate, 1-3 g / L diammonium citrate, 1-3 g / L dipotassium hydrogen phosphate, 0.2-0.3 g / L manganese sulfate, and 0.5-1.5 g / L Tween; more preferably, it comprises 30 g / L glucose, 10 g / L soybean peptone, 15 g / L bovine bone peptone, 10 g / L yeast extract, 5 g / L anhydrous sodium acetate, 2 g / L diammonium citrate, 2 g / L dipotassium hydrogen phosphate, 0.25 g / L manganese sulfate, and 1 g / L Tween; the fermentation medium is preferably prepared with water. The fermentation temperature of this invention is preferably 36-38℃, the alkali content is controlled at 5.0, and the fermentation time is preferably 10-12 hours. The centrifugation conditions of this invention are preferably 8000 rpm for 90 minutes. The freeze-drying protectant of this invention preferably comprises 200 g / L skim milk, 200 g / L trehalose, and 60 g / L sucrose, and the freeze-drying protectant is prepared with distilled water. The bacterial sludge and freeze-drying protectant of this invention are mixed at a mass-to-volume ratio of 1 g:1 mL. The freeze-drying process of this invention includes pre-freezing, primary drying, and desorption drying; the pre-freezing condition is preferably -40℃ for 4 hours; the primary drying involves controlling the temperature sequentially at -40℃, -35℃, -30℃, -25℃, -20℃, -15℃, -10℃, -5℃, and 0℃, with the preferred conditions being -40℃ for 1 hour, -35℃ for 2 hours, -30℃ for 4 hours, -25℃ for 4 hours, -20℃ for 4 hours, -15℃ for 4 hours, -10℃ for 4 hours, -5℃ for 4 hours, and 0℃ for 3 hours; the desorption drying involves controlling the temperature sequentially at 6℃, 12℃, and 24℃, with the preferred conditions being 6℃ for 4 hours, 12℃ for 3 hours, and 24℃ for 12 hours.

[0038] This invention also provides the application of the above-mentioned compound microbial powder in the production of digestive enzymes and / or short-chain fatty acids, wherein the digestive enzymes include lipases, proteases, and / or amylases. The compound microbial powder of this invention enables the synergistic growth of bacterial strains, increases the number of viable bacteria after culture, and significantly increases the yield of lipases, proteases, amylases, and short-chain fatty acids, thereby improving the degradation rate of dietary fiber. Significant synergistic effects exist among the various bacterial strains.

[0039] The present invention also provides the application of the above-mentioned Bifidobacterium breve BD003 or Bifidobacterium breve BD003 freeze-dried powder or compound bacterial powder in any of the following: (1) preparing health food or medicine that helps to lubricate the intestines and promote bowel movements; (2) preparing health food or medicine that helps to regulate the intestinal flora; (3) preparing health food or medicine that helps to aid digestion; (4) preparing medicine that improves the level of gastrointestinal regulatory peptides.

[0040] This invention also provides a product that helps with bowel movements, the product comprising the aforementioned Bifidobacterium breve BD003 or lyophilized powder or compound powder of Bifidobacterium breve BD003. The product of this invention includes health foods and pharmaceuticals; the product may also include optional excipients or carriers used in health foods or pharmaceuticals.

[0041] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0042] The strains used in specific embodiments of the present invention are described below: *Lactobacillus casei* GG002, accession number CGMCC No. 29392, disclosed in patent CN202411686473.1; *Lactobacillus plantarum* LZ010, accession number CGMCC No. 24258, disclosed in patent CN202211522892.2; *Lactobacillus plantarum* LZ026, accession number CGMCC No. 22832, disclosed in patent CN202310709086.4; *Lactobacillus fermentum* LF028, accession number CGMCC No. 22833, disclosed in patent CN202210216462.1; *Lactobacillus reuteri* LL029, accession number CGMCC No. 23648, disclosed in patent CN202211522999.7; *Bifidobacterium lactis* BR001, accession number CGMCC... No. 23665 is disclosed in patent CN202211193880.X; Lactobacillus rhamnosus GS044, with accession number CGMCC No. 33713, is disclosed in patent CN202511061670.9; Lactobacillus paracasei GF045, with accession number CGMCC No. 26482, is disclosed in patent CN202310335478.9; and Bifidobacterium longum BC012, with accession number CGMCC No. 23663, is disclosed in patent CN202211522885.2.

[0043] Unless otherwise specified, the following embodiments are all conventional methods.

[0044] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0045] Example 1 Isolation and identification of Bifidobacterium breve BD003: The collected breast milk samples were serially diluted and spread onto MRS solid medium containing 2% calcium carbonate, and anaerobically incubated at 37°C for 48 hours. Single colonies with calcium-dissolving zones were picked and subjected to streak purification for three generations to obtain purified bacterial strains.

[0046] Observe the morphology of the purified strain in the culture medium; colony morphology is as follows: Figure 1 As shown; the results showed that the culture medium contained tiny, round, white colonies that were raised, smooth, and had neat edges (entire).

