Microbial preparation and use thereof

Abstract

This application discloses a microbial preparation and its application, belonging to the field of microbial technology. The microbial preparation contains *Bifidobacterium lactis* strains, including strains B-1 and / or B-6, which were isolated from the intestines of healthy breastfed infants aged 10 months and 6 months, respectively. Both strains meet safety standards, exhibiting no hemolysis, no production of biogenic amines or nitrites, and excellent resistance to gastric acid and bile salts, antibacterial activity, bacterial adhesion, and passage stability. Using zebrafish and mouse constipation models, it was confirmed that B-1 in the preparation upregulates 5-HT, repairs the intestinal barrier, and enhances overall intestinal motility; while B-6 optimizes the intestinal flora, increases short-chain fatty acids, and increases stool volume.

Description

Technical Field

[0001] This application belongs to the field of microbial technology, and more specifically, relates to a microbial preparation and its application. Background Technology

[0002] Constipation is a common gastrointestinal functional disorder in clinical practice. Typical symptoms include decreased bowel movement frequency, hard stools, and difficulty in defecation. Prolonged constipation can significantly reduce patients' quality of life and easily lead to various complications such as intestinal inflammation and anorectal diseases. Currently, chemical drugs used clinically to improve constipation generally have drawbacks such as slow onset of action, large individual differences in efficacy, and the potential for long-term continuous use to cause intestinal dependence, accompanied by varying degrees of toxic side effects, thus limiting drug safety. As a result, research has become a hot topic regarding the use of probiotics to regulate intestinal physiological function and replace traditional laxatives.

[0003] Gut microecological homeostasis is a core condition for maintaining normal peristalsis and metabolism in the digestive tract. Constipation patients commonly exhibit a gut microbiota imbalance, characterized by a significant decrease in the abundance of beneficial bacteria and the abnormal proliferation of opportunistic pathogens. Numerous animal experiments and clinical studies have confirmed that exogenous probiotic supplementation can reshape the gut microbiota structure, fundamentally improving the causes of constipation, making it a green and safe intervention for constipation. *Bifidobacterium animalis* subsp. *lactamase* is one of the most common and dominant Bifidobacteria in the intestines of infants and young children. Its abundance shows a significant downward trend with age, a pattern that closely matches the increasing incidence of constipation with age in the general population. Due to its good safety profile, it has become a popular strain for the development of functional probiotics.

[0004] However, the commercially available Bifidobacterium animalis subsp. lactis strains still have some areas that urgently need optimization: First, their probiotic properties are insufficient, and their functions such as laxative, antibacterial, and intestinal barrier repair are difficult to work synergistically; Second, their stress resistance is significantly weak, with poor resistance to gastric acid and bile salts, low in vivo survival rate, poor strain passage stability, and low fermentation proliferation efficiency, which is not conducive to large-scale, high-density production; Third, most Bifidobacterium strains ferment alone, resulting in yogurt with low flavor acceptance, often accompanied by a pungent acetic acid taste or unpleasant aftertaste.

[0005] Therefore, there is an urgent need for a novel strain of Bifidobacterium animalis lactis that possesses excellent laxative activity, high stress resistance and production properties, and excellent fermentation flavor for the production of microbial preparations. Summary of the Invention

[0006] The purpose of this application is to provide a microbial preparation and its application, which contains a strain of Bifidobacterium animalis subsp. lactis that has excellent growth characteristics, stress resistance, antibacterial properties, and the ability to promote intestinal peristalsis and improve constipation. It is also odorless and has a good flavor in probiotic preparations, fermented dairy products or related products, and can further effectively fill the technical gap of the unbalanced characteristics of existing strains, and meet the actual application needs of the probiotic industry.

[0007] To achieve the above objectives, this application provides a microbial preparation containing live bacteria of Bifidobacterium animalis subsp. lactis and / or its fermentation metabolites.

[0008] Specifically, a strain of Bifidobacterium lactis (Bifidobacterium animalis) Bifidobacterium animalis subsp .lactis B-1, Bifidobacterium animalis subsp. lactis strain B-1 ( Bifidobacterium animalis subsp lactis B-1) has the accession number CGMCC No.38186 and was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 30, 2026.

[0009] Specifically, a strain of Bifidobacterium lactis (Bifidobacterium animalis) Bifidobacterium animalis subsp .lactis B-6, Bifidobacterium animalis subsp. lactis strain B-6 ( Bifidobacterium animalis subsp lactis B-6) has the accession number CGMCC No.38187 and was deposited at the China General Microbiological Culture Collection Center on March 30, 2026.

[0010] Furthermore, Bifidobacterium B-1 strain was derived from the intestines of healthy, exclusively breastfed 10-month-old infants, and Bifidobacterium B-6 strain was derived from the intestines of healthy, exclusively breastfed 6-month-old infants. The isolation method was as follows: Fresh intestinal contents samples were collected from healthy, exclusively breastfed infants, serially diluted with sterile diluent, and plated onto MRS-LM selective solid medium supplemented with L-cysteine ​​hydrochloride and mupirocin lithium. The samples were cultured under anaerobic conditions. Single colonies were repeatedly picked and streaked 3-4 times to obtain a single purified strain with uniform genetic traits. The identification method was as follows: The genome of the purified target strains was extracted, and the 16S rRNA gene was amplified using universal primers and sequenced. The 16S rRNA nucleotide sequence of B-1 is shown in Seq.1, and the 16S rRNA nucleotide sequence of B-6 is shown in Seq.2. The sequencing results were compared with the NCBI database using BLAST homology. The sequences of the two strains were consistent with those of Bifidobacterium lactis subsp. animalis strains (Bifidobacterium lactis) recorded in Genebank. Bifidobacterium animalis subsp . lactisBoth strains showed a homology similarity exceeding 99.8%, confirming their classification as *Bifidobacterium animalis* subsp. *lactobacter* at the molecular level. Morphological characteristics were as follows: When inoculated into MRS-LM solid medium for anaerobic culture, the colonies were milky white and raised, with a smooth, glossy surface and regular, hairless edges. After anaerobic activation culture in MRS-LM liquid medium, the bacterial cells appeared rod-shaped under a light microscope.

[0011] Furthermore, both Bifidobacterium B-1 and Bifidobacterium B-6 strains of this application exhibit good resistance to gastric acid and bile salts. Gastric acid resistance: Their resistance to gastric acid is comparable to or better than that of commercially available Bifidobacterium lactis subsp. animalis strains of the same type. After digestion in simulated gastric acid at pH 2.0 for 0.5 h, the survival rate of Bifidobacterium B-1 strain was 40.40% ± 7.32%, and the survival rate of Bifidobacterium B-6 strain was 61.73% ± 10.62%, both higher than the survival rate of commercial strain BB12 (26.32% ± 7.02%) under the same conditions. After digestion in simulated gastric acid at pH 3.0 for 2.0 h, the survival rate of Bifidobacterium B-1 strain was 93.65% ± 4.62%, and the survival rate of Bifidobacterium B-6 strain was 86.65% ± 2.73%, comparable to or better than the survival rate of commercial strain BB12 (87.84% ± 6.27%) under the same conditions. Bile salt tolerance: The bile salt tolerance of the strains is comparable to or better than that of commercially available Bifidobacterium lactis strains of the same type: ① Growth curves obtained from MRS-L liquid medium with different concentrations of porcine bile salts show that the growth curves of each strain are basically consistent in MRS-L liquid medium without bile salts. In MRS-L medium with different concentrations of porcine bile salts, under the same conditions, the time to reach the stationary phase of Bifidobacterium B-1, Bifidobacterium B-6, and BB12 strains is basically the same, but the growth rate and turbidity of the bacterial solution in the stationary phase are significantly better for Bifidobacterium B-1 and Bifidobacterium B-6 than for BB12; ② After digestion in simulated intestinal fluid with 0.1% porcine bile salts for 2 hours, both Bifidobacterium B-6 and Bifidobacterium B-1 strains showed excellent tolerance, and their survival rates were significantly higher than those of the commercial control strain BB12. This indicates that the Bifidobacterium B-1 and Bifidobacterium B-6 strains of this application have good potential for intestinal survival and colonization.

[0012] Furthermore, both the Bifidobacterium B-1 and Bifidobacterium B-6 strains of this application exhibit good antibacterial activity against intestinal pathogens (in vitro antibacterial performance), inhibiting the growth and proliferation of bacteria including, but not limited to, Escherichia coli and Staphylococcus aureus. Their inhibitory effects on Escherichia coli and Staphylococcus aureus are comparable. Compared to the commercially available Bifidobacterium lactis subsp. BB12 strain, the fermentation supernatant of Bifidobacterium B-1 and Bifidobacterium B-6 strains shows superior inhibition against Escherichia coli (Gastrointestinal tract infection).- Staphylococcus aureus (G) + All of them have significant inhibitory effects, showing good potential probiotic properties.

[0013] Furthermore, the Bifidobacterium B-1 and Bifidobacterium B-6 strains of this application belong to the subspecies of Bifidobacterium animalis (Bifidobacterium lactis), which is listed in the National Health Commission's "List of Microbial Strains that Can Be Used in Food" and possesses the legal qualification for use as a food ingredient. A series of in vitro safety experiments verified that both strains were negative for hemolysis, indicating no risk of hemolysis; the strains do not synthesize harmful biological amines such as histamine and putrescine; the nitrate reduction experiment was negative, and the metabolic process does not generate toxic nitrite byproducts. Simultaneously, the drug resistance spectrum detection results of the strains comply with the relevant provisions of the "GB31615.2-2025 National Food Safety Standard for Food-Use Microbial Strains Safety Evaluation Procedures," and there is no risk of horizontal transfer of acquired drug resistance genes. In summary, the Bifidobacterium B-1 and Bifidobacterium B-6 strains of this application meet safety standards and can be safely used in the development of food, health products, feed, and pharmaceutical-related products.

[0014] Furthermore, the Bifidobacterium B-1 and Bifidobacterium B-6 strains of this application possess both cell hydrophobicity and self-aggregation properties. Cell hydrophobicity: The surface hydrophobicity of the strains was determined using an organic reagent adsorption method, with the commercially available strain BB12 as a control. Experimental results showed that in the n-hexane system, the hydrophobicity of the Bifidobacterium B-1 and Bifidobacterium B-6 strains of this application was similar to that of BB12, approximately 50%; in the xylene system, the hydrophobicity of the Bifidobacterium B-1 strain was superior to that of BB12. The overall hydrophobicity of both strains was within the excellent range of 40%–70%, indicating that the strains have excellent intestinal mucosal adhesion properties, easily forming biofilms in the intestine, which is beneficial for intestinal colonization and exerting antibacterial effects. Self-aggregation performance: Using commercial BB12 as a control, the self-aggregation experiment of the strains was carried out. The early self-aggregation rate of Bifidobacterium B-1 and Bifidobacterium B-6 strains of this application was significantly better than that of the control strain, and the self-aggregation rate gradually increased with the extension of the standing time; after standing for 16 hours, the self-aggregation rate of all three groups of strains was close to 90%. The excellent self-aggregation characteristics can improve the survival stability of the strains in the gastrointestinal environment and further ensure the effective colonization of the strains in the intestine.

[0015] Furthermore, the Bifidobacterium B-1 and Bifidobacterium B-6 strains of this application exhibit growth and passage stability, with an optimal growth temperature range of 36℃ to 42℃. When cultured in basic MRS-L liquid medium, the strains reach the stable growth phase in 10-12 hours, during which the viable cell count in the fermentation broth remains at 1×10⁻⁶. 8 CFU / mL ~ 1×10 9 CFU / mL. After more than 10 consecutive subculturings, the changes in the lag phase, logarithmic phase, and stationary phase of the strain's growth curve, as well as the OD... 600The values ​​show no significant fluctuations, the genetic traits are stable, and it is suitable for continuous industrial-scale production.

