Composite probiotic, application thereof and composite probiotic powder

Abstract

The invention relates to the technical field of microecological preparations, in particular to a compound probiotic, application thereof and compound probiotic powder. The invention provides a lactobacillus acidophilus DH-La-166 beneficial to relaxing bowel, and the preservation number of the lactobacillus acidophilus DH-La-166 is CGMCC (China General Microbiological Culture Collection Center) No. 13077. The invention also provides a compound probiotic containing the bacterial strain. The compound probiotics can promote each other and cooperate with each other in the growth process, enhance intestinal peristalsis of constipation patients and regulate intestinal flora. Experiments show that the bacterial powder prepared from the compound probiotics can significantly improve the water content, the excrement discharge time, the excrement discharge amount and the intestinal tract movement speed of excrement of mice with constipation, so that the effect of treating constipation is achieved, the safety is high, side effects are avoided, a scientific basis and a technical path are provided for precise development of functional foods and pharmaceutical preparations, and the method is worthy of popularization and application. And a new effective means is provided for prevention and adjuvant therapy of constipation.

Description

Technical Field

[0001] This invention relates to the technical field, and more particularly to a compound probiotic, its application, and a compound probiotic powder. Background Technology

[0002] Constipation, a common digestive disorder, is experiencing a rising global incidence, particularly among the elderly, post-operative patients, and those with unbalanced diets. Current treatments primarily rely on stimulant laxatives (such as senna and magnesium sulfate), bulk-forming laxatives (such as dietary fiber), and lubricants (such as liquid paraffin). However, long-term use can lead to side effects such as drug dependence, electrolyte imbalance, intestinal nerve damage, and loose stools, and fails to address the core pathological mechanism of gut microbiota dysbiosis. Probiotics, on the other hand, are a class of live microorganisms beneficial to the host. They colonize the human gut and reproductive system, producing definite health benefits and improving the host's microecological balance, thus exerting beneficial effects on the gut. Increasing evidence shows that gut microbiota can significantly improve or treat constipation or constipation-related symptoms, including gut microbiota such as Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, and Bifidobacterium animalis.

[0003] With the widespread application of probiotics in the food, pharmaceutical, and health product industries, market demand for highly active, multi-strain compound probiotic products continues to rise. Compound probiotics are produced through fermentation using synthetic microbiome technology, screening for naturally compatible combinations of various probiotics. The significance lies in leveraging the synergistic effects of natural microbial communities: different strains can complement each other's metabolic pathways, improving substrate utilization (such as breaking down complex carbohydrates) and enhancing product diversity (such as organic acids and prebiotics). Simultaneously, the symbiotic relationship between the microbial communities can inhibit contamination by other microorganisms, stabilizing the fermentation system. In the food and health product industries, this directly optimizes product function and quality, and because it originates from nature, it is easily commercialized.

[0004] The study of synthetic microbiomes involves integrating different microbial strains to efficiently, stably, and safely handle more complex tasks, achieving goals that single strains cannot accomplish, thereby meeting a wider range of needs. It is not simply a collection of many independent microorganisms; rather, it is a complex ecosystem formed through interactions such as resource competition, symbiotic nutrition, quorum sensing, and horizontal gene transfer. Its significance lies in the precise regulation of the fermentation process: it allows for the targeted design of strain interaction patterns (such as nutrient allocation and signal transduction), eliminating interspecies competition and maximizing the synthesis efficiency of target products. Simultaneously, it enables the analysis of redundancy and synergistic mechanisms in mixed fermentation, providing theoretical support for optimizing fermentation parameters and promoting the upgrading of probiotic mixed fermentation from empirical to precise and engineered approaches. Compared to single strains, synthetic microbial communities can better adapt to environmental changes, cooperate metabolically, catalyze more complex reactions, achieve synergistic promotion of probiotic functions, avoid competitive inhibition, maximize the functional effects of probiotics, and expand their application potential in industrial fermentation, medicine, and other fields.