[0047] The microstructure of the purified strain was photographed using an electron microscope. The microstructure is shown in the image below. Figure 2 As shown in the figure; the results show that the bacteria appear as rod-shaped or forked rod-shaped under a microscope.

[0048] The purified strain was sent to Shanghai Sangon Biotech Co., Ltd. for 16S rDNA sequencing. Comparison with the NCBI database confirmed that the screened strain was *Bifidobacterium breve*. Bifidobacterium breve It was named Bifidobacterium breve BD003.

[0049] Bifidobacterium breve BD003 was deposited on May 22, 2024 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 30721.

[0050] Experimental Example 1 1. Digestive enzyme production capacity of each strain Sample preparation: The test strains (Bifidobacterium breve BD003, Lactobacillus rhamnosus GS066, Lactobacillus paracasei GF027, Lactobacillus casei GG002, Lactobacillus plantarum LZ010, Lactobacillus plantarum LZ015, Lactobacillus plantarum LZ026, Lactobacillus fermentum LF028, Lactobacillus reuteri LL029, and Bifidobacterium lactis BR001) were inoculated into MRS medium at an inoculum of 1% (v / v) and cultured anaerobically at 37℃ for 48 h. The fermentation broth of each strain was then collected.

[0051] Detection: The fermentation broth was processed and tested according to the kit operation procedure. The kits used were lipase (LPS) assay kit (microplate method) (Nanjing Jiancheng), total protease (t-Pro) enzyme-linked immunosorbent assay kit (Gilead Sciences), and α-amylase (AMS) test kit (microplate method) (Nanjing Jiancheng).

[0052] Using MRS medium as a blank control, the enzyme activity of each strain was calculated according to the two-point calibration method provided in the kit.

[0053] Table 1. Content of digestive enzymes in fermentation broth of different bacterial strains

[0054] Note: In the table, different uppercase letters indicate highly significant differences (P < 0.01), and different lowercase letters indicate significant differences (P < 0.05).

[0055] The results showed that several strains exhibited better protease production than amylase production, which in turn out to be better than lipase production. Specifically, strain LZ015 showed the best lipase production (2.62 U / mL), strain GS066 showed the best protease production (31.98 U / mL), and strain GF027 showed the best amylase production (12.21 U / mL). In contrast, strain BD003 showed weak protease production and essentially no ability to produce either amylase or lipase.

[0056] 2. Dietary fiber decomposition capacity of each strain Prepare the dietary fiber screening medium: 4.0g dietary fiber (1.0g inulin, 1.0g pectin, 2.0g wheat bran fiber), 5.0g peptone, 3.0g yeast extract, 3.0g beef extract, 1.0g Tween 80, 0.5g cysteine, 2.0g dipotassium hydrogen phosphate, 2.0g disodium citrate, 0.2g magnesium sulfate, 0.05g calcium chloride, add water to a final volume of 1L, adjust the pH to 6.8-7.0, and then sterilize.

[0057] Sample preparation: The test strains (Bifidobacterium breve BD003, Lactobacillus rhamnosus GS066, Lactobacillus paracasei GF027, Lactobacillus casei GG002, Lactobacillus plantarum LZ010, Lactobacillus plantarum LZ015, Lactobacillus plantarum LZ026, Lactobacillus fermentum LF028, Lactobacillus reuteri LL029, and Bifidobacterium lactis BR001) were inoculated into dietary fiber screening medium at an inoculation rate of 1% (v / v) and cultured anaerobicly at 37℃ for 48 h. The fermentation broth of each strain was collected and centrifuged (8000 rpm, 15 min) to obtain the supernatant.

[0058] Detection: The dietary fiber residue and short-chain fatty acid content in the fermentation supernatant were detected using a dietary fiber detection kit (purchased from Shanghai Jinpan Biotechnology Co., Ltd.) and an SCFA detection kit (purchased from Shanghai Baililai Biotechnology Co., Ltd.). A blank culture medium was used as a blank control.

[0059] Table 2. Dietary fiber degradation rate and short-chain fatty acid yield of different strains

[0060] Note: In the table, different uppercase letters indicate highly significant differences (P < 0.01), and different lowercase letters indicate significant differences (P < 0.05).

[0061] The results showed that the dietary fiber degradation rate and total short-chain fatty acid content of strain BD003 were significantly higher than those of other strains, and the dietary fiber degradation rate and total short-chain fatty acid content were positively correlated.

[0062] Example 2 Preparation of lyophilized Bifidobacterium breve BD003 powder: Culture medium (per liter): lactose 10g, glucose 10g, sucrose 5g, soybean peptone 10g, bovine bone peptone 10g, beef extract 5g, yeast extract 10g, anhydrous sodium acetate 5g, diammonium citrate 2g, dipotassium hydrogen phosphate 2g, manganese sulfate 0.25g, Tween 1g.