[0016] Furthermore, the Bifidobacterium B-1 and Bifidobacterium B-6 strains of this application possess in vivo laxative effects. The laxative function of the strains was verified using a zebrafish constipation model for initial screening and a mouse constipation model. Zebrafish experiment results: Both Bifidobacterium B-1 and Bifidobacterium B-6 strains of this application can upregulate the content of the key intestinal peristalsis-promoting neurotransmitter 5-HT, accelerating intestinal emptying. Mouse in vivo experiment results: Both strains can improve loperamide-induced constipation, accelerate intestinal peristalsis, and increase fecal output; among them, Bifidobacterium B-1 strain showed a comprehensive and stable improvement effect, with significant advantages in shortening the time to first black stool and increasing small intestinal propulsion rate, focusing on enhancing intestinal motility; Bifidobacterium B-6 strain had a later onset of action, primarily increasing stool volume, with limited effect on promoting small intestinal peristalsis, focusing on regulating fecal output.

[0017] Furthermore, animal physiological and biochemical assays and gut microbiota analysis confirmed that the two strains improved intestinal dysfunction through multiple target pathways, with significant differences in their mechanisms of action: Bifidobacterium B-1 strain mainly exerted its effects by significantly increasing colonic 5-HT expression and repairing the intestinal mucosal barrier; while Bifidobacterium B-6 strain focused on optimizing the composition of the gut microbiota and promoting the production of short-chain fatty acids, thus reshaping the gut microbiota homeostasis from a metabolic perspective.

[0018] Specifically, this application also provides a high-density fermentation method for culturing *Bifidobacterium lactis* strains (Bifidobacterium B-1 or Bifidobacterium B-6), comprising the following steps: inoculating the seed culture of *Bifidobacterium B-1* or *Bifidobacterium B-6* into a fermentation medium, controlling the temperature at 36℃~42℃, the initial pH at 6.5, maintaining a constant pH of 5.8 during fermentation, and obtaining a viable cell count of up to 1×10⁻⁶ in the fermentation broth. 9 CFU / mL ~ 1×10 10 The CFU / mL concentration is 10 to 100 times higher than that of ordinary MRS-L medium.

[0019] Furthermore, the fermentation medium contains: a carbon source, a nitrogen source, inorganic salts, buffer salts, water, and growth promoters. Even further, the carbon source is selected from one or any combination of lactose, glucose, sucrose, maltose, fructose, xylose, trehalose, and whey powder; the nitrogen source is selected from one or any combination of peptone, yeast extract / yeast extract, beef extract / beef extract, soybean peptone, wheat peptone, and casein hydrolysate; the inorganic salts are selected from sodium acetate, sodium citrate, magnesium sulfate, manganese sulfate, ammonium sulfate, ferrous sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, and copper sulfate. One or more of zinc sulfate, calcium chloride, magnesium chloride, sodium bicarbonate, and potassium carbonate; growth promoting factors selected from Tween 80, L-cysteine ​​hydrochloride, glutathione, vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, folic acid, adenine, guanine, cytosine, uracil, inosine, adenosine, whey protein hydrolysate, soybean peptides, fructooligosaccharides, galactooligosaccharides, maltodextrin, isomaltooligosaccharides, inulin, various amino acids, and hydrolyzed proteins.

[0020] Preferred high-density fermentation medium includes: glucose 15 g / L, galactooligosaccharides 5 g / L, yeast extract 30 g / L, potassium dihydrogen phosphate 1.8 g / L, disodium hydrogen phosphate 2.2 g / L, anhydrous sodium acetate 4.4 g / L, L-cysteine ​​hydrochloride 0.4 g / L, magnesium sulfate 0.3 g / L, manganese sulfate 0.3 g / L, and Tween 80 0.5 g / L.

[0021] Specifically, this application also provides a freeze-dried bacterial powder containing the aforementioned *Bifidobacterium animalis* subsp. *lactamase* strains (Bifidobacterium B-1 and / or Bifidobacterium B-6 strains), wherein the viable count of the original bacterial powder in the freeze-dried bacterial powder is not less than 1 × 10⁻⁶. 10 CFU / g, meaning the number of viable bacteria in the produced original bacterial powder is not less than 1×10⁻⁶. 10 The number of live bacteria can be adjusted according to production needs when preparing freeze-dried bacterial powder or producing downstream products.

[0022] Furthermore, the freeze-dried bacterial powder is prepared by centrifuging the fermentation broth containing Bifidobacterium animalis subsp. lactis strain to obtain bacterial sludge, mixing and emulsifying it with a freeze-drying protectant, and then freeze-drying it under vacuum to obtain probiotic freeze-dried bacterial powder containing Bifidobacterium B-1 strain or Bifidobacterium B-6 strain of this application.

[0023] Furthermore, the freeze-drying protectant used in the freeze-drying of bacterial fermentation cells to prepare bacterial powder can significantly improve the freeze-drying survival rate and product storage stability. The bacterial powder prepared using this protectant has significant advantages in cold chain storage scenarios of 4~15℃, and can also be adapted to medium and long-term circulation at room temperature (25℃). It can buffer temperature fluctuations in storage and transportation, effectively delay the decay of probiotic live bacteria, and ensure the stability of live bacteria during the product's shelf life. The freeze-drying protectant raw materials are selected from one or more of the following: skim milk powder, whey protein, α-lactalbumin, sodium caseinate, lactoferrin, gelatin, soy protein, yeast extract, hydrolyzed whey protein, glutathione, sucrose, lactose, galactose, trehalose, maltose, galactooligosaccharides, maltodextrin, isomaltooligosaccharides, xylooligosaccharides, fructooligosaccharides, inulin, polydextrose, maltodextrin, resistant dextrin, mannitol, sorbitol, xylitol, erythritol, gum arabic, xanthan gum, guar gum, sodium alginate, carrageenan, sodium ascorbate, L-cysteine ​​hydrochloride, monosodium glutamate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and sodium citrate.

[0024] Specifically, this application also provides a composition, which is a multifunctional probiotic composition. The composition contains the aforementioned *Bifidobacterium lactis* strains (Bifidobacterium B-1 and / or Bifidobacterium B-6 strains), and a pharmaceutically or food-grade acceptable carrier. The composition possesses pharmacological and physiological activities such as promoting bowel movements, repairing the intestinal epithelial barrier, regulating intestinal flora structure, increasing the content of short-chain fatty acids in the intestine, and improving intestinal dysfunction. Based on application scenarios, it is divided into three main application areas: food (including general food, health products, and special medical foods), pet food (including pet food and livestock feed additives), and veterinary drugs / microbial drugs. The strain addition amount is 1×10⁻⁶. 4 CFU / g ~ 1×10 11 CFU / g.

[0025] Furthermore, firstly, in the food industry, the composition can be processed into ordinary food, health food, and special medical purpose formula food. The dosage form of the composition can be divided into three forms: solid, liquid, and semi-solid. Ordinary food includes dairy products, solid beverages, snacks, meal replacement foods, or grain foods; health food includes at least one of dietary fiber, probiotics, prebiotics, postbiotics, and medicinal and edible ingredients; medical purpose formula food is an enteral nutrition preparation for postoperative or elderly patients with constipation, or a special formula food for infants with functional constipation. In the above products, the preferred addition amount of Bifidobacterium B-1 strain or Bifidobacterium B-6 strain is 1×10⁻⁶. 6 CFU / g ~ 1×10 11 The CFU / g can also be adjusted according to the formula and stability, subject to the product execution standard; no live bacteria count requirement is required for inactivated products.

[0026] Secondly, in the fields of livestock and pet feed, the composition can be formulated into pet food and livestock feed additives to improve intestinal flora imbalance, constipation, and digestive disorders in pets such as dogs and cats, as well as pigs, cattle, sheep, and poultry. Product forms include pet staple food, pet snacks, freeze-dried probiotic powder for pets, pet health products, prescription pet food or pet nutritional supplements, feed premixes, pelleted feed additives, and drinking water probiotic preparations. The strain addition in the above products is controlled at 1×10⁻⁶. 4 CFU / g ~ 1×10 9 The CFU / g can also be adjusted according to the formula and stability, subject to the product execution standard; no live bacteria count requirement is required for inactivated products.

[0027] Thirdly, in the field of pharmaceutical microbial preparations, the composition can be used to prepare oral pharmaceutical preparations and enteric-coated capsules, and can be combined with conventional pharmaceutical excipients (fillers, binders, disintegrants, lubricants, wetting agents, flavoring agents, pH adjusters, etc.); the effective content of the strain in the drug is 1×10⁻⁶. 7 CFU / g ~ 1×10 11 CFU / g. Specifically, fillers include lactose, mannitol, and starch; binders include hydroxypropyl methylcellulose and povidone; disintegrants include crospovidone and sodium carboxymethyl starch; lubricants include magnesium stearate and talc; wetting agents include polysorbate and sodium lauryl sulfate; flavoring agents include steviol glycosides and lemon flavoring; and pH adjusters include citric acid and sodium bicarbonate.

[0028] Specifically, this application also provides the application of a microbial preparation containing Bifidobacterium animalis subsp. lactis strain in the preparation of products for relieving constipation, promoting bowel movements, repairing intestinal barrier damage, regulating intestinal flora and / or increasing the content of short-chain fatty acids in the intestine.

[0029] Furthermore, the product relieves constipation and repairs intestinal barrier damage by upregulating colonic 5-hydroxytryptamine levels, promoting the repair of the intestinal mucus barrier and tight junction barrier, and inhibiting the growth of Escherichia coli and / or Staphylococcus aureus.

[0030] Specifically, the microbial preparation of this application is a fermented dairy product, which is prepared by the following method: using fresh milk as a substrate, inoculating it with freeze-dried Bifidobacterium lactis strain 10 for fermentation, fermenting at a constant temperature of 37℃~42℃ until the acidity reaches 70°T~80°T, breaking the milk and cooling to 4℃, followed by refrigeration and ripening to obtain the fermented dairy product; wherein, the inoculation amount is 10 6 CFU / g ~10 7 CFU / g.

[0031] In summary, this application has the following beneficial effects: 1. The bacterial agent containing *Bifidobacterium animalis* subsp. *lactamase* strains provided in this application comprises *Bifidobacterium* B-1 strain and / or *Bifidobacterium* B-6 strain, isolated from the intestines of healthy breastfed infants aged 10 months and 6 months, respectively. The strains are classified as *Bifidobacterium animalis* subsp. *lactamase* strains and are included in the national "List of Microbial Strains that Can Be Used in Food". In vitro safety experiments confirmed that both strains showed negative hemolysis reactions and did not produce harmful biogenic amines or nitrites during metabolism. The strains' drug resistance spectrum conforms to the "GB31615.2-2025 National Food Safety Standard: Safety Evaluation Procedures for Microbial Strains for Food Use," with no risk of lateral transmission of acquired drug resistance genes. The source is clear, the genetic background is well-defined, and they can be safely applied to the development of food, health products, feed, and pharmaceutical products.

[0032] 2. The *Bifidobacterium animalis* subsp. *lactamase* strain provided in this application exhibits superior or equal resistance to gastric acid and bile salts compared to the commercial control strain BB12. Furthermore, it can promote strain proliferation under low-concentration bile salt environments. It reaches a stable growth phase after 10-12 hours of culture in conventional MRS-L liquid medium, achieving a viable bacterial concentration of up to 1×10⁻⁶. 8 CFU / mL ~ 1×10 9 CFU / mL; Fermentation was carried out using the high-density fermentation medium specifically described in this application. The culture entered a stationary phase after 8-10 hours, and the viable cell concentration could be stably increased to no less than 1×10⁻⁶. 9 CFU / mL, up to 1×10 10 CFU / mL. The strain exhibits no decline in growth characteristics after more than 10 generations of continuous passage, demonstrating excellent genetic stability. The bacterial powder prepared in combination with the freeze-drying protectant of this application shows significant advantages in cold chain storage at 4~15℃, while also being suitable for medium- to long-term circulation at room temperature (25℃). It can buffer temperature fluctuations during storage and transportation, effectively delay the decay of live probiotics, and ensure the stability of live bacteria during the product's shelf life.

[0033] 3. The *Bifidobacterium lactis* strain described in this application effectively inhibits the proliferation of common intestinal pathogens such as *Escherichia coli* and *Staphylococcus aureus*. The bacteria exhibit excellent hydrophobicity and self-aggregation properties, demonstrating outstanding adhesion and colonization ability to the intestinal mucosa. In vivo pharmacological experiments in zebrafish and mice showed that both strains can upregulate intestinal 5-HT levels, accelerate intestinal emptying, and improve constipation. They can also repair the intestinal mucosal barrier, optimize the disordered intestinal flora composition, and increase the content of short-chain fatty acids in the intestine. The *Bifidobacterium* B-1 strain focuses on enhancing intestinal motility, while the *Bifidobacterium* B-6 strain is more focused on improving fecal excretion. The two strains have different pathways of action and can both be used independently for intestinal regulation. Synergistic use of both may achieve multi-target intestinal regulation, with an overall laxative effect superior to existing commercial strains.