[0005] Currently, the production of compound probiotic preparations mostly employs a process of fermenting individual strains separately and then combining them, with mixed fermentation processes being less common. Both methods have certain limitations. Single-strain fermentation requires independent process optimization, resulting in low equipment utilization and high production costs (culture medium costs account for over 30%). Furthermore, single-strain fermentation relies on the metabolic pathways of a single strain. For example, while *Lactobacillus acidophilus* can efficiently produce lactic acid to regulate intestinal pH, it lacks the ability to degrade complex polysaccharides and cannot activate the specific utilization of oligosaccharides by *Bifidobacterium animalis*. Although *Lactobacillus rhamnosus* has advantages in mucosal adhesion, its production of short-chain fatty acids is insufficient when cultured alone, making it difficult to effectively improve intestinal motility in patients with constipation. *Bifidobacterium animalis* has stringent nutritional requirements, necessitating the addition of components such as vitamin B12. Moreover, single-strain fermented products have limited functionality and cannot meet the market demand for compound probiotics.

[0006] Technical challenges facing mixed fermentation include: First, traditional mixed fermentation suffers from interspecies metabolic antagonism, and the metabolic coupling mechanisms between cross-phylum strains are not fully understood. For example, oxygen competition between Bifidobacterium animalis (obligatory anaerobic) and facultative anaerobic lactic acid bacteria leads to a decrease in viable cell count of over 40%. Second, the culture medium composition cannot meet the nutritional needs of multiple microorganisms. For instance, Lactobacillus plantarum requires a high nitrogen source to promote protease secretion, while Bifidobacterium animalis prefers carbohydrate metabolism in a low-oxygen environment, making it difficult to balance in traditional MRS media. Third, the fermenter process parameters (such as dissolved oxygen, pH, and temperature) lack dynamic control, resulting in a shortened exponential growth phase and increased cell autolysis rate.

[0007] Single-strain fermentation requires multiple fermentation devices, resulting in long production cycles, high costs, and difficulties in controlling compatibility between strains. Furthermore, the compounding process easily leads to loss of bacterial activity. In existing mixed fermentation technologies, different probiotics often exhibit growth antagonism due to differences in growth conditions (such as nutritional requirements, pH tolerance range, and metabolite interactions), leading to low cell density (live counts often below 10). 9 The concentration of CFU / mL is insufficient to meet the requirements of high-density production. Furthermore, pH fluctuations, nutrient competition, and the accumulation of metabolic waste during fermentation further inhibit cell proliferation, limiting the industrial-scale efficiency of compound probiotics.

[0008] Furthermore, the compound probiotic powders obtained from single-strain fermentation and mixed fermentation have the following different problems: The functional advantages of compound probiotic powders combined after single-strain fermentation lie in the controllability of strain activity and the targeting of functions. Single-strain fermentation can independently optimize the culture conditions of each strain, ensuring that each strain proliferates in the optimal environment and maximizing the activity and functional stability of a single strain; at the same time, the strain ratio can be precisely adjusted according to needs to achieve targeted combination of functional modules. However, this method lacks the synergistic domestication between strains, and after mixing, the functional superposition effect may be limited due to the mutual inhibition of metabolites and nutrient competition, or even the loss of activity. On the other hand, the core advantage of compound probiotic powders obtained by mixed fermentation comes from the synergistic evolution and metabolic network reconstruction of strains during the co-culture process. First, in co-fermentation, bacterial strains can optimize metabolic efficiency through interactions (such as cross-feeding and signal transduction). For example, enzymes secreted by one strain can assist another strain in breaking down complex substrates, improving overall nutrient utilization and enhancing the environmental adaptability of the bacterial powder (such as tolerance to gastric acid and bile). Second, co-culture induces strains to synthesize unique secondary metabolites (such as novel bacteriocins and short-chain fatty acids), endowing the bacterial powder with richer functional activities (such as synergistic antibacterial activity and regulation of intestinal barrier function). Furthermore, long-term co-culture can reduce interspecies competition, forming a stable microbial community structure and ensuring functional consistency of the bacterial powder during storage and colonization in the host. Compared to single-strain combinations, if the strains are properly matched, the function of co-fermented bacterial powder will more closely resemble the synergistic effect of a natural micro-ecosystem, potentially achieving a "1+1>2" functional amplification, and demonstrating superior performance in maintaining intestinal homeostasis and improving host health. Therefore, developing a co-fermentation technology for compound probiotics that can achieve efficient synergistic growth of multiple strains, increase live bacteria density, and balance functional synergy has become a key breakthrough direction for the current development of the probiotic industry. Summary of the Invention