[0063] Fermentation was carried out in a fermentation tank at 37℃ with an alkali control of 5.0 for 14 hours. The fermented product was then harvested and centrifuged (8000 rpm, 90 min) to obtain mycelial sludge. This sludge was mixed with a freeze-drying protectant (200 g / L skim milk, 200 g / L trehalose, and 60 g / L sucrose, prepared with distilled water) at a mass-to-volume ratio of 1 g:1 mL. The mixture was then freeze-dried (pre-freezing at -40℃ for 4 h, primary drying at -40℃ for 1 h, -35℃ for 2 h, -30℃ for 4 h, -25℃ for 4 h, -20℃ for 4 h, -15℃ for 4 h, -10℃ for 4 h, -5℃ for 4 h, and 0℃ for 3 h; desorption drying at 6℃ for 4 h, 12℃ for 3 h, and 24℃ for 12 h) to obtain freeze-dried powder.

[0064] Experimental Example 2 1. Preparation of mycelium powder (1) Preparation of Bifidobacterium breve BD003 bacterial powder: The freeze-dried powder prepared in Example 2 was diluted with maltodextrin according to the number of viable bacteria in the powder to obtain 1×10 11 CFU / g of bacterial powder.

[0065] (2) Preparation of Lactobacillus rhamnosus GS066 bacterial powder: Culture medium (per liter): 30g glucose, 15g beef extract, 10g tryptone, 10g yeast extract, 5g anhydrous sodium acetate, 2g diammonium citrate, 2g dipotassium hydrogen phosphate, 0.25g manganese sulfate, 1g Tween.

[0066] Fermentation was carried out in a fermentation tank at 37℃ with an alkali control of 5.0 for 12 hours. The fermented product was then harvested and centrifuged (8000 rpm, 90 min) to obtain mycelial sludge. This sludge was mixed with a freeze-drying protectant (200 g / L skim milk, 200 g / L trehalose, and 60 g / L sucrose, prepared with distilled water) at a mass-to-volume ratio of 1 g:1 mL. The mixture was then freeze-dried (pre-freezing at -40℃ for 4 hours, followed by primary drying at -40℃ for 1 hour, -35℃ for 2 hours, -30℃ for 4 hours, -25℃ for 4 hours, -20℃ for 4 hours, -15℃ for 4 hours, -10℃ for 4 hours, -5℃ for 4 hours, and 0℃ for 3 hours; followed by desorption drying at 6℃ for 4 hours, 12℃ for 3 hours, and 24℃ for 12 hours). The resulting product was diluted with maltodextrin based on the viable count of the mycelial powder to obtain a concentration of 1×10⁻⁶. 11 CFU / g bacterial powder (lyophilized powder).

[0067] (3) Preparation of Lactobacillus paracasei GF027 bacterial powder: Culture medium (per liter): 30g glucose, 10g soybean peptone, 15g tryptone, 10g yeast extract, 5g anhydrous sodium acetate, 2g diammonium citrate, 2g dipotassium hydrogen phosphate, 0.25g manganese sulfate, 1g Tween.

[0068] Fermentation was carried out in a fermentation tank at 37℃ with an alkali control of 5.0 for 12 hours. The fermented product was then harvested and centrifuged (8000 rpm, 90 min) to obtain mycelial sludge. This sludge was mixed with a freeze-drying protectant (200 g / L skim milk, 200 g / L trehalose, and 60 g / L sucrose, prepared with distilled water) at a mass-to-volume ratio of 1 g:1 mL. The mixture was then freeze-dried (pre-freezing at -40℃ for 4 hours, followed by primary drying at -40℃ for 1 hour, -35℃ for 2 hours, -30℃ for 4 hours, -25℃ for 4 hours, -20℃ for 4 hours, -15℃ for 4 hours, -10℃ for 4 hours, -5℃ for 4 hours, and 0℃ for 3 hours; followed by desorption drying at 6℃ for 4 hours, 12℃ for 3 hours, and 24℃ for 12 hours). The resulting product was diluted with maltodextrin based on the viable count of the mycelial powder to obtain a concentration of 1×10⁻⁶. 11 CFU / g bacterial powder (lyophilized powder).

[0069] (4) Preparation of Lactobacillus plantarum LZ015 bacterial powder: Culture medium (per liter): 30g glucose, 10g soybean peptone, 15g bovine bone peptone, 10g yeast extract, 5g anhydrous sodium acetate, 2g diammonium citrate, 2g dipotassium hydrogen phosphate, 0.25g manganese sulfate, 1g Tween.

[0070] Fermentation was carried out in a fermentation tank at 37℃ with an alkali control of 5.0 for 12 hours. The fermented product was then harvested and centrifuged (8000 rpm, 90 min) to obtain mycelial sludge. This sludge was mixed with a freeze-drying protectant (200 g / L skim milk, 200 g / L trehalose, and 60 g / L sucrose, prepared with distilled water) at a mass-to-volume ratio of 1 g:1 mL. The mixture was then freeze-dried (pre-freezing at -40℃ for 4 hours, followed by primary drying at -40℃ for 1 hour, -35℃ for 2 hours, -30℃ for 4 hours, -25℃ for 4 hours, -20℃ for 4 hours, -15℃ for 4 hours, -10℃ for 4 hours, -5℃ for 4 hours, and 0℃ for 3 hours; followed by desorption drying at 6℃ for 4 hours, 12℃ for 3 hours, and 24℃ for 12 hours). The resulting product was diluted with maltodextrin based on the viable count of the mycelial powder to obtain a concentration of 1×10⁻⁶. 11 CFU / g bacterial powder (lyophilized powder).