[0034] 4. The probiotic preparation prepared from the *Bifidobacterium animalis* subsp. *lactamase* strain described in this application exhibits good heat resistance and can retain a high level of viable bacteria even when stored at 37°C. Addressing the industry challenge of balancing viability, processing efficiency, and edible quality in traditional probiotic fermented foods, the *Bifidobacterium* strain provided in this application can be used alone as a starter culture in yogurt preparation, resulting in a product with high viable bacteria count, excellent flavor, and good texture. When applied to fermented dairy product production, it results in a short fermentation cycle, odorless products, and stable and uniform product texture. This effectively overcomes the technical shortcomings of single probiotic strains in terms of performance, providing a reliable strain resource for the development of high-quality probiotic fermented products.

[0035] In summary, the Bifidobacterium B-1 and Bifidobacterium B-6 strains of this application make up for the shortcomings of existing commercial Bifidobacterium strains in terms of coordination in probiotic efficacy, production characteristics and sensory quality. They have extremely high industrial application value in the fields of general food, health food, special medical food, livestock feed, pet supplies or probiotic pharmaceuticals. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 These are colony images and microscopic images of *Bifidobacterium animalis* subsp. *lactum* as presented in Example 2 of this application; wherein, Figure 1 In the diagram, A represents a colony diagram. Figure 1 In this diagram, B represents a microscope image.

[0038] Figure 2 This is a comparative diagram of the acid resistance characteristics of *Bifidobacterium animalis* subsp. *lactum* proposed in Example 3 of this application; wherein, Figure 2 In this context, A represents the characteristics of strain B-1. Figure 2 The B in the text refers to the characteristics of strain B-6.

[0039] Figure 3 This is the growth curve of Bifidobacterium lactis subsp. animalis proposed in Example 4 of this application in MRS liquid culture medium with different concentrations of bile salts.

[0040] Figure 4 This is a diagram showing the results of the antibacterial experiment on Escherichia coli by the fermentation supernatant of Bifidobacterium lactis according to Example 5 of this application; wherein, Figure 4 In this context, A represents the control result of strain B-1. Figure 4 The B in the figure represents the control result of strain B-6.

[0041] Figure 5 This is a diagram showing the antibacterial test results of the fermentation supernatant of *Bifidobacterium animalis* subsp. *lactamase* against *Staphylococcus aureus* as presented in Example 5 of this application; wherein, Figure 5 In this context, A represents the control result of strain B-1. Figure 5 The B in the figure represents the control result of strain B-6.

[0042] Figure 6 This refers to the hemolytic identification and double-layer plate staining results of *Bifidobacterium animalis* subsp. *lactamella* as described in Example 6 of this application; wherein, Figure 6 In this context, A represents the result of the hemolytic test. Figure 6 In this context, B represents the color development result of a double-layer plate.

[0043] Figure 7 The results are from the nitrate reduction experiment of Bifidobacterium lactis subsp. animalis proposed in Example 6 of this application.

[0044] Figure 8 The results of the hydrophobicity experiment of *Bifidobacterium lactis* subsp. *anisole* in xylene and n-hexane, as proposed in Example 7 of this application, are as follows: Figure 8 In this context, A indicates the hydrophobicity of strain B-1. Figure 8 The B in the text indicates the hydrophobicity of strain B-6.

[0045] Figure 9 These are the experimental results of the self-coagulation rate of *Bifidobacterium animalis* subsp. *lactum* at different times, as presented in Example 7 of this application; wherein, Figure 9 In this context, A represents the self-aggregation property of strain B-1. Figure 9 The B in the text indicates the self-aggregation of strain B-6.

[0046] Figure 10 This is a continuous passage growth curve of *Bifidobacterium animalis* subsp. *lactum* as proposed in Example 8 of this application; wherein, Figure 10 In this text, A represents the growth curve of strain B-1 after successive passages. Figure 10 In this text, B represents the growth curve of B-6 through continuous subculturing. Figure 10 In the figure, C represents the growth curve of viable bacteria count of B-1 at different generations. Figure 10 In the figure, D represents the growth curve of viable bacteria number of B-6 at different generations.

[0047] Figure 11 This refers to the relative fluorescence intensity experiment results of zebrafish fed Nile red for 8 hours as described in Example 9 of this application; wherein, Figure 11 In this context, A represents the relative fluorescence intensity of the zebrafish intestinal contents after 8 hours. Figure 11 In this text, B represents the contents of zebrafish intestines under a fluorescence microscope after 8 hours.

[0048] Figure 12These are the experimental results of zebrafish 5-HT content proposed in Example 9 of this application; wherein, Figure 12 In this context, A represents the result for strain B-1. Figure 12 The B in the figure represents the result of strain B-6.

[0049] Figure 13 This is a graph showing the results of the basic indicators in the mouse experiment proposed in Example 9 of this application; wherein, Figure 13 In the diagram, AC represents the changes in body weight, food intake, and water intake of mice in group B-1, respectively, while CF represents the changes in body weight, food intake, and water intake of mice in group B-6, respectively.

[0050] Figure 14 These are the results of the measurement of defecation time and fecal particle number in the mouse experiment proposed in Example 9 of this application; wherein, Figure 14 In the table, A and B represent the time it takes for the first black feces to be expelled in mice in groups B-1 and B-6, respectively, and C and D represent the number of black feces in groups B-1 and B-6 after 5 hours, respectively.

[0051] Figure 15 This is a photograph of the mouse ink intestinal propulsion experiment proposed in Example 9 of this application.

[0052] Figure 16 These are data graphs from the mouse ink intestinal propulsion experiment and colonic 5-HT content experiment proposed in this application; among them, Figure 16 In this context, A represents the data from the mouse ink intestinal propulsion experiment in Example 9. Figure 16 In this context, B represents the experimental data on colonic 5-HT content in Example 10.

[0053] Figure 17 These are images of HE-stained sections and AB-PAS-stained sections of mouse colon as presented in Example 10 of this application.

[0054] Figure 18 This refers to the immunofluorescence detection results of intestinal mucosal barrier-related proteins proposed in Example 10 of this application; wherein, Figure 18 In the diagram, A represents the fluorescence signal intensity map, B represents the protein expression level of group B-1, and C represents the protein expression level of group B-6.

[0055] Figure 19 This is the α diversity index assessment result of the mouse gut microbiota analysis proposed in Example 11 of this application.

[0056] Figure 20 This is the result of β-diversity-PCA analysis of mouse gut microbiota proposed in Example 11 of this application.

[0057] Figure 21This is a bar chart of the community composition of the mouse gut microbiota analysis proposed in Example 11 of this application; wherein, Figure 21 In this context, A represents the horizontal composition of a gate, and B represents the horizontal composition of a genus.

[0058] Figure 22 This is a graph showing the results of short-chain fatty acid content in the intestines of mice in group B-1, as proposed in Example 12 of this application.

[0059] Figure 23 This is a graph showing the results of short-chain fatty acid content in the intestines of mice in group B-6 as proposed in Example 12 of this application.

[0060] Figure 24 This is a schematic diagram of the growth curve of viable bacteria count in a fermenter of Bifidobacterium animalis subsp. lactis proposed in Example 13 of this application. Detailed Implementation

[0061] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0062] Preservation of biological materials: Bifidobacterium B-1 strain ( Bifidobacterium animalis subsp lactis B-1), categorized and named Bifidobacterium animalis subsp lactis It was deposited on March 30, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 38186. The depositary address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0063] Bifidobacterium B-6 strain ( Bifidobacterium animalis subsp lactis B-6), categorized and named Bifidobacterium animalis subsp lactisIt was deposited on March 30, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 38187. The depositary address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0064] The technical solutions described above in this application will be explained in detail below with reference to specific embodiments.

[0065] Example 1 This embodiment illustrates the screening and identification of Bifidobacterium animalis subspecies Bifidobacterium B-1 and Bifidobacterium B-6.

[0066] 1.1 Strains Isolation and Screening: The strains in this embodiment were obtained from fresh feces of healthy infants in Chengdu, Sichuan Province. Strain B-1 was derived from the intestines of a 10-month-old healthy breastfed infant, and strain B-6 was derived from the intestines of a 6-month-old healthy breastfed infant. The culture media used in this application were: MRS liquid medium (also known as MRS broth medium, purchased from Guangdong Huankai Microbial Technology Co., Ltd., catalog number 027312); MRS solid medium, i.e., MRS liquid medium with 2wt% agar added; MRS-L liquid medium, i.e., MRS liquid medium with 0.5 g / L L-cysteine ​​hydrochloride added; and MRS-LM solid medium, i.e., MRS solid medium with 0.5 g / L L-cysteine ​​hydrochloride and 50 mg / L mupirocin lithium added.

[0067] The separation methods include: (1) collecting the sample and placing it in a sterile storage tube containing 30% glycerol; (2) serially diluting the sample with sterile physiological saline (10 ppm). -1 Up to 10 -7 (2) Take 50 μL to 100 μL of each gradient dilution and spread it on MRS-LM solid medium. After the plate is dried, invert it and place it in the anaerobic workstation. Incubate at 37℃ for 24 h to 72 h. (3) After the colony grows, pick a single colony and streak it on MRS-LM solid medium for purification. Repeat the above operation to purify 3 to 4 times to obtain a purified single strain.

[0068] 1.2 Molecular biological identification: Purified single colonies were inoculated into 5 mL of MRS-L liquid medium and anaerobically cultured at 37℃ for 12-24 h until the stationary phase. 1.5 mL of the culture was collected, centrifuged (12000 rpm, 1 min), and the supernatant was discarded. Genomic DNA was extracted from the centrifuged bacterial sludge using a Sangon Biotech bacterial genomic extraction kit. PCR amplification was performed using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1541R (5′-AAGGAGGTGATCCAGCC-3′). The amplified products were sent to Sangon Biotech for sequencing. The 16S rRNA nucleotide sequence of strain B-1 is shown in Seq.1, and the 16S rRNA nucleotide sequence of strain B-6 is shown in Seq.2. The sequencing results were compared with the NCBI database using BLAST homology. The sequences of the two strains were compared with those in the Genebank database. Bifidobacterium animalis subsp lactis Both strains showed a homology similarity of >99.8%, and molecular biological identification results confirmed that they belonged to the subspecies of Bifidobacterium animalis.

[0069] Seq.1:

[0070] Seq.2:

[0071] Example 2 This embodiment is used to observe the colony morphology and microscopic morphology of the strains. Specifically, it includes: inoculating strains B-1 and B-6 onto MRS-LM solid medium, anaerobic culture, and observing the colony morphology: the colonies are generally milky white and raised, with a smooth and glossy surface, and neat, burr-free edges. (See attached image for morphology details.) Figure 1 A. Using an inoculation loop, pick a single colony purified in Example 1 and inoculate it into 5 mL of MRS-L liquid medium. Incubate anaerobicly at 37°C for 12-24 hours until the stationary phase (first-generation activated culture). Take 4% of the first-generation activated culture and inoculate it into fresh MRS-L liquid medium. Incubate anaerobicly at 37°C for 12-24 hours until the stationary phase (second-generation activated culture). Subsequent passages are performed in this manner. Examine the fermentation broth of the bacteria after three generations of activation in MRS-L liquid medium under an optical microscope. The bacteria are mainly rod-shaped. The microscopic results are shown in [Figure 1]. Figure 1 The B in the text; depending on the number of passages, culture environment, and dormancy / activation state of the bacteria, the strain can also exhibit various morphologies such as V-shape, Y-shape, and comma shape.