[0009] In view of this, the present invention provides a compound probiotic, its application, and a compound probiotic powder.

[0010] This invention isolates a strain of Lactobacillus acidophilus DH-La-166 from a fecal sample of a healthy human, with the accession number CGMCC No. 13077.

[0011] The present invention also provides a compound probiotic, including the aforementioned Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium animalis, and Lactobacillus plantarum; wherein the Lactobacillus acidophilus includes Lactobacillus acidophilus DH-La-166 as described in claim 1 and / or Lactobacillus acidophilus DH213 with accession number CGMCC NO.11228.

[0012] In some implementations, the ratio of viable counts of Lactobacillus acidophilus, Bifidobacterium animalis, Lactobacillus rhamnosus, and Lactobacillus plantarum is (1~4):(1~4):(1~4):(1~4). In some specific embodiments, the ratio of viable counts is 1:1:3:3, 1:1.3:2.9:3.2, 1:1:1:1, 3:3:3:2, 2:2:1:1, or 3:3:2:1.

[0013] In some preferred embodiments, the compound probiotics include Lactobacillus acidophilus DH-La-166, Bifidobacterium animalis DH216, Lactobacillus rhamnosus DH-Lr-121, and Lactobacillus plantarum DH-Lp-22. The viable counts of Lactobacillus acidophilus DH-La-166, Bifidobacterium animalis DH216, Lactobacillus rhamnosus DH-Lr-121, and Lactobacillus plantarum DH-Lp-22 are as described above and will not be repeated here.

[0014] In some implementations, the compound probiotics include Lactobacillus acidophilus DH213, Bifidobacterium animalis DH216, Lactobacillus rhamnosus DH-Lr-121, and Lactobacillus plantarum DH-Lp-22. The proportions of each strain are as described above and will not be repeated here.

[0015] The preservation number of the animal Bifidobacterium DH216 is CGMCC NO.13232;

[0016] The preservation number of *Lactobacillus rhamnosus* DH-Lr-121 is CGMCC NO. 13076;

[0017] The preservation number of the *Lactobacillus plantarum* DH-Lp-22 is CGMCC NO.13074.

[0018] The compound probiotics provided by this invention are not simply an addition of strains, but a combination of strains that can promote each other and synergistically improve constipation, as confirmed by mixed culture and pharmacodynamic studies.

[0019] This invention involves the mixed fermentation of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, and Bifidobacterium animalis. Based on the ecological theory of probiotic compounding (such as cross-feeding, functional complementarity, and niche competition mechanisms), it aims to construct a multi-strain synergistic system, breaking through the functional limitations of single strains.

[0020] 1) By utilizing a cross-feeding mechanism, Lactobacillus plantarum degrades complex polysaccharides to generate oligosaccharides, which are then specifically utilized by Bifidobacterium animalis, thus expanding the overall nutritional niche of the microbial community; 2) By leveraging the complementary effect, the mucosal adhesion ability of Lactobacillus rhamnosus is combined with the antibacterial metabolites (such as lactic acid and hydrogen peroxide) of Lactobacillus acidophilus to enhance intestinal colonization competitiveness and pathogen inhibition effect; 3) Based on the coupling of metabolic modules of strains with large phylogenetic distances (such as cross-phylum synergy between Lactobacillus and Bifidobacterium), resource competition among closely related strains is avoided, a stable symbiotic network is constructed, and the synergistic production of beneficial metabolites such as short-chain fatty acids is achieved; 4) Based on the compound microbial powder prepared by the above-mentioned mixed fermentation method of this invention, this invention verifies that it has a significant laxative effect.