[0071] 2. Strains were grouped according to their proportions. Each bacterial powder is mixed according to a mass ratio (the mass ratio is the same as the ratio of live bacteria).

[0072] Table 3. Strains and their ratios grouped

[0073] 3. Statistical analysis of viable cell counts in cultures with different formulation ratios Each bacterial powder was inoculated separately or mixed according to the proportions in Table 3, and then inoculated into MRS medium at a total inoculation amount of 1% (0.1g bacterial powder: 10ml medium). The medium was incubated at 37℃ for 48h, and viable cell counts were performed.

[0074] viable cell count results as follows Figure 3 As shown in the figure, different uppercase letters indicate highly significant differences (P < 0.01), and different lowercase letters indicate significant differences (P < 0.05). The results show that, under the same inoculum size, the co-culture group with the ratio of strain 1 had the highest final number of viable bacteria, which was higher than that of the strains cultured alone, indicating that the ratio of strains in group 1 can enable the strains to grow synergistically.

[0075] 4. Digestive enzyme production capacity of co-cultured strains The various bacterial powders were mixed according to the proportions in Table 3, and then inoculated into MRS medium at an inoculum volume of 1% (0.1g bacterial powder: 10ml medium). The mixtures were incubated at 37℃ for 48 hours, and the fermentation broth was collected. The fermentation broth was then processed according to the detection method described in Example 1 and analyzed. The results are as follows: Table 4. Digestive enzyme content in fermentation broths of strains with different ratios

[0076] Note: In the table, different uppercase letters indicate highly significant differences (P < 0.01), and different lowercase letters indicate significant differences (P < 0.05).

[0077] The results showed that group 1 had the highest yields of lipase, protease, and amylase, and these yields were significantly different from those of other groups.

[0078] 5. Co-culture dietary fiber degradation rate The various bacterial powders were mixed according to the proportions in Table 3, and then inoculated into dietary fiber screening medium (formula same as in Example 1) at a total inoculum volume of 1% (0.1g bacterial powder: 10ml medium). The mixture was incubated at 37℃ for 48h, and the fermentation broth was collected. The fermentation broth was centrifuged (8000rpm, 15min) and the supernatant was collected. The supernatant was tested according to the kit method in Example 1. The results are as follows: Table 5. Fiber degradation rate and short-chain fatty acid yield of strains with different ratios

[0079] Note: In the table, different uppercase letters indicate highly significant differences (P < 0.01), and different lowercase letters indicate significant differences (P < 0.05).

[0080] The results showed that groups 1 and 5 in the culture group had higher dietary fiber degradation rates, which were significantly different from other groups. Groups 1 and 5 in the culture group also had higher short-chain fatty acid production, which were significantly different from other groups.

[0081] 6. Calculate the synergy coefficient The synergy coefficient (SC) was calculated based on the results of single-strain experiments and co-culture experiments, using the following formula: ; ; in, This represents the actual measured value after mixed culture; This represents the expected value calculated based on the effect of a single strain and its weight in the mixed system; This represents the detection value when strain i is cultured alone; This indicates the weight of strain i in the mixed system; i represents different strains.

[0082] Synergy coefficient (SC) of strains under different ratios, as follows Figure 4 As shown in the results, the SC values ​​in each group were greater than or equal to 1, indicating that the four strains had synergistic or additive effects in the production of digestive enzymes and the degradation of cellulose. Among them, the synergistic effect of the strains in group 1 was better, that is, the synergistic effect of strains LZ026:GS066:GS027:BD003 was the best when the ratio was 1:1:1:1.

[0083] Example 3 A compound bacterial powder is prepared by mixing Lactobacillus rhamnosus GS066 freeze-dried powder, Lactobacillus paracasei GF027 freeze-dried powder, Lactobacillus plantarum LZ015 freeze-dried powder and Bifidobacterium breve BD003 freeze-dried powder in a live bacteria ratio of 1:1:1:1.

[0084] The preparation method of Bifidobacterium breve BD003 lyophilized powder is the same as in Example 2.

[0085] Preparation method of lyophilized Lactobacillus rhamnosus GS066 powder: Prepare the culture medium (per liter): 30g glucose, 15g beef extract, 10g tryptone, 10g yeast extract, 5g anhydrous sodium acetate, 2g diammonium citrate, 2g dipotassium hydrogen phosphate, 0.25g manganese sulfate, and 1g Tween.

[0086] Fermentation was carried out in a fermentation tank at 37℃ with an alkali control of 5.0 for 12 hours. The fermented product was then harvested and centrifuged (8000 rpm, 90 min) to obtain mycelial sludge. This sludge was mixed with a freeze-drying protectant (200 g / L skim milk, 200 g / L trehalose, and 60 g / L sucrose, prepared with distilled water) at a mass-to-volume ratio of 1 g:1 mL. The mixture was then freeze-dried (pre-freezing at -40℃ for 4 hours, followed by primary drying at -40℃ for 1 hour, -35℃ for 2 hours, -30℃ for 4 hours, -25℃ for 4 hours, -20℃ for 4 hours, -15℃ for 4 hours, -10℃ for 4 hours, -5℃ for 4 hours, and 0℃ for 3 hours; followed by desorption drying at 6℃ for 4 hours, 12℃ for 3 hours, and 24℃ for 12 hours) to obtain freeze-dried powder.