[0072] Example 3 This example was used to verify the resistance of strains B-1 and B-6 to simulated gastric acid. The experiment was conducted according to the group standard "T / CNHFA435-2024 Test Method for Gastric Juice Tolerance of Probiotic Preparations". The results are shown below. Figure 2 The experimental results showed that after digestion in simulated gastric acid at pH 2.0 for 0.5 h, the survival rate of strain B-1 was 40.40%±7.32%, and the survival rate of strain B-6 was 61.73%±10.62%, both higher than the survival rate of commercial strain BB12 (26.32%±7.02%) under the same conditions. After digestion in simulated gastric acid at pH 3.0 for 2.0 h, the survival rate of strain B-1 was 93.65%±4.62%, and the survival rate of strain B-6 was 86.65%±2.73%, while the survival rate of commercial strain BB12 under the same conditions was 87.84%±6.27%.

[0073] Example 4 This example is used to test the bile salt tolerance and simulated intestinal fluid tolerance of strains B-1 and B-6.

[0074] 4.1. Growth curves tested in culture media with different concentrations of bile salts: The B-1 and B-6 strains of this application were cultured with the commercially available animal Bifidobacterium lactis subsp. BB12 in MRS-L liquid medium supplemented with different concentrations of porcine bile salts (0 wt%, 0.01 wt%, 0.03 wt%, 0.05 wt%, 0.07 wt%, and 0.09 wt%). The growth curve results are shown in [Figure number missing]. Figure 3 .

[0075] The experimental method was as follows: After obtaining the activated second-generation solution according to Example 2, the bacterial solution was inoculated into 5 mL of MRS-L liquid culture medium containing different concentrations of porcine bile salts at an inoculation rate of 4%. After shaking and mixing, the liquid was immediately added to the corresponding 100-well plate of the automatic growth curve measuring instrument, with 250 μL added to each well. Each sample was prepared in 5 replicates. The culture was set at 37℃, and the OD was measured every 30 min. 600 The oscillation lasted for 15 seconds before and after the measurement. The final results are plotted with time on the x-axis and OD on the y-axis. 600 Presented as the vertical axis.

[0076] The experimental results were as follows: In MRS-L liquid medium without added bile salts, the growth curves of all strains were basically consistent. In MRS-L liquid medium with different concentrations of porcine bile salts, under the same conditions, the time to reach the stationary phase for *Bifidobacterium animalis* subsp. lactis B-1 and B-6 strains was basically the same as that for BB12. However, the growth rate and stationary phase turbidity of strains B-1 and B-6 were significantly better than those of BB12. Furthermore, the stationary phase turbidity of *Bifidobacterium animalis* subsp. lactis B-1 strain was slightly higher in low-concentration (0.01%) bile salt medium than in the bile-free medium, indicating that *Bifidobacterium animalis* subsp. lactis B-1 strain not only has good bile salt tolerance but may even utilize bile salts to promote its own growth and proliferation to some extent.

[0077] 4.2. Testing the survival rate after digestion of simulated intestinal fluid containing bile salts: The concentration of bile salts in the human small intestine is usually between 0.03% and 0.3%. The experiment used simulated intestinal fluid containing 0.1% bile salts. The specific formula was: 6.8g potassium dihydrogen phosphate, 10g trypsin, 1g porcine bile salts, pH adjusted to 6.8, and RO water added to 1L.

[0078] The experimental method was as follows: 1 mL of activated third-generation fresh bacterial culture was mixed with 9 mL of simulated intestinal fluid containing 0.1% bile salts and then anaerobically cultured at 37℃. The viable bacterial counts at 0h and 2h were determined according to GB 4789.35-2023 National Food Safety Standard for Microbiological Examination of Food (Lactic Acid Bacteria Examination). The number of viable bacteria after bile salt digestion at 2h and the survival rate were calculated. The survival rate was calculated as (2h viable bacterial count / 0h viable bacterial count) × 100%. The bacterial strains used were *Bifidobacterium animalis* subsp. lactis B-1, B-6, and BB12. The results are shown in Table 1.

[0079] Table 1. Viable bacterial count and survival rate after 2 hours of digestion with simulated intestinal fluid containing 0.1% bile salts.

[0080] Experimental results: The survival rates of strains B-1 and B-6 after 2 hours of treatment were 2.13% and 7.58%, respectively, which were significantly higher than those of BB12 (0.04%). This indicates that these two strains have stronger adaptability to the intestinal bile salt environment and have the potential to colonize in vivo and exert probiotic effects. They can be selected as highly resistant candidate strains for the next stage of screening.

[0081] Example 5 In this embodiment, the perforation method was used, with commercial strain BB12 as a positive control and MRS-L liquid medium (adjusted to the pH of the supernatant after fermentation with dilute hydrochloric acid) as a negative control, to investigate the inhibitory activity of fermentation supernatants of strains B-1 and B-6 against Escherichia coli and Staphylococcus aureus.

[0082] 5.1. Antibacterial experiment against Escherichia coli: Activated indicator bacteria Escherichia coli (ATCC 25922) were mixed into nutrient agar at 45℃~50℃ (final concentration 10). 6 After shaking well, prepare nutrient agar plates containing *E. coli* (CFU / mL). Take activated second-generation B-1 and B-6 bacterial cultures, centrifuge at 12000 rpm for 1 min at 4℃, and collect the fermentation supernatant for later use. After punching wells, add 200 μL of the fermentation supernatant of the test strain to each well, and incubate aerobically at 37℃ for 12-18 h. Measure the diameter of the inhibition zone with calipers. The experimental results are shown in [Figure number missing]. Figure 4 .

[0083] Experimental results: The inhibition zones of strains B-1 and B-6 were 14.69 mm and 14.82 mm, respectively, significantly higher than those of the corresponding negative control groups (11.91 mm and 12.72 mm); the inhibition zones of the positive control BB12 were 16.46 mm and 15.81 mm, respectively. This indicates that strains B-1 and B-6 have similar antibacterial abilities and possess excellent anti-Escherichia coli activity.

[0084] 5.2. Antibacterial test against Staphylococcus aureus: The experimental procedure was the same as that for Escherichia coli, except that the indicator bacterium was changed to Staphylococcus aureus (ATCC 6538). The experimental results are shown in […]. Figure 5 The experimental results showed that the inhibition zones of strains B-1 and B-6 were 24.60 mm and 23.41 mm, respectively, significantly higher than their negative controls (8.10 mm and 7.93 mm). The inhibition zones of the positive control BB12 were 32.42 mm and 32.80 mm, respectively. This indicates that both strains B-1 and B-6 can effectively inhibit the proliferation of Staphylococcus aureus, demonstrating good antibacterial potential.

[0085] Example 6 This embodiment conducts safety tests on strains B-1 and B-6 from four dimensions: hemolysis test, amine production test, nitrate reduction test, and antibiotic susceptibility test, to verify the compliance of the strains with food safety standards.

[0086] 6.1 Hemolysis Test: The experiment was conducted using Columbia 5% defibrinated sheep blood agar plates (purchased from Bikman Biotechnology). Staphylococcus aureus ATCC6538 was used as a positive control, and a blank control group was set up. Strains B-1 and B-6 were anaerobically cultured at 37℃ for 24-48 hours, while the positive strain was aerobically cultured for 12-18 hours. Corresponding blank controls were established for both culture conditions (streaked with sterile culture medium). The experimental results are shown in [Figure number missing]. Figure 6 The results showed that the positive strain colonies had obvious clear zones (β-hemolysis), while the blank, B-1, and B-6 colonies had no hemolytic zones. This indicates that both strains are non-hemolytic strains, do not produce hemolytic toxins, have good safety, and meet the hemolytic safety standards for food microorganisms.

[0087] 6.2 Experiment on the Formation of Harmful Biogenic Amines: Lactic acid bacteria with decarboxylase activity can produce biogenic amines with potential safety risks through the decarboxylation reaction of amino acids. The experiment used a double-layer plate colorimetric method to verify whether *Bifidobacterium lactis* subsp. *lactam* B-1 and B-6 possess decarboxylases that produce common biogenic amines (histamine, cadaverine, tyramine, phenylethylamine, tryptamine, putrescine). The experiment followed the method and culture medium components described in 1.3.2 of Lu Shiling's "Research on the Isolation Methods of Biogenic Amine-Producing Enteric Bacteria and Lactic Acid Bacteria in Traditional Sausages" to prepare the lower layer of the double-layer plate. *Escherichia coli* (ATCC25922) was used as a positive control, and a blank control was used as a reference. *Bifidobacterium* was cultured anaerobicly (24h~48h) at 37℃, while *Escherichia coli* was cultured aerobically (12h~18h). Corresponding blank controls (streaked with sterile culture medium) were established for each culture condition. The experimental results are shown in [Figure 1]. Figure 6 The results of double-layer agar development of B-type amine-producing strains showed that the streaked plate of *E. coli* quickly changed from orange-yellow to purple after the upper culture agar containing bromocresol purple was poured in, indicating that this strain can produce basic amines. In contrast, the blank control and the experimental groups of *Bifidobacterium animalis* subsp. lactis strains B-1 and B-6 remained orange-yellow. Therefore, it can be concluded that *Bifidobacterium animalis* subsp. lactis strains B-1 and B-6 do not produce histamine, tyramine, cadaverine, tryptamine, phenylethylamine, or putrescine, and their amino acid decarboxylase activity is very low, thus they do not produce basic amines and are safe for consumption.

[0088] 6.3 Nitrate Reduction Experiment: Using the commercially available nitrate biochemical identification tubes (075320) from Huankai and the matching nitrate reduction reagent (029070), the presence of nitrate reductase in *Bifidobacterium animalis* subsp. *lactamase* B-1 and B-6 strains was confirmed. *Escherichia coli* (ATCC25922) was used as a positive control, and BB12 as a negative control. B-1 and B-6 bacterial cultures in the logarithmic growth phase (second generation) were diluted to an appropriate concentration (10...). 8 After determining the CFU / mL level, 50 μL of bacterial suspension was added to a microfluidic bottle and incubated at 37°C for 18-24 hours. A colorimetric solution was then added. If the liquid turned red, the result was positive. If the liquid was colorless or light pink, a small amount of zinc powder was added for confirmation. If no color change occurred after adding zinc powder, the result was positive, indicating that nitrates were strongly reduced to nitrogen gas and removed from the system. If the liquid turned red, the result was negative. The experimental results are shown below. Figure 7 The positive control turned red upon color development, while the blank, B-1 strain, B-6 strain, and BB12 were initially colorless but turned red after the addition of zinc powder, proving that B-1 and B-6 strains do not contain nitrate reductase and do not produce harmful nitrite metabolites.

[0089] 6.4 Antibiotic Susceptibility Testing: According to Appendix E, "Determination of Antimicrobial Resistance of Food-Use Bacterial Strains (Microbroth Dilution Method)," of the National Food Safety Standard GB31615.2-2025, the required antibiotics for Bifidobacterium spp. (ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, chloramphenicol) were determined. Antibiotic susceptibility testing was conducted on the strains using the disk diffusion method (KB method) according to CLSI (Clinical and Laboratory Standards Institute) standards. The experimental method was as follows: The activated bacterial culture from the second generation to the logarithmic phase was diluted with sterile physiological saline to 0.5 McFarland turbidity. 100 μL of the diluted solution was spread onto the surface of an MRS-L solid culture plate and allowed to stand for 5 min for absorption. Using sterile forceps, antibiotic susceptibility testing strips, which had been removed from a 4°C freezer and equilibrated at room temperature for 30 minutes, were affixed to the surface of MRS-L solid plates. One blank susceptibility testing strip and three identical strips were placed on each plate as parallel plates. The plates were then incubated at 37°C for 24–48 hours in an anaerobic workstation. The diameter of the inhibition zone (mm) was measured using calipers, accurate to 0.1 mm, with the completely transparent edge of the inhibition zone as the standard. The experimental results are shown in Table 2, where S indicates sensitivity, I indicates intermediate resistance, and R indicates resistance.

[0090] Table 2. Inhibition zones and drug susceptibility testing of different antibiotics on bacterial strains.

[0091] This shows that the drug susceptibility spectrum of strains B-1 and B-6 is basically consistent with that of commercial BB12: sensitive to vancomycin, erythromycin, chloramphenicol, ampicillin, and clindamycin; inherently resistant to gentamicin, kanamycin, streptomycin, and ciprofloxacin; strain B-1 is resistant to tetracycline, and strain B-6 is intermediately resistant to tetracycline, with no acquired resistance mutations and no risk of horizontal transmission of resistance genes.