[0021] The present invention also provides a culture medium combination for culturing the *Lactobacillus acidophilus* or the compound probiotics described herein. This culture medium combination includes a seed culture medium and / or a fermentation culture medium.

[0022] The seed culture medium formula is selected from any of the following:

[0023] Peptone 3~15g / L, beef extract 3~15g / L, yeast extract 2~10g / L, diammonium hydrogen citrate 0.5~5g / L, glucose 5~25g / L, Tween-80 0.5~3g / L, sodium acetate 2~10g / L, dipotassium hydrogen phosphate 1~5g / L, magnesium sulfate 0.1~1.5g / L, manganese sulfate 0.1~1g / L, cysteine ​​0.1~2g / L, and water;

[0024] Ingredients: 3-15 g / L peptone, 3-15 g / L beef extract, 2-10 g / L yeast extract, 0.5-5 g / L diammonium citrate, 5-25 g / L glucose, 0.5-3 g / L Tween-80, 2-10 g / L sodium acetate, 1-5 g / L dipotassium hydrogen phosphate, 0.1-1.5 g / L magnesium sulfate, 0.1-1 g / L manganese sulfate, 0.1-2 g / L tomato extract, and water;

[0025] Tryptone 3~15g / L, beef extract 3~15g / L, yeast extract 2~10g / L, diammonium hydrogen citrate 0.5~5g / L, glucose 5~25g / L, Tween-80 0.5~3g / L, sodium acetate 2~10g / L, dipotassium hydrogen phosphate 1~5g / L, magnesium sulfate 0.1~1.5g / L, manganese sulfate 0.1~1g / L, and water;

[0026] Soy protein peptone 3~15g / L, beef extract 3~15g / L, yeast extract 2~10g / L, diammonium hydrogen citrate 0.5~5g / L, glucose 5~25g / L, Tween-80 0.5~3g / L, sodium acetate 2~10g / L, dipotassium hydrogen phosphate 1~5g / L, magnesium sulfate 0.1~1.5g / L, manganese sulfate 0.1~1g / L, and water.

[0027] In some specific embodiments, the formulation of the seed culture medium is selected from any one of the following (1) to (4):

[0028] (1) 5 g / L peptone, 5 g / L beef extract, 5 g / L yeast extract, 2 g / L diammonium citrate, 15 g / L glucose, 0.5-31 g / L Tween 80, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, 0.5 g / L manganese sulfate, 1 g / L cysteine ​​and water.

[0029] (2) 5 g / L peptone, 5 g / L beef extract, 5 g / L yeast extract, 2 g / L diammonium citrate, 15 g / L glucose, 0.5~31 g / L Tween-80, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, 0.5 g / L manganese sulfate, 1 g / L tomato extract and water.

[0030] (3) 5 g / L tryptone, 5 g / L beef extract, 5 g / L yeast extract, 2 g / L diammonium citrate, 15 g / L glucose, 0.5~31 g / L Tween-80, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, 0.5 g / L manganese sulfate and water.

[0031] (4) 5 g / L soybean peptone, 5 g / L beef extract, 5 g / L yeast extract, 2 g / L diammonium citrate, 15 g / L glucose, 0.5~31 g / L Tween-80, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, 0.5 g / L manganese sulfate and water.

[0032] In some embodiments, the fermentation medium comprises water and the following components:

[0033] Peptone 15-40 g / L, beef extract 10-30 g / L, yeast extract 5-30 g / L, glucose 5-30 g / L, sucrose 10-30 g / L, fructooligosaccharides 1-10 g / L, Tween-80 0.5-3 g / L, sodium acetate 2-10 g / L, dipotassium hydrogen phosphate 1-5 g / L, magnesium sulfate 0.1-1.5 g / L, manganese sulfate 0.1-1 g / L, calcium carbonate 5-15 g / L, cysteine ​​0.5-5 g / L, and tomato extract 0.5-5 g / L.