[0087] Preparation method of lyophilized Lactobacillus paracasei GF027 powder: Prepare the culture medium (per liter): 30g glucose, 10g soybean peptone, 15g tryptone, 10g yeast extract, 5g anhydrous sodium acetate, 2g diammonium citrate, 2g dipotassium hydrogen phosphate, 0.25g manganese sulfate, and 1g Tween.

[0088] Fermentation was carried out in a fermentation tank at 37℃ with an alkali control of 5.0 for 12 hours. The fermented product was then harvested and centrifuged (8000 rpm, 90 min) to obtain mycelial sludge. This sludge was mixed with a freeze-drying protectant (200 g / L skim milk, 200 g / L trehalose, and 60 g / L sucrose, prepared with distilled water) at a mass-to-volume ratio of 1 g:1 mL. The mixture was then freeze-dried (pre-freezing at -40℃ for 4 hours, followed by primary drying at -40℃ for 1 hour, -35℃ for 2 hours, -30℃ for 4 hours, -25℃ for 4 hours, -20℃ for 4 hours, -15℃ for 4 hours, -10℃ for 4 hours, -5℃ for 4 hours, and 0℃ for 3 hours; followed by desorption drying at 6℃ for 4 hours, 12℃ for 3 hours, and 24℃ for 12 hours) to obtain freeze-dried powder.

[0089] Preparation method of lyophilized Lactobacillus plantarum LZ015 powder: Prepare the culture medium (per liter): 30g glucose, 10g soybean peptone, 15g bovine bone peptone, 10g yeast extract, 5g anhydrous sodium acetate, 2g diammonium citrate, 2g dipotassium hydrogen phosphate, 0.25g manganese sulfate, and 1g Tween.

[0090] Fermentation was carried out in a fermentation tank at 37℃ with an alkali control of 5.0 for 12 hours. The fermented product was then harvested and centrifuged (8000 rpm, 90 min) to obtain mycelial sludge. This sludge was mixed with a freeze-drying protectant (200 g / L skim milk, 200 g / L trehalose, and 60 g / L sucrose, prepared with distilled water) at a mass-to-volume ratio of 1 g:1 mL. The mixture was then freeze-dried (pre-freezing at -40℃ for 4 hours, followed by primary drying at -40℃ for 1 hour, -35℃ for 2 hours, -30℃ for 4 hours, -25℃ for 4 hours, -20℃ for 4 hours, -15℃ for 4 hours, -10℃ for 4 hours, -5℃ for 4 hours, and 0℃ for 3 hours; followed by desorption drying at 6℃ for 4 hours, 12℃ for 3 hours, and 24℃ for 12 hours) to obtain freeze-dried powder.

[0091] Experimental Example 3 1. Preparation of mycelium powder (1) Preparation of Lactobacillus plantarum LZ010 bacterial powder: Culture medium (per liter): 30g glucose, 10g soybean peptone, 15g bovine bone peptone, 10g yeast extract, 5g anhydrous sodium acetate, 2g diammonium citrate, 2g dipotassium hydrogen phosphate, 0.25g manganese sulfate, 1g Tween.

[0092] Fermentation was carried out in a fermentation tank at 37℃ with an alkali control of 5.0 for 12 hours. The fermented product was then harvested and centrifuged (8000 rpm, 90 min) to obtain mycelial sludge. This sludge was mixed with a freeze-drying protectant (200 g / L skim milk, 200 g / L trehalose, and 60 g / L sucrose, prepared with distilled water) at a mass-to-volume ratio of 1 g:1 mL. The mixture was then freeze-dried (pre-freezing at -40℃ for 4 hours, followed by primary drying at -40℃ for 1 hour, -35℃ for 2 hours, -30℃ for 4 hours, -25℃ for 4 hours, -20℃ for 4 hours, -15℃ for 4 hours, -10℃ for 4 hours, -5℃ for 4 hours, and 0℃ for 3 hours; followed by desorption drying at 6℃ for 4 hours, 12℃ for 3 hours, and 24℃ for 12 hours). The resulting product was diluted with maltodextrin based on the viable count of the mycelial powder to obtain a concentration of 1×10⁻⁶. 11 CFU / g bacterial powder (lyophilized powder).

[0093] (2) Preparation of Lactobacillus rhamnosus GS044 bacterial powder: Prepare the culture medium (per liter): 30g glucose, 15g beef extract, 10g tryptone, 10g yeast extract, 5g anhydrous sodium acetate, 2g diammonium citrate, 2g dipotassium hydrogen phosphate, 0.25g manganese sulfate, and 1g Tween.