[0092] In summary, strains B-1 and B-6 do not cause hemolysis, do not produce bioamines or nitrites, and their drug resistance indicators meet the national standards for food strain safety, making them safe for use in the development of food, feed, and pharmaceutical products.

[0093] Example 7 This embodiment is used to detect the surface hydrophobicity and self-aggregation properties of the bacterial strain. Both *Bifidobacterium animalis* subsp. *lactam* B-1 and B-6 strains exhibit good surface hydrophobicity and self-aggregation ability. Strong hydrophobicity helps the bacteria adhere to the intestinal mucosa, reducing the probability of them being flushed out by intestinal contents; good self-aggregation promotes bacterial aggregation, enhancing the stability and tolerance of the bacteria in the gastrointestinal environment.

[0094] 7.1 Hydrophobicity test of bacterial cells: The commercial BB12 strain was used as a control. The strain was cultured in MRS-L liquid medium at 37℃ for 12-24 hours. The obtained fermentation broth was centrifuged at 6000 rpm for 5 minutes, the supernatant was discarded, and the broth was washed twice with physiological saline and resuspended. The OD of the bacterial suspension was adjusted. 600 To a value of 0.5 ± 0.02. Take 3 mL of bacterial culture and add 2 mL of n-hexane and xylene respectively, vortex for 2 min, let stand at room temperature for 0 h and 0.5 h, and then measure the OD of the lower aqueous phase. 600 Three parallel samples were prepared for each group. The formula for calculating hydrophobicity is: Hydrophobicity (%) = (1 - OD) / (1 - OD) t / OD0)×100%, OD t OD after standing for t hours 600 Value, OD0 is the OD value after standing for 0 hours. 600 Values, experimental results are shown in Figure 8 .

[0095] Experimental results: In the n-hexane system, the hydrophobicity of strain B-1 was 53.21%, that of strain B-6 was 52.18%, and that of BB12 was 52.63%; in the xylene system, the hydrophobicity of strain B-1 was 64.93%, that of BB12 was 51.75%, and that of strain B-6 was 42.01%.

[0096] 7.2 Autoaggregation assay: The commercial BB12 strain was used as a control. The strain was cultured in MRS-L liquid medium at 37℃ for 12-24 hours. The resulting fermentation broth was centrifuged at 6000 rpm for 5 minutes, the supernatant was discarded, and the broth was washed twice with physiological saline and resuspended. The OD of the bacterial suspension was adjusted. 600 To 0.5 ± 0.02. Take 5 mL of bacterial suspension and place it in an anaerobic environment at 37℃. Take samples at 0 h, 4 h, 8 h, and 16 h to measure the OD of the upper layer. 600 The formula for calculating the self-condensation rate is: Self-condensation rate (%) = [(OD0 - OD200)] t ) / OD0]×100%, OD t OD after standing for t hours 600 Value, OD0 is the OD value after standing for 0 hours. 600 Values, experimental results are shown in Figure 9 .

[0097] Experimental results showed that after 4 hours of static incubation, the autoaggregation rates of strains B-1 (49.58%) and B-6 (52.25%) were higher than those of BB12 (38.16%). After 8 hours of static incubation, the autoaggregation rates of strains B-1 (80.81%), B-6 (83.00%), and BB12 (76.83%) were all observed. After 16 hours of static incubation, the autoaggregation rates of all three groups were approximately 90%. This indicates that the early aggregation performance of strains B-1 and B-6 in this application is superior to that of commercial strains, and their overall colonization potential is excellent.

[0098] Example 8 This embodiment investigates the optimal growth temperature of the strain. Using commercially available BB12 as a control, a 10-generation subculturing experiment was conducted. The genetic stability of the strain was evaluated through growth curve monitoring and plate viable counts. The optimal growth temperature range for the strain is 36℃~42℃.

[0099] The experimental procedure included: inoculating the bacterial strain from an MRS-LM streak plate into 5 mL of MRS-L liquid medium, designated as generation 1; subsequent passages were continuously subcultured at a 4% inoculum volume, for a total of 10 generations. Fresh bacterial culture from each generation was aliquoted into 100-well plates compatible with the automated growth curve analyzer, with 250 μL added to each well, 5 replicates per group. The sealed plates were placed in the plate slots of the automated growth curve analyzer for OD (Organizational Deposition) analysis. 600 Continuous measurement. The incubation temperature was set to 37℃, and OD was measured every 30 minutes. 600 The plates were oscillated for 15 seconds before and after each test. Additionally, the 3rd and 10th generations were selected, and plate viable counts were performed every 2 hours according to GB 4789.35-2023 National Food Safety Standard for Microbiological Examination of Food (Lactic Acid Bacteria Examination). Detailed experimental results can be found in [link to experimental results]. Figure 10(Data from generations 11 to 20 are not shown). Among them, the online growth curve detection results of strains B-1 and B-6 from generation 1 to generation 10 show the growth trend, logarithmic phase onset time, and maximum OD. 600 The numerical values ​​showed no significant attenuation or shift; the logarithmic phase of the strain was concentrated between 2 and 8 hours, and it entered the stationary phase after 10 to 12 hours of culture, with the peak OD value... 600 Approximately 1.2, indicating that the genetic traits of both strains are stable. Plate viable count results: The viable counts of strains B-1 and B-6 during the plateau phase were consistently significantly higher than those of the control strain BB12; the viable count of strain B-1 during the third generation stable phase reached 1×10⁻⁶. 9 CFU / mL, still maintained at 5×10⁻⁶ after ten generations. 8 CFU / mL; the viable counts of B-6 in the third and tenth generation stable phases were 5 × 10⁻⁶. 8 CFU / mL and 3×10 8 CFU / mL. In contrast, the viable cell count of BB12 at the plateau phase of both the third and tenth generations was lower than that of the strain described in this application. This indicates that strains B-1 and B-6 not only exhibit stable growth performance during subculturing, but also consistently demonstrate superior viable cell counts compared to the commercial strain BB12, thus possessing greater potential for industrial fermentation.

[0100] Example 9 In this embodiment, in vivo efficacy experiments were conducted using a zebrafish juvenile constipation model and a BALB / c mouse constipation model. The laxative activity of strains B-1 and B-6 was verified from multiple dimensions, including intestinal emptying, neurotransmitters, growth indicators, defecation status, and small intestinal propulsion, in order to clarify the characteristics of the two strains.

[0101] 9.1 Validation of the Zebrafish Constipation Model: Using 5-day-fleshed (dpf) zebrafish juveniles, a constipation model induced by loperamide hydrochloride was established. The effects of strains B-1 and B-6 on improving intestinal function were evaluated from two dimensions: intestinal emptying function (Nile Red staining) and neurotransmitter levels (5-HT content). Specifically, healthy zebrafish juveniles developed to 5 dpf (day 5 post-fertilization) were selected and subjected to a constant temperature cycle of 28℃, 14h light / 10h darkness, without feeding throughout the experiment. The zebrafish were randomly divided into four groups: control group, model group, B-1 treatment group, and B-6 treatment group, with 15 fish in each group and three biological replicates. The control group was kept in RO water throughout the experiment without any modeling treatment. The model group used loperamide hydrochloride (10 μg / mL) to induce a constipation model without strain intervention. After modeling with loperamide hydrochloride, the B-1 and B-6 treatment groups were treated with strains B-1 and B-6, respectively.

[0102] The specific processing procedure is as follows: (1) Modeling stage (16h~17h): The model group, B-1 treatment group and B-6 treatment group were placed in a solution containing loperamide hydrochloride; the control group was placed in RO water.

[0103] (2) Maintenance of modeling stage (7h~8h): The model group, B-1 treatment group and B-6 treatment group were replaced with fresh loperamide hydrochloride solution; the control group was replaced with RO water.

[0104] (3) Intervention / Recovery Phase (16h~17h): The control group was replaced with RO water; the model group was rinsed with RO water 3 times and then replaced with RO water; the B-1 treatment group and the B-6 treatment group were rinsed with RO water 3 times and then replaced with a bacterial suspension containing the corresponding strain (1×10⁻⁶). 5 Intervention was carried out using CFU / mL.

[0105] Nile Red Staining: Nile red is a lipophilic fluorescent dye that can label lipid substances in the intestine. Higher fluorescence intensity indicates more retained contents and poorer intestinal emptying function. This application uses Nile red staining to evaluate the intestinal emptying function of zebrafish, including: 8 hours into the intervention phase, Nile red working solution was added to the solutions of each group to a final concentration of 0.5 μg / mL, and the mixture was soaked in the dark for 2 hours to allow the dye to enter the intestine and label the intestinal contents. After staining, the mixture was washed three times with RO water to remove residual dye from the body surface, and then the solution was replaced with the corresponding group's solution for another 8 hours of soaking. Afterward, the Nile red fluorescence signal in the zebrafish intestine was observed using a fluorescence microscope (excitation wavelength 488 nm, emission wavelength 570 nm), and images were acquired. Image analysis software was used to quantitatively analyze the fluorescence intensity in the intestine. With the fluorescence intensity of the model group as 100%, the fluorescence intensity of the intestinal contents in each group relative to the model group was calculated to reflect the intestinal emptying rate. Results are shown below. Figure 11 The results showed that the relative fluorescence intensity of the B-1 treatment group was 76.22%, significantly lower than both the model group (100%) and the normal control group (82.24%), indicating that strain B-1 could not only reverse the emptying inhibition caused by loperamide but also further enhance intestinal peristalsis, with a better emptying effect than normal levels. The relative fluorescence intensity of the B-6 treatment group was 78.69%, also significantly lower than both the model group and the control group, demonstrating good emptying-promoting activity. Therefore, both strains B-1 and B-6 have good potential to improve intestinal function.

[0106] 5-HT Content: 5-HT is a key regulator of intestinal peristalsis, with approximately 90% synthesized in the intestine. It promotes intestinal smooth muscle contraction and accelerates emptying of contents by activating the myenteric plexus. This application determined the serotonin (5-HT) content, including: after the strain intervention, zebrafish from each group were collected, washed three times with RO water to remove residual fluid from the body surface, and then stored in pre-cooled PBS buffer at -20°C. The zebrafish samples were processed according to the 5-HT detection kit instructions, and the 5-HT content was determined using enzyme-linked immunosorbent assay (ELISA). Results are shown below. Figure 12 .Depend on Figure 12 It can be seen that the 5-HT content in the B-1 treatment group was 0.294 μg / μL, which was not only significantly higher than that in the model group, but also higher than that in the normal control group. This indicates that the B-1 strain can effectively reverse the decrease in 5-HT levels caused by the constipation model induced by loperamide hydrochloride, and even has an upregulatory effect exceeding the baseline level. The 5-HT content in the B-6 treatment group was 0.199 μg / μL, which was also significantly higher than that in the model group and close to that in the normal control group, showing a good regulatory effect.

[0107] The above results indicate that both strains B-1 and B-6 can effectively upregulate loperamide-induced 5-HT levels in zebrafish, improving intestinal neurotransmitter imbalance. Combined with the Nile Red staining emptying rate experiment results, this suggests that both strains can promote intestinal peristalsis by upregulating 5-HT levels, thereby improving intestinal emptying disorders in a constipation model and possessing the potential for alleviating constipation symptoms.

[0108] 9.2 Validation of mouse constipation model: The intervention effects of Bifidobacterium strains B-1 and B-6 on mice with constipation induced by loperamide hydrochloride were systematically validated. The analysis was carried out from three core dimensions: basal growth status, defecation function, and intestinal motility, providing experimental evidence for the in vivo laxative and probiotic properties of the strains.

[0109] 9.2.1 Experimental Methods: Male BALB / c mice (initially 6 weeks old) with an initial weight of 20±4g were used as the research subjects. After a 7-day acclimatization period, they were randomly divided into 5 groups (n=10) for a 14-day modeling and intervention period. The experimental protocol is shown in Table 3. During this period, the mice were administered loperamide hydrochloride twice daily by gavage (10 mg / kg for loperamide hydrochloride, 5 mg / kg for domperidone, and 1×10 mg / kg for B-1 / B-6). 10 The mice were monitored regularly for CFU / kg, and their body weight, food intake, and water consumption were recorded. Defecation time, number of fecal particles, and fecal weight were also monitored regularly. After a small intestinal motility test on day 15, the mice were euthanized and dissected for sampling, followed by physiological and biochemical analysis.