[0034] In some specific embodiments, the fermentation medium consists of water and the following components:

[0035] The ingredients are: peptone 30 g / L, beef extract 10 g / L, yeast extract 15 g / L, glucose 10 g / L, sucrose 15 g / L, fructooligosaccharides 5 g / L, Tween-80 1 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.1 g / L, calcium carbonate 10 g / L, cysteine ​​3 g / L, and tomato extract 3 g / L.

[0036] The present invention also provides a fermentation method, comprising:

[0037] The Lactobacillus acidophilus or the compound probiotics described in this invention are inoculated into the seed culture medium in the culture medium combination and cultured to obtain seed liquid.

[0038] The seed culture is inoculated into the fermentation medium for fermentation.

[0039] First, the compound probiotics of this invention are inoculated into the seed culture medium for seed culture. Preferably, the four strains of the compound probiotics are inoculated separately into the seed culture medium for seed culture. Specifically, the seed culture medium and culture method used for the four strains are as follows:

[0040] Lactobacillus rhamnosus DH-Lr-121: Modified MRS medium containing 0.5% tryptone. The culture method included: inoculating Lactobacillus rhamnosus DH-Lr-121 into the medium, culturing at 37°C for 18 hours, and then subculturing twice.

[0041] Bifidobacterium animalis DH216: Modified MRS medium containing 0.1% cysteine. The culture method included inoculation with Bifidobacterium animalis DH216 and anaerobic culture at 37°C for 20 hours.

[0042] Lactobacillus acidophilus DH-La-166: Modified MRS medium containing 0.1% tomato extract. The culture method included inoculating with Lactobacillus acidophilus DH-La-166 and incubating statically at 37°C for 21 hours.

[0043] Lactobacillus plantarum DH-Lp-22: Modified MRS medium containing 0.5% soybean peptone. The culture method included inoculation with Lactobacillus plantarum DH-Lp-22 and incubation at 37°C for 14 hours.

[0044] After the above culture, seed cultures of four bacteria were obtained.

[0045] After obtaining the seed culture, the seed culture is inoculated into a fermentation medium for fermentation culture. The fermentation includes an early fermentation stage and a middle and late fermentation stage; the early fermentation stage is from 0 to 3 hours of fermentation, and the temperature during the early fermentation stage is 38 to 40°C, specifically 38°C, 39°C, or 40°C; the middle and late fermentation stage is from 3 hours of fermentation to the end of fermentation, and the temperature during the middle and late fermentation stage is 35 to 37°C, specifically 35°C, 36°C, or 37°C; the pH of the fermentation is 5.0 to 8.0, specifically 5.0, 6.0, 7.0, or 8.0.

[0046] Furthermore, the fermentation process also includes a step of adding a carbon source. The amount of carbon source added is such that the sugar concentration in the fermentation broth is 0.05~10 g / L; specifically, the sugar concentration can be 0.05 g / L, 2 g / L, 4 g / L, 6 g / L, 8 g / L, or 10 g / L.

[0047] Furthermore, the fermentation process also includes a step of adding a nitrogen source; the amount of nitrogen source added is such that the concentration of amino acids in the fermentation broth is 0.05~5 g / L, and the specific concentration of amino acids can be 0.05 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L or 5 g / L.

[0048] The present invention also provides fermentation products obtained by any of the above-described fermentation methods.

[0049] The present invention also provides the use of the Lactobacillus acidophilus, the compound probiotics, or the fermentation products described above in the preparation of products that help with bowel movements.