[0094] Fermentation was carried out in a fermentation tank at 37℃ with an alkali control of 5.0 for 12 hours. The fermented product was then harvested and centrifuged (8000 rpm, 90 min) to obtain mycelial sludge. This sludge was mixed with a freeze-drying protectant (200 g / L skim milk, 200 g / L trehalose, and 60 g / L sucrose, prepared with distilled water) at a mass-to-volume ratio of 1 g:1 mL. The mixture was then freeze-dried (pre-freezing at -40℃ for 4 hours, followed by primary drying at -40℃ for 1 hour, -35℃ for 2 hours, -30℃ for 4 hours, -25℃ for 4 hours, -20℃ for 4 hours, -15℃ for 4 hours, -10℃ for 4 hours, -5℃ for 4 hours, and 0℃ for 3 hours; followed by desorption drying at 6℃ for 4 hours, 12℃ for 3 hours, and 24℃ for 12 hours). The resulting product was diluted with maltodextrin based on the viable count of the mycelial powder to obtain a concentration of 1×10⁻⁶. 11 CFU / g bacterial powder (lyophilized powder).

[0095] (3) Preparation of Lactobacillus paracasei GF045 bacterial powder: Prepare the culture medium (per liter): 30g glucose, 10g soybean peptone, 15g tryptone, 10g yeast extract, 5g anhydrous sodium acetate, 2g diammonium citrate, 2g dipotassium hydrogen phosphate, 0.25g manganese sulfate, and 1g Tween.

[0096] Fermentation was carried out in a fermentation tank at 37℃ with an alkali control of 5.0 for 12 hours. The fermented product was then harvested and centrifuged (8000 rpm, 90 min) to obtain mycelial sludge. This sludge was mixed with a freeze-drying protectant (200 g / L skim milk, 200 g / L trehalose, and 60 g / L sucrose, prepared with distilled water) at a mass-to-volume ratio of 1 g:1 mL. The mixture was then freeze-dried (pre-freezing at -40℃ for 4 hours, followed by primary drying at -40℃ for 1 hour, -35℃ for 2 hours, -30℃ for 4 hours, -25℃ for 4 hours, -20℃ for 4 hours, -15℃ for 4 hours, -10℃ for 4 hours, -5℃ for 4 hours, and 0℃ for 3 hours; followed by desorption drying at 6℃ for 4 hours, 12℃ for 3 hours, and 24℃ for 12 hours). The resulting product was diluted with maltodextrin based on the viable count of the mycelial powder to obtain a concentration of 1×10⁻⁶. 11 CFU / g bacterial powder (lyophilized powder).

[0097] (4) Preparation of Bifidobacterium longum BC012 bacterial powder: Culture medium (per liter): lactose 10g, glucose 10g, sucrose 5g, soybean peptone 10g, bovine bone peptone 10g, beef extract 5g, yeast extract 10g, anhydrous sodium acetate 5g, diammonium citrate 2g, dipotassium hydrogen phosphate 2g, manganese sulfate 0.25g, Tween 1g.

[0098] Fermentation was carried out in a fermentation tank at 37℃ with an alkali control of 5.0 for 14 hours. The fermented product was then harvested and centrifuged (8000 rpm, 90 min) to obtain mycelial sludge. This sludge was mixed with a freeze-drying protectant (200 g / L skim milk, 200 g / L trehalose, and 60 g / L sucrose, prepared with distilled water) at a mass-to-volume ratio of 1 g:1 mL. The mixture was then freeze-dried (pre-freezing at -40℃ for 4 h, primary drying at -40℃ for 1 h, -35℃ for 2 h, -30℃ for 4 h, -25℃ for 4 h, -20℃ for 4 h, -15℃ for 4 h, -10℃ for 4 h, -5℃ for 4 h, and 0℃ for 3 h; desorption drying at 6℃ for 4 h, 12℃ for 3 h, and 24℃ for 12 h). The resulting product was diluted with maltodextrin based on the viable count of the mycelial powder to obtain a concentration of 1×10⁻⁶. 11 CFU / g bacterial powder (lyophilized powder).

[0099] The preparation methods for Bifidobacterium breve BD003 powder, Lactobacillus rhamnosus GS066 powder, Lactobacillus paracasei GF027 powder, and Lactobacillus plantarum LZ015 powder are the same as in Experiment 2.

[0100] 2. Strains were grouped according to their proportions. The various bacterial powders are mixed according to the following mass ratios (the mass ratio has the same relationship as the live bacteria count ratio): Formula group: Bifidobacterium breve BD003 powder: Lactobacillus rhamnosus GS066 powder: Lactobacillus paracasei GF027 powder: Lactobacillus plantarum LZ015 powder = 1:1:1:1.

[0101] Lactobacillus plantarum LZ010 replacement group: Bifidobacterium breve BD003 powder: Lactobacillus rhamnosus GS066 powder: Lactobacillus paracasei GF027 powder: Lactobacillus plantarum LZ010 powder = 1:1:1:1.

[0102] Lactobacillus rhamnosus GS044 replacement group: Bifidobacterium breve BD003 powder: Lactobacillus rhamnosus GS044 powder: Lactobacillus paracasei GF027 powder: Lactobacillus plantarum LZ015 powder = 1:1:1:1.