[0110] Table 3 Grouping and Intervention Programs

[0111] 9.2.2 Basic experimental indicators: During the experiment, the body weight, 24-hour food intake, and water intake of mice in each group were monitored regularly. The results are as follows: Figure 13 The results showed that all mice survived the 14-day intervention period, with no abnormalities in weight or food intake, and no deaths or acute poisoning. This indicates that the high-dose, short-term continuous oral administration of B-1 / B-6 strains demonstrated good safety and no acute toxicity. Initially, there was no significant difference in body weight among the groups, with consistent baseline levels. During the experiment, the body weight of all groups showed a slow growth trend; the model group showed slightly slower weight gain than the control group. The weight change trends of the B-1, B-6, and DPLT groups were not significantly different from those of the control and model groups, indicating that the strain intervention did not adversely affect the growth of the mice. Monitoring of food and water intake showed that the dietary behavior of the mice fluctuated over time, but remained within the normal physiological range, with no statistically significant differences between groups. These results demonstrate that strains B-1 and B-6 have good safety at the experimental doses and did not negatively affect the growth and dietary behavior of the model mice.

[0112] 9.2.3 Determination of defecation time and number of fecal particles: The first black stool test was conducted on days 7 and 14 of the experiment. The time of first black stool expulsion and the number of black stool particles within 5 hours were recorded for each group of mice. The experimental method was as follows: Except for the control group mice which were administered 0.2 mL of PBS buffer by gavage, all other groups were administered 0.2 mL of loperamide hydrochloride by gavage. One hour later, the control and model groups were administered 0.2 mL of ink by gavage; the bacterial strain treatment group and the positive drug group were administered 0.2 mL of ink containing the corresponding test substance by gavage. After ink administration, mice were placed individually in metabolic cages with a tray lined with white filter paper underneath. Timing was recorded from the start of ink administration, recording the time of the first black stool expulsion for each mouse, and the total number of black stool particles within 5 hours after the first black stool. The results are as follows: Figure 14 .

[0113] Results on the time to first black stool excretion: The time to first black stool excretion in the model group on day 7 (127 min) and day 14 (124 min) was significantly longer than that in the control group (56.5 min / 51.5 min), indicating that loperamide hydrochloride successfully inhibited intestinal peristalsis in mice, and the constipation model was effectively established. Furthermore, the excretion time in the model group remained almost unchanged on days 7 and 14, indicating that the constipation model remained stable throughout the experimental period. The B-1 treatment group had an excretion time of 95 min on day 7, approximately 25% shorter than the model group (127 min), comparable to the DPLT group (92 min); on day 14, the excretion time decreased to 74 min, significantly shorter than the model group (124 min) and also significantly shorter than the DPLT group (105 min), indicating that the improvement effect of strain B-1 became increasingly significant with prolonged intervention, exceeding that of positive control drugs. The time to first melena in the B-6 treatment group on day 7 was 115 minutes, slightly shorter than that in the model group (127 minutes), indicating a weaker improvement. On day 14, the time decreased to 92.5 minutes, approximately 26% shorter than that in the model group (124 minutes), showing a significantly enhanced improvement. This indicates that the effect of strain B-6 also increases with the duration of intervention. These results demonstrate that both strains B-1 and B-6 of this application can effectively shorten the time to first melena in constipation model mice and promote intestinal peristalsis.

[0114] Fecal pellet count results: On day 7, the number of black stool pellets over 5 hours did not differ significantly among the groups, indicating that the constipation model's initial effect was mainly a prolonged defecation time. However, as the intervention progressed (to day 14), the decrease in stool volume caused by constipation became more pronounced, with a significant reduction in the number of black stool pellets in each group, the model group showing the greatest reduction. Both strains B-1 and B-6 in the experimental groups improved stool volume. On day 7, the B-1 treatment group had 217 pellets, with no significant difference from the model and control groups; on day 14, the number was 118 pellets, slightly higher than the model group (101 pellets), but comparable to the control group (117 pellets), indicating that strain B-1 could improve the reduced stool volume caused by the model, restoring it to normal levels. On day 7, the number of particles in the B-6 treatment group was 204, which was not significantly different from other groups; on day 14, the number of particles was 134, which was significantly higher than that in the model group (101 particles) and the control group (117 particles), indicating that the B-6 strain can not only reverse the reduction in defecation volume caused by the model, but also significantly increase the amount of defecation within 5 hours.

[0115] In summary, both strains B-1 and B-6 of this application exhibit certain functional characteristics in promoting bowel movements. The B-1 group showed a significantly shorter time to first black stool, and the number of black stool particles within 5 hours returned to normal, indicating improvements in intestinal motility and stool volume. The B-6 group showed a shorter time to first black stool, but a significantly increased number of black stool particles within 5 hours, indicating a stronger promoting effect on intestinal motility, with stool volume recovering to or even exceeding normal levels. Furthermore, the improvement effects of both strains B-1 and B-6 were more pronounced on day 14, suggesting that the effects of the strains increase with the duration of intervention, consistent with the characteristic of probiotics exerting their effects by regulating the intestinal microecology.

[0116] (4) Determine the intestinal propulsion rate of mice On day 15 of the experiment, a small intestinal motility test was conducted to detect the ink propulsion rate of mice in each group. Ink preparation: Accurately weigh 100g of gum arabic, add 800mL of water, and boil until the solution is clear. Weigh 50g of activated charcoal (powder) and add it to the above solution, then boil three times. After the solution cools, add water to make up to 1000mL, store in a refrigerator at 4℃, and shake well before use.

[0117] Mice were fasted for 12 hours before the experiment, but allowed free access to water. At the start of the experiment, except for the control group, all other groups of mice were gavaged with loperamide hydrochloride solution to induce the model. One hour later, each group of mice was gavaged with 0.2 mL of ink suspension containing the corresponding test substance (ink for the control and model groups, and ink suspension containing the corresponding test substance for the test strain and DPLT groups). Thirty minutes after gavage, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg), blood was collected from the eyeballs, and the mice were euthanized by cervical dislocation. The abdominal cavity was quickly opened, and the small intestine from the pylorus to the ileocecal junction was separated and cut off. After being gently straightened, the total length L0 of the small intestine (pylorus to ileocecal junction) and the length L of the ink propelled from the tip were measured. x (From the pylorus to the ink front), calculate the ink propulsion rate in the small intestine using the following formula: Small intestine propulsion rate (%) = L x / L0×100%. The result is as follows: Figure 15 and Figure 16 The results showed that the propulsion rate of the model group (46.39%) was significantly lower than that of the control group (92.43%), indicating that loperamide had a significant inhibitory effect on small intestinal peristalsis; the propulsion rate of the DPLT group (59.23%) was significantly higher than that of the model group (46.39%), indicating that the positive control drug could improve small intestinal peristalsis; the propulsion rate of the B-1 group was 63.23%, significantly higher than that of the model group (46.39%), and also higher than that of the DPLT group (59.23%); the propulsion rate of the B-6 group was 47.91%, only slightly higher than that of the model group (46.39%), indicating that the B-6 strain had a limited promoting effect on small intestinal peristalsis.

[0118] In summary, strain B-1 effectively and stably reversed the inhibition of small intestinal motility by loperamide, with its effect even showing a trend of being superior to the positive control drug. Combined with the first black stool test, strain B-1 simultaneously shortened defecation time and increased stool volume, indicating that it comprehensively improves the peristaltic function of both the small and large intestines, possibly exerting its effect through regulating intestinal smooth muscle contraction and promoting neurotransmitter release. On the other hand, strain B-6 had almost no direct promoting effect on small intestinal motility. However, in the first black stool test, strain B-6 significantly increased the number of black stool particles after 5 hours, suggesting that the action site of strain B-6 may be more biased towards the large intestine stage, promoting defecation by regulating water absorption and improving stool characteristics, rather than directly accelerating small intestinal motility. In other words, strain B-1's function is more inclined towards promoting intestinal motility, while strain B-6's function is more inclined towards regulating fecal excretion.

[0119] Example 10 This example is used to study the mechanism of laxative effect of strains B-1 and B-6 in the mouse experiment of Example 9.

[0120] 10.1 Increasing 5-HT levels in the colon of constipated mice: Enzyme-linked immunosorbent assay (ELISA) was used to quantitatively detect 5-HT levels in colonic tissue to investigate the potential mechanisms by which strains B-1 and B-6 improve constipation. 5-HT levels in mouse colonic tissue were detected using the Mouse 5-HT ELISA KIT kit from Shanghai Langton Biotechnology Co., Ltd. The results are shown below. Figure 16 The results showed that the colonic 5-HT content in the model group mice was 22.22 μg / μL, significantly lower than the 56.00 μg / μL in the control group, indicating that loperamide successfully inhibited colonic 5-HT levels, consistent with the slowed intestinal peristalsis. The 5-HT content in the B-1 treatment group was 67.53 μg / μL, significantly higher than the model and control groups, suggesting that strain B-1 can strongly upregulate colonic 5-HT levels, and its high standard deviation indicates large individual differences in response; the 5-HT content in the B-6 treatment group was 45.51 μg / μL, also significantly higher than the model group, and close to the DPLT group level. Combining the results of the small intestinal ink propulsion rate and the first black stool experiment, it is speculated that strain B-1 comprehensively promotes small and large intestinal peristalsis by significantly upregulating colonic 5-HT levels; while strain B-6 mainly improves large intestinal function by moderately upregulating colonic 5-HT levels, with a weaker direct promoting effect on small intestinal peristalsis. The above results indicate that both strains of bacteria can relieve constipation by regulating intestinal 5-HT levels.

[0121] 10.2 Repair of Cellular and Intestinal Barrier Damage: The pathophysiological process of functional constipation is not only characterized by intestinal motility disorders, but also accompanied by significant damage to the intestinal mucosal barrier and abnormal cell structure. Prolonged retention of feces in the colon can lead to mechanical damage to the colonic mucosa, goblet cell dysfunction, and downregulation of tight junction protein expression, thereby disrupting the intestinal mucus barrier and tight junction barrier, exacerbating constipation symptoms. This experiment aimed to verify the repair effects of Bifidobacterium animalis strains B-1 and B-6 on colonic structural damage and intestinal barrier function in constipated mice, providing histomorphological evidence for their probiotic mechanism in improving constipation. Specifically, after euthanasia of mice, distal colonic tissue was rapidly separated, rinsed thoroughly with pre-cooled PBS buffer, and intestinal contents were removed. The colonic tissue blocks were fixed in 4% paraformaldehyde solution for 24 h, followed by conventional gradient ethanol dehydration, xylene clearing, and paraffin embedding. Finally, continuous colonic tissue sections with a thickness of 5 μm were prepared using a paraffin microtome and dried for later use.

[0122] 10.2.1 H&E Staining (Observation of Cell Morphology and Tissue Structure): Prepared colon tissue sections were dewaxed to water and then stained with hematoxylin-eosin (HE) staining solution: hematoxylin staining for 5 min, followed by rinsing with water; differentiation with 1% hydrochloric acid alcohol for 30 s, followed by blueing for 5 min; staining with eosin for 1 min, and then dehydrating with graded ethanol, clearing with xylene, and mounting with neutral resin. The crypt morphology, mucosal thickness, tissue structure integrity, and inflammatory cell infiltration of the colon tissue of each group of mice were observed using an optical microscope. The H&E staining results are shown in the figure below. Figure 17 In the control group, the colon structure of mice was intact, with a regular star-shaped intestinal lumen, abundant mucosal folds, neatly arranged crypts, and sufficient goblet cells, showing no obvious inflammatory infiltration. In the model group, the intestinal lumen showed significant eccentric dilation, marked fecal retention, disappearance of mucosal folds and disordered crypts on the compressed side, accompanied by extensive inflammatory cell infiltration and severe mucosal damage. In the DPLT group, intestinal dilation and fecal retention were significantly improved compared to the model group; mucosal folds and crypt structures partially recovered, and inflammatory infiltration was reduced, but not completely restored to normal. In the B-1 group, the colon morphology was similar to the control group, with a regular intestinal lumen, abundant and intact folds, and good crypt and goblet cell structure, with only a few scattered inflammatory cells, and the tissue damage was basically repaired. In the B-6 group, the colon structure was improved compared to the model group; intestinal dilation was reduced, folds and crypts were more regularly arranged, and inflammatory infiltration was reduced. Therefore, compared with the control group, the model group showed obvious intestinal dilation, eccentric fecal retention, and mucosal damage with inflammatory infiltration in the colon; the colonic structure was improved to varying degrees after intervention with positive drugs, B-1 strain, and B-6 strain.