[0103] Lactobacillus paracasei GF045 replacement group: Bifidobacterium breve BD003 powder: Lactobacillus rhamnosus GS066 powder: Lactobacillus paracasei GF045 powder: Lactobacillus plantarum LZ015 powder = 1:1:1:1.

[0104] Bifidobacterium longum BC012 replacement group: Bifidobacterium longum BC012 powder: Lactobacillus rhamnosus GS066 powder: Lactobacillus paracasei GF027 powder: Lactobacillus plantarum LZ015 powder = 1:1:1:1.

[0105] 3. Digestive enzyme production capacity and dietary fiber degradation rate of co-cultured strains After mixing the various bacterial powders according to the ratio in step 2, inoculate them into MRS medium at a total inoculum of 1% (0.1g bacterial powder: 10ml medium) and incubate at 37℃ for 48h. Collect the fermentation broth. After treating the fermentation broth according to the test kit method in Experiment Example 1, test the digestive enzyme production capacity.

[0106] After mixing the various bacterial powders according to the ratio in step 2, inoculate them into dietary fiber screening medium (formula same as in Experiment 1) at a total inoculum of 1% (0.1g bacterial powder: 10ml medium). Incubate at 37℃ for 48h, collect the fermentation broth, centrifuge (8000rpm, 15min) and collect the supernatant. Analyze the supernatant according to the detection method of the kit in Experiment 1.

[0107] The results are as follows: Table 6. Digestive enzyme content and dietary fiber degradation rate in fermentation broths of strains with different ratios

[0108] Note: In the table, different uppercase letters indicate highly significant differences (P < 0.01), and different lowercase letters indicate significant differences (P < 0.05).

[0109] The results showed that after each major component in the formula was replaced, the yields of lipase, protease, and amylase all decreased significantly (P<0.01), and the degradation rate of dietary fiber decreased significantly (P<0.01). This indicates that the ratio of *Bifidobacterium breve* BD003 powder, *Lactobacillus rhamnosus* GS066 powder, *Lactobacillus paracasei* GF027 powder, and *Lactobacillus plantarum* LZ015 powder has a synergistic effect, and each component is irreplaceable.

[0110] Example 4 A compound bacterial powder differs from Example 3 in that it is prepared by mixing Lactobacillus rhamnosus GS066 freeze-dried powder, Lactobacillus paracasei GF027 freeze-dried powder, Lactobacillus plantarum LZ015 freeze-dried powder, and Bifidobacterium breve BD003 freeze-dried powder in a live bacteria ratio of 3:3:4:10.

[0111] Test Example 4 The experimental bacterial powder was the composite bacterial powder prepared in Example 3.

[0112] Male BALB / c mice were randomly divided into six groups: a blank control group, a model group, a positive control group (using Bifidobacterium tetrad live bacteria tablets), a low-dose experimental bacterial powder group, a medium-dose experimental bacterial powder group, and a high-dose experimental bacterial powder group, with 10 mice in each group. Except for the blank control group, all other groups were administered loperamide (5 mg / kg) daily by gavage to establish a constipation model; the positive control group was administered Bifidobacterium tetrad live bacteria tablets by gavage. The experimental bacterial powder for each dosage group was diluted with physiological saline and administered by gavage. The gavage volume for the low, medium, and high-dose groups was 2 × 10⁻⁶ total live bacteria counts. 7 CFU / each, 2×10 8 CFU / each, 2×10 9 CFU / animal, all administered by gavage once daily for 21 days.

[0113] During the experiment, the number of fecal pellets, the time of the first black stool, and the quality of black stool were recorded. After the last administration, the intestinal ink propulsion rate was measured, and serum and small intestinal tissue were collected to detect the activity of gastrointestinal regulatory peptides (MTL, Gas, SP, SS, VIP, ET-1).

[0114] Table 7. Effects of experimental bacterial powder on stool quality, number of stool pellets, and time to first stool pellet excretion in mice.

[0115] Note: In the table, ## and ### represent P<0.01 and P<0.001 respectively compared with the blank control group; , , These represent P<0.05, P<0.01, and P<0.001 compared to the model group, respectively.

[0116] The results showed that, compared with the blank control group, the model group mice exhibited a significant decrease in the quality and number of black feces excreted within 5 hours (P<0.001), and a significant prolongation in the time to excretion of the first black feces (P<0.01), indicating successful model establishment. Compared with the model group, the experimental medium / high dose groups showed an increase in the quality and number of black feces excreted within 5 hours (P<0.001), and a shortened time to excretion of the first black feces (P<0.001), which was superior to the positive control group.

[0117] Table 8. Effects of experimental bacterial powder on small intestinal motility in mice.

[0118] Note: In the table, ## and ### represent P<0.01 and P<0.001 respectively compared with the blank control group; , P<0.01 and P<0.001, respectively, compared to the model group.