[0123] 10.2.2 AB-PAS staining (observation of goblet cells and mucus barrier): Colon tissue sections were stained with AB-PAS. After dewaxing to water, Alcian Blue (AB) staining solution was added for 15 min, followed by washing with water. Periodic acid (PAS) staining solution was then added for 15 min, followed by washing with water. Schiff's reagent was used for development for 15 min. The sections were then dehydrated with a series of ethanol solutions, cleared with xylene, and mounted with neutral resin. The number of goblet cells, the depth of mucin staining, and the distribution of the mucus barrier were observed under a light microscope. The AB-PAS staining results are shown in [Figure number missing]. Figure 17 , Figure 17 The blue / blue-purple signal represents the mucin secreted by goblet cells and is a core indicator for evaluating intestinal mucus barrier function. Results showed that in the control group, mice had sufficient and densely distributed goblet cells in the colonic crypts, with strong and deep blue-purple staining signals and a continuous and intact mucus layer, indicating normal intestinal mucus barrier function. In the model group, the number of goblet cells was significantly reduced, the blue-purple staining signal was significantly weakened and sparsely distributed, and a large area of ​​goblet cell absence was visible in the left mucosal layer, indicating severe disruption of mucus layer continuity, suggesting that loperamide-induced constipation can lead to severe damage to intestinal mucus barrier function. In the DPLT group, the number of goblet cells was significantly increased compared to the model group, the blue-purple staining signal was significantly enhanced, and the mucus layer continuity was significantly improved compared to the model group, but not completely restored to the control level. In the B-1 treatment group, the number of colonic goblet cells was significantly increased compared to the model group, the blue-purple staining depth and distribution uniformity were significantly improved, and the integrity of the mucus barrier was significantly restored, suggesting that strain B-1 can effectively promote goblet cell mucin secretion and repair constipation-induced intestinal mucus barrier damage, with a repair effect comparable to the positive control drug domperidone. The number of goblet cells and the intensity of blue-purple staining signal were increased in the B-6 treatment group compared with the model group, and the continuity of the mucus layer was improved, suggesting that the B-6 strain also has a certain repair effect on the mucus barrier.

[0124] 10.2.3 Immunofluorescence detection of intestinal mucosal barrier-related proteins: MUC2 is a core mucin secreted by colonic goblet cells, constituting the intestinal mucus layer and maintaining the integrity of the chemical barrier; Claudin-1 / Occludin / ZO-1 are all tight junction proteins that maintain the integrity of the intestinal physical barrier. Sections of colonic tissue were dewaxed to water and antigen retrieval was performed. Primary antibodies against mucin Muc2 and tight junction proteins Occludin / Claudin / ZO-1 were added and incubated overnight at 4°C. After washing, secondary fluorescent antibody was added, and the nuclei were stained with DAPI. After mounting, the slides were observed using a laser confocal microscope, and the fluorescence intensity was quantitatively analyzed using ImageJ software to reflect the expression levels of intestinal barrier-related proteins. The results are shown in [Figure number missing]. Figure 18The results showed that, compared with the control group, the fluorescence signal intensity of MUC2 protein in the colon tissue of the model group mice was significantly reduced and its distribution was disordered, with large areas of signal loss in the crypt region, indicating a reduction in the number of goblet cells and impaired mucus secretion function. The fluorescence signals of tight junction proteins Claudin-1, Occludin, and ZO-1 were also significantly weakened, with disrupted signal continuity and disordered intercellular junction structures, indicating that the model group mice suffered dual damage to both the mucus barrier and the epithelial barrier. After treatment with the positive drug DPLT, the expression levels of the above proteins in the mouse colon significantly recovered. Specifically, the expression level of MUC2 was significantly upregulated compared with the model group (relative expression level 1.34), and significantly higher than that of the control group. The fluorescence signal in the crypt region recovered to be continuous and uniform, indicating a significant improvement in goblet cell function. The expression levels of Claudin-1, Occludin, and ZO-1 also recovered significantly compared with the model group, with a more continuous and complete signal distribution, indicating effective repair of the intestinal epithelial barrier function. The B-1 treatment group showed a similar repair effect to the DPLT group. The relative expression level of MUC2 protein in this group was 1.31, which was not significantly different from that in the DPLT group. The fluorescence signal was widely distributed in the crypt region, and the number of goblet cells and mucus secretion function were well restored. The expression levels of Claudin-1 and ZO-1 were 1.05 and 1.16, respectively, slightly higher than the control group and significantly higher than the model group. The signal distribution was continuous and regular, and the integrity of the epithelial barrier was effectively restored. The expression level of Occludin also increased significantly compared with the model group, reaching 0.83, which was comparable to the level in the DPLT group. The relative expression level of MUC2 protein in the B-6 group was 1.17, which was higher than that in the control group, but significantly lower than that in the B-1 and DPLT groups. The expression levels of Claudin-1 and ZO-1 were 0.97 and 1.07, respectively, which recovered to levels close to those in the control group. The expression level of Occludin was 0.88, slightly higher than that in the model group, but still lower than that in the B-1 and DPLT groups. The fluorescence imaging results were consistent with the quantitative data. Although the signal intensity of the B-6 group was higher than that of the model group, the signal continuity between the crypt apex and epithelial cells was still weaker than that of the B-1 and DPLT groups. These results indicate that treatment with strain B-1 significantly improved intestinal barrier damage, and its repair effect on the mucus barrier and tight junctions was comparable to that of the positive drug DPLT, demonstrating good intestinal barrier protection. Treatment with strain B-6 could, to some extent, improve barrier damage in the model group.

[0125] Example 11 Fresh fecal samples were collected from mice in each group in Example 9, immediately flash-frozen in liquid nitrogen, and then stored at -80°C. The samples were sent to Meiji Biotechnology for 16S rRNA high-throughput sequencing and microbial diversity analysis. Alpha diversity analysis, β diversity analysis, and species composition analysis were performed on the results to systematically evaluate the regulatory effects of the strains on the structure and homeostasis of the intestinal microbial community in constipated mice. The results are shown in […]. Figures 19-21 .

[0126] The alpha diversity index was used to assess the richness and diversity of the gut microbiota in each group of mice. Sequencing results showed that the coverage index of each group was greater than 0.98, indicating good coverage of the sequencing data, which could reflect the true composition of the microbiota in the samples, and the differences between groups were significant (P=0.00535). Ace index analysis showed that the microbiota richness of the DPLT group was significantly higher than that of the control group (P<0.05), and the richness of the model group, B-1 group, and B-6 group was also higher than that of the control group (P=0.0338). Shannon index results showed that the microbiota diversity of the model group was lower than that of the control group, the DPLT group recovered to near normal levels, and the diversity of the B-1 group and B-6 group was higher than that of the model group, with the B-1 group showing the best performance. Simpson index results showed that the index of the model group was significantly higher than that of the control group (P<0.05), indicating uneven distribution of the microbiota; the indices of the DPLT group, B-1 group, and B-6 group were all significantly reduced, indicating improved microbiota evenness, with the DPLT group showing the best performance, followed by the B-1 group. The above results indicate that Bifidobacterium animalis subsp. lactis B-1 strain can effectively restore the diversity of intestinal flora in constipated mice and improve the imbalance of the microecological system.

[0127] Principal component analysis (PCA) was used to analyze the β-diversity of the gut microbiota in each group of mice. PCA at the OTU level showed that principal component 1 and principal component 2 explained 13.24% and 10.46% of the microbiota structure variation, respectively, for a combined explained rate of 23.7%. PermanoVA analysis indicated highly significant differences in microbiota structure among the groups (R0.05). 2 =0.50139, P=0.001). The control and model samples were clearly separated, indicating a significant change in the gut microbiota structure after modeling. The microbiota structure of the B-1 treatment group partially overlapped with that of the model group, while the B-6 treatment group formed independent clusters, suggesting that B-6 has a significant regulatory effect on the gut microbiota structure. The DPLT group samples were more dispersed, with greater intra-group variability.

[0128] like Figure 21As shown in Figure A, at the phylum level, the gut microbiota of mice in each group mainly consisted of Firmicutes and Bacteroidetes. Compared with the control group, the abundance of Firmicutes was significantly increased and the abundance of Bacteroidetes was significantly decreased in the model group, and the Firmicutes / Bacteroidetes ratio was significantly increased, indicating a significant dysregulation of the gut microbiota structure. After treatment with DPLT, B-1, and B-6, the abundance of Firmicutes showed a decreasing trend, while the abundance of Bacteroidetes showed a recovering trend. Among them, the B-6 group showed the most significant regulatory effect, and its microbiota structure was closer to that of the control group. Figure 21 As shown in Figure B, at the genus level, *Lactobacillus* is the dominant genus in each group. The abundance of *Lactobacillus* was significantly increased and the diversity of the gut microbiota decreased in the model group; the abundance of *Lactobacillus* was significantly decreased in all treatment groups, and the microbiota structure was closer to the "diversity distribution" of the control group, with the B-6 group showing the most significant recovery in microbiota structure. These results indicate that the B-6 treatment of this application can effectively regulate the composition of the intestinal microbiota and improve the dysbiosis induced by the model. In summary, the *Bifidobacterium animalis* subsp. *lactamase* B-1 and B-6 strains of this application can improve constipation by regulating intestinal microecological imbalance. Specifically, this manifests as effectively improving the aforementioned microbiota imbalance, restoring microbiota richness and diversity, reconstructing the intestinal microbiota structure, and regulating the abundance of key phyla and genera.

[0129] Example 12 To investigate the effects of *Bifidobacterium lactis* strains B-1 and B-6 on the metabolism of short-chain fatty acids (SCFAs) in the intestines of constipated mice, targeted metabolomics was used to quantitatively analyze eight SCFAs in feces. Fresh fecal samples from mice in each group (Example 9) were collected, flash-frozen in liquid nitrogen, and stored at -80°C. The samples were then sent to Meiji Biotechnology for analysis, and the results are shown below. Figure 22 and Figure 23 .

[0130] Compared with the control group, the levels of acetic acid, propionic acid, butyric acid, and total SCFAs in the feces of mice in the model group were significantly reduced, with the total SCFAs content decreasing by about 30%, indicating that the intestinal flora's ability to ferment and produce SCFAs was impaired under constipation, leading to metabolic homeostasis imbalance. Intervention with the positive control drug DPLT significantly reversed these changes, resulting in a significant recovery of acetic acid, butyric acid, and total SCFAs levels compared to the model group. The total SCFAs content increased by about 52% compared to the model group, restoring levels close to or higher than those in the control group. Intervention with *Bifidobacterium animalis* subsp. lactis B-1 strain also significantly improved the abnormal SCFAs metabolism in the model group, with acetic acid, butyric acid, and total SCFAs levels increasing by about 35%, 41%, and 31% respectively compared to the model group, restoring levels indistinguishable from the control group. This indicates that strain B-1 can effectively promote intestinal SCFAs production and restore metabolic homeostasis. In comparison, strain B-6 showed a more significant improvement, with acetic acid, propionic acid, and total SCFAs levels increasing by approximately 72%, 86%, and 70%, respectively, compared to the model group. The total SCFAs content was significantly higher than in the control group and other treatment groups, suggesting that B-6 has a stronger promoting effect on intestinal SCFAs synthesis and can effectively restore intestinal metabolic function in constipated mice. In conclusion, both strains B-1 and B-6 can significantly improve SCFAs metabolic disorders in constipated mice.