[0119] The results showed no difference in the total length of the small intestine among the groups (P<0.05). Compared with the blank control group, the ink propulsion length and rate in the small intestine of the model group were both reduced (P<0.001), indicating that small intestinal peristalsis was inhibited after gavage administration of loperamide hydrochloride suspension. Compared with the model group, the ink propulsion rate in the small intestine of the medium / high dose groups of the experimental bacterial powder was significantly increased (P<0.001), and the ink propulsion rate in the small intestine of the low dose group of the experimental bacterial powder and the positive control group was significantly increased (P<0.01). The medium / high dose groups of the experimental bacterial powder were more effective than the positive control in promoting intestinal peristalsis.

[0120] The effect of experimental bacterial powder on gastrointestinal regulatory peptides in mouse serum, such as Figure 5 As shown in the figure, #, ##, and ### represent P<0.05, P<0.01, and P<0.001 respectively compared with the blank control group. , , "Compared with the model group, P < 0.05, P < 0.01, and P < 0.001" represent the results. The results showed that gastrointestinal regulatory peptides MTL, GAS, SP, SS, VIP, and ET-1 are important indicators for evaluating gastrointestinal motility. Compared with the blank control group, the serum concentrations of MTL, GAS, and SP in the model group mice were significantly decreased (P < 0.001, P < 0.01), while the serum concentrations of SS, VIP, and ET-1 were significantly increased (P < 0.05, P < 0.01). Compared with the model group, the serum concentrations of MTL, GAS, and SP in the medium / high dose groups of the experimental bacterial powder were significantly increased (P < 0.05, P < 0.01, P < 0.001), while the serum concentrations of SS, VIP, and ET-1 were significantly decreased (P < 0.05, P < 0.01, P < 0.001), which was superior to the positive control group.

[0121] MTL, GAS, and SP are excitatory peptide neurotransmitters that can accelerate intestinal peristalsis and shorten the transit time of intestinal contents, while ET-1, SS, and VIP are inhibitory neurotransmitters that can relax the gastrointestinal tract and prolong the transit time of food in the gastrointestinal tract. Therefore, the experimental bacterial powder of this invention can promote defecation by affecting the balance between excitatory and inhibitory peptide neurotransmitters.

[0122] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of Bifidobacterium breve BD003, characterized in that, The Bifidobacterium breve (B. breve) Bifidobacteriumbreve ) BD003 has a preservation number of CGMCC No. 30721.

2. A method of preparing Bifidobacterium breve BD003 lyophilized powder, characterized by, The process includes the following steps: inoculating the Bifidobacterium breve BD003 of claim 1 into a fermentation medium for fermentation, centrifuging the fermentation broth to obtain bacterial sludge, mixing the bacterial sludge with a freeze-drying protectant, and freeze-drying to obtain freeze-dried Bifidobacterium breve BD003 powder.

3. The production method according to claim 2, characterized by, The fermentation medium comprises: lactose 8-12 g / L, glucose 8-12 g / L, sucrose 4-6 g / L, soybean peptone 8-12 g / L, bovine bone peptone 8-12 g / L, beef extract 4-6 g / L, yeast extract 8-12 g / L, anhydrous sodium acetate 4-6 g / L, diammonium citrate 1-3 g / L, dipotassium hydrogen phosphate 1-3 g / L, manganese sulfate 0.2-0.3 g / L, and Tween 0.5-1.5 g / L.

4. The lyophilized Bifidobacterium breve BD003 powder prepared by the preparation method according to any one of claims 2 to 3.

5. A composite bacterial powder, characterized by, The compound bacterial powder includes freeze-dried powder of *Lactobacillus rhamnosus* GS066, freeze-dried powder of *Lactobacillus paracasei* GF027, freeze-dried powder of *Lactobacillus plantarum* LZ015, and freeze-dried powder of *Bifidobacterium breve* BD003 as described in claim 4; the preservation number of *Lactobacillus rhamnosus* GS066 is CGMCC No. 29393; the preservation number of *Lactobacillus paracasei* GF027 is CGMCC No. 22831; and the preservation number of *Lactobacillus plantarum* LZ015 is CGMCC No. 29395.

6. The compound microbial powder according to claim 5, characterized in that, The ratio of viable bacteria in the compound bacterial powder is (15~25):(15~25):(15~25):(25~50).

7. The use of the compound microbial powder according to any one of claims 5 to 6 in the production of digestive enzymes and / or short-chain fatty acids.

8. The application according to claim 7, characterized in that, The digestive enzymes include lipases, proteases, and / or amylases.

9. The application of the *Bifidobacterium breve* BD003 according to claim 1, or the freeze-dried powder of *Bifidobacterium breve* BD003 according to claim 4, or the compound bacterial powder according to any one of claims 5-6, in any of the following, characterized in that, (1) Prepare health foods or medicines that help with bowel movements; (2) Prepare health foods or medicines that help regulate the intestinal flora; (3) Prepare health foods or medicines that aid digestion; (4) Prepare drugs that improve the level of gastrointestinal regulatory peptides.

10. A product that helps to lubricate the intestines and promote bowel movements, characterized in that, The product comprises Bifidobacterium breve BD003 as described in claim 1, or the lyophilized powder of Bifidobacterium breve BD003 as described in claim 4, or the compound bacterial powder as described in any one of claims 5 to 6.

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