[0131] Example 13 High-density culture was conducted in a 5L fermenter using the modified fermentation medium described in this application. First, seed culture of strain B-1, which had undergone two generations of continuous activation in MRS-L liquid medium, was prepared: activation conditions were 4% inoculum and incubation at 37℃ for 12-16 hours. Subsequently, the seed culture was inoculated into the fermenter at a 4% inoculum. Fermentation parameters included: fermentation temperature of 37℃, initial pH of 6.5, and a constant pH of 5.8 throughout fermentation. Fermentation broth was collected at 0h, 2h, 4h, 6h, 8h, 10h, 12h, and 14h, and viable cell counts were determined according to GB4789.34 National Food Safety Standard for Microbiological Examination of Food – Identification of Bifidobacteria. The 14h fermentation broth was collected for subsequent centrifugation, emulsification, and freeze-drying. The formulation of the high-density fermentation medium for strain B-1 in this application is as follows: glucose 15 g / L, galactooligosaccharides 5 g / L, yeast extract 30 g / L, potassium dihydrogen phosphate 1.8 g / L, disodium hydrogen phosphate 2.2 g / L, anhydrous sodium acetate 4.4 g / L, L-cysteine ​​0.4 g / L, magnesium sulfate 0.3 g / L, manganese sulfate 0.3 g / L, and Tween 80 0.5 g / L. A growth curve was plotted based on the measured viable cell count, and the results are shown below. Figure 24 .

[0132] The results showed that the growth rate and peak viable count of Bifidobacterium animalis B-1 were significantly improved in the modified liquid culture medium compared with the static fermentation in the ordinary MRS-L liquid culture medium. The viable count reached its peak and stabilized at approximately 5 × 10⁻⁶ after 8 hours of dynamic fermentation. 9 CFU / mL.

[0133] Example 14 14.1 Optimization of the freeze-drying protectant: The high-density fermentation process of Example 13 was used to prepare Bifidobacterium animalis subsp. lactis B-1 fermentation broth. After fermentation, the broth was centrifuged at 4°C and 6000 r / min for 20 min. Bottom bacterial sludge and part of the supernatant were collected. 25 g of supernatant was added to every 100 g of bacterial sludge to obtain a diluted bacterial sludge. The diluted bacterial sludge and freeze-drying protectant were mixed and emulsified at a ratio of 1:1.5 and then dispensed into sterile freeze-drying trays for freeze-drying. After freeze-drying, the bacterial powder was placed in sterile aluminum foil bags, and viable bacteria were counted according to GB4789.34 National Food Safety Standard for Microbiological Examination of Food: Identification of Bifidobacteria. The protectant composition was: 11 wt% skim milk powder, 8 wt% xylooligosaccharides, 3 wt% trehalose, 1 wt% mannitol, and 0.05 wt% L-cysteine ​​hydrochloride.

[0134] The freeze-drying process employed a segmented vacuum freeze-drying technique: In the pre-freezing stage, the temperature was lowered to -40°C at a rate of 0.5°C / min and held for 2 hours; in the first drying stage, the temperature was increased to -25°C at a rate of 0.5°C / min, with a vacuum of 150 μbar, and dried for 54 hours; in the second drying stage, the temperature was increased to 25°C at a rate of 0.2°C / min, with a vacuum of 30 μbar, and dried for 6 hours. The freeze-drying process yielded *Bifidobacterium animalis* subsp. *lactamase* B-1 freeze-dried bacterial powder. The B-1 freeze-dried bacterial powder prepared using the optimized freeze-drying process described in this embodiment achieved a viable count of 5 × 10⁻⁶. 11 The CFU / g is significantly higher than that of unoptimized conventional pure skim milk powder, with high bacterial survival rate and low freeze-drying loss rate.

[0135] 14.2. Stability of Bacterial Powder: The B-1 freeze-dried bacterial powder was stored in sealed containers at 4℃ (simulated closed refrigeration), 15℃ (simulated open refrigeration), 25℃ (simulated ambient temperature), and 37℃ (accelerated test). Samples were taken at regular intervals of 0, 1, 2, and 3 months to determine the viable cell count and calculate the survival rate, thus evaluating the storage stability of the bacterial powder. The test results are shown in Table 4. The B-1 bacterial powder was prepared using the freeze-drying protectant specified in this application, while the BB12 bacterial powder was purchased from the market. Both were packaged in the same aseptic aluminum foil bags using heat sealing.

[0136] Table 4. Viable cell count and survival rate of strains at different storage temperatures

[0137] As shown in Table 4, the self-made Bifidobacterium animalis subsp. lactis B-1 freeze-dried powder and the commercially available BB12 bacterial powder both exhibit good storage stability at storage and transportation temperatures of 4℃, 15℃, and 25℃. Under accelerated high-temperature conditions of 37℃, the viable cells of both strains showed a significant decrease. The difference in the storage performance of the bacterial powder is directly related to the tolerance characteristics of the strains themselves and the matching freeze-drying protectant system.

[0138] After 3 months of storage at 4℃ and 15℃, the survival rate of both strains was greater than 88%, demonstrating excellent activity retention under low-temperature conditions. At 4℃ with sealed storage, the survival rate of B-1 was higher than that of BB12 throughout the entire storage period. After 3 months of storage, the survival rate of B-1 was 92.86%, while that of BB12 was 88.20%. At 15℃, the difference narrowed, with the survival rates of B-1 at 93.21% and BB12 at 90.73% after 3 months of storage, though B-1 remained slightly higher than the control strain. At 25℃, the cell death rate slightly increased. After 1 month of storage, the survival rate of B-1 was 93.21%, slightly higher than that of BB12 (92.42%). After 3 months of storage, the survival rate of B-1 was 77.00%, while that of BB12 was 68.82%. Under these temperature conditions, the lyophilization protectant maintained a stable glassy framework, effectively protecting the cells and suitable for ambient temperature circulation. Under extreme high-temperature conditions of 37℃, the cell death rate significantly accelerated. After one month of storage, the survival rate of B-1 was 52.96%, and that of BB12 was 49.16%; after two months of storage, the survival rate of B-1 was 29.44%, and that of BB12 was 23.40%; after three months of storage, the survival rate of B-1 was 19.86%, and that of BB12 was 18.99%. High temperatures can destroy the glassy protective structure formed by the protective agent, causing the bacteria to lose their protective coating and the number of viable bacteria to decline rapidly. Throughout the accelerated storage period, the viable bacterial survival rate of B-1 was not lower than that of the control strain BB12.

[0139] In summary, strain B-1 combined with the freeze-drying protectant system of this application exhibits good viable bacteria retention capacity under cold chain storage conditions of 4℃~15℃. It is also suitable for short-term circulation at room temperature of 25℃, which can buffer temperature fluctuations during storage and transportation to a certain extent, delay the decay of probiotic viable bacteria, and help ensure the viable bacteria stability of the product during its shelf life.

[0140] Example 15 Fermented milk was prepared using the freeze-dried Bifidobacterium animalis subsp. lactis B-1 lyophilized bacterial powder prepared in Example 14 as the fermentation starter and fresh milk with a protein content of 3.0 as the substrate. White sugar (5%) was slowly added to the fresh milk (95%), and the mixture was stirred at 600 rpm for 5 minutes, then hydrated at 55°C for 30 minutes. After hydration, homogenization was performed (65°C, 20 MPa), followed by pasteurization (95°C, 5 minutes), and then cooled to 37°C for inoculation. The B-1 freeze-dried bacterial powder was inoculated at 0.01% (w / w) (inoculation amount was 10...). 6 CFU / g ~107 After stirring evenly, the mixture was fermented at constant temperatures of 37°C and 42°C until the acidity reached 70°T~80°T. Then, the emulsion was broken and the mixture was rapidly cooled to 4°C before being refrigerated for further maturation. Acidity, time, pH, flavor, and texture were recorded, and the results are shown in Table 5.

[0141] Table 5 B-1 Fermented Milk Parameter Records

[0142] The results showed that the *Bifidobacterium lactis* subsp. *species* from this application can participate in dairy product fermentation. The B-1 strain exhibited a higher acid production rate at 42℃ than at 37℃. Fermentation at 42℃ reached its optimal acidity in 7-8 hours, compared to 11-12 hours at 37℃. The fermentation time at 42℃ was shortened by approximately 40%, significantly improving production efficiency and making the fermentation temperature suitable for industrial production. The acidity increase during the ripening process was slow. Furthermore, after reaching the optimal acidity range, both strains maintained a viable count ≥1×10⁻⁶. 8 The CFU / g concentration increased 10-100 times compared to the initial inoculum. Strain B-1 exhibits a pure flavor with no off-flavors, possessing only a slight astringent taste characteristic of Bifidobacterium-fermented dairy products, without any unpleasant flavors. At 37°C, it has a uniform texture and moderate viscosity; at 42°C, only a small amount of fine particles appear, which does not affect product quality. It demonstrates good compatibility with dairy applications. Further development could allow for multi-stage temperature control of fermentation or be combined with texture-improving starter cultures such as those that secrete extracellular polysaccharides to provide better product manufacturing solutions. In summary, the strain described in this application can serve as an excellent fermentation strain for use as a single or compound starter culture in the industrial production of probiotic fermented dairy products, endowing the product with unique sensory qualities and a superior probiotic experience, demonstrating broad application prospects.

[0143] Although preferred embodiments of the present application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are limited to all changes and modifications that include the preferred embodiments and fall within the scope of the embodiments of the present application.

Claims

1. A microbial preparation, characterized in that, Contains live bacteria of *Bifidobacterium animalis* subsp. *lactamase* and / or its fermentation metabolites, wherein the *Bifidobacterium animalis* subsp. *lactamase* strain includes *Bifidobacterium animalis* subsp. *lactamase* strain (… Bifidobacterium animalis subsp .lactis B-1 and / or the Bifidobacterium animalis subsp. lactis strain is a Bifidobacterium animalis subsp. lactis strain ( Bifidobacterium animalis subsp .lactis )B-6.

2. The microbial preparation according to claim 1, characterized in that, The Bifidobacterium animalis subspecies Lactobacillus strain B-1 has the accession number CGMCC No.38186 and was deposited at the China General Microbiological Culture Collection Center on March 30, 2026.

3. The microbial preparation according to claim 1, characterized in that, The Bifidobacterium animalis subspecies Lactobacillus strain B-6 has the accession number CGMCC No.38187 and was deposited at the China General Microbiological Culture Collection Center on March 30, 2026.

4. The microbial preparation according to claim 1, characterized in that, The microbial preparation is a freeze-dried bacterial powder, and the viable count in the original freeze-dried bacterial powder is not less than 1×10⁻⁶. 10 CFU / g.

5. The microbial preparation according to claim 4, characterized in that, The freeze-dried bacterial powder is prepared by centrifuging a fermentation broth containing the Bifidobacterium lactis strain to obtain bacterial sludge, then mixing and emulsifying it with a freeze-drying protectant and freeze-drying it.

6. The microbial preparation according to claim 1, characterized in that, The microbial preparation is a composition comprising the Bifidobacterium lactis strain and a pharmaceutically or food-grade acceptable carrier. The composition is a food, health product, special medical food, pet food, livestock feed, or microbial drug, and the amount of strain added is 1×10⁻⁶. 4 CFU / g ~ 1×10 11 CFU / g.

7. The microbial preparation according to claim 1, characterized in that, The microbial preparation is a fermented dairy product, which is prepared by the following method: using fresh milk as a substrate, inoculating it with freeze-dried Bifidobacterium lactis strain 10 for fermentation, maintaining a constant temperature of 37°C~42°C until the acidity reaches 70°T~80°T, breaking the milk and cooling to 4°C, followed by refrigeration and maturation to obtain the fermented dairy product; wherein, the inoculation amount is 10 6 CFU / g ~10 7 CFU / g.

8. The microbial preparation according to claim 1, characterized in that, The *Bifidobacterium lactis* strain was obtained through high-density fermentation, including the following steps: The seed culture of the Bifidobacterium lactis strain was inoculated into a fermentation medium for fermentation. The fermentation medium contains: carbon source, nitrogen source, inorganic salts, buffer salts, water, and growth promoters; the fermentation temperature is controlled at 36℃~42℃, the initial pH is 6.5, and the pH is maintained at a constant 5.8 during fermentation until the viable cell count in the fermentation broth reaches 1×10⁻⁶. 9 CFU / mL ~ 1×10 10 CFU / mL.

9. The application of the microbial preparation according to any one of claims 1-8, characterized in that, The microbial preparation is formulated into a product for relieving constipation, promoting bowel movements, repairing intestinal barrier damage, regulating intestinal flora, and / or increasing the content of short-chain fatty acids in the intestine.

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