[0050] This invention also provides products that help with bowel movements, comprising the *Lactobacillus acidophilus*, the compound probiotics, or the fermentation products described in this invention, as well as acceptable excipients. This invention does not impose special restrictions on the types of excipients; any type that complies with relevant regulations and standards and does not affect the activity and function of the probiotics is acceptable. Specific selection can be made according to the product dosage form requirements. For example, when preparing probiotic powder, maltodextrin, gum arabic, dietary fiber, etc., can be used as protective agents or carriers to improve the storage stability and intestinal colonization ability of the powder; when preparing probiotic tablets or capsules, microcrystalline cellulose, magnesium stearate, etc., can be added as fillers and lubricants to ensure formulation formability and ease of administration; skim milk powder, trehalose, and ascorbic acid can also be added, and the mixture can be freeze-dried to produce probiotic powder.

[0051] This invention also provides a compound probiotic powder, which is obtained by freeze-drying the following raw materials:

[0052] This invention relates to the compound probiotics or fermentation product as described above, skim milk powder, trehalose, and ascorbic acid. In some embodiments, the preferred mass proportions of each ingredient are as follows: 30-100 parts compound probiotics or the fermentation product, 10 parts skim milk powder, 5 parts trehalose, and 0.5 parts ascorbic acid. In some specific embodiments, the mass proportions of each ingredient are specifically selected from one of the following combinations:

[0053] Combination 1: 30 parts compound probiotics or the fermentation product mentioned above, 10 parts skim milk powder, 5 parts trehalose and 0.5 parts ascorbic acid;

[0054] Combination 2: 50 parts of compound probiotics or the fermentation product mentioned above, 10 parts of skim milk powder, 5 parts of trehalose and 0.5 parts of ascorbic acid;

[0055] Combination 3: 100 parts of compound probiotics or the fermentation product mentioned above, 10 parts of skim milk powder, 5 parts of trehalose and 0.5 parts of ascorbic acid.

[0056] The present invention also provides a method for preparing the compound probiotic powder, comprising: mixing the compound probiotic or the fermentation product, skim milk powder, trehalose and ascorbic acid of the present invention, and sequentially pre-freezing, vacuum drying, and pulverizing to obtain the compound probiotic powder.

[0057] In the above preparation method, the components are first mixed evenly, and then the mixed material is pre-frozen and vacuum dried. The pre-freezing includes: pre-freezing at -80℃ for 12 hours; the pre-freezing temperature is -75~-85℃, specifically -75℃, -80℃, or -85℃; the pre-freezing time is 10~15 hours, specifically 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, or 15 hours. The vacuum drying includes: drying under a vacuum of -55℃ and 5 Pa for 48 hours. The vacuum drying temperature is specifically -50~-60 degrees Celsius; the vacuum degree is specifically 3~10 Pa, specifically 3 Pa, 5 Pa, 8 Pa, or 10 Pa; the vacuum drying time is 40~60 hours, specifically 40 hours, 45 hours, 50 hours, 55 hours, or 60 hours. After the above vacuum drying, freeze-dried material is obtained. The freeze-dried material is then pulverized to obtain the compound probiotic powder of the present invention. The pulverized material is 80-120 mesh, specifically 80, 90, 100, 110, or 120 mesh.

[0058] This invention provides a strain of *Lactobacillus acidophilus* DH213, which is beneficial for bowel movement and defecation, with the preservation number CGMCC No. 11228. This invention also provides a compound probiotic containing this strain. This compound probiotic can promote and synergistically enhance intestinal peristalsis and regulate intestinal flora in patients with constipation during its growth. Experiments show that the probiotic powder prepared using this compound probiotic can significantly increase the water content, fecal excretion time, fecal volume, and intestinal peristalsis speed in constipated mice, thereby achieving a therapeutic effect on constipation. It also features high safety and no side effects, providing a scientific basis and technical pathway for the precision development of functional foods and pharmaceutical preparations, and offering a new and effective means for the prevention and adjuvant treatment of constipation.

[0059] Biological Preservation Instructions

[0060] Biological material: DH-La-166, classified as Lactobacillus acidophilus, was deposited on September 30, 2016, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC NO.13077.

[0061] Biological material: DH213, classified and named Lactobacillus acidophilus, was deposited on August 11, 2015, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC NO.11228.

[0062] Biological material: DH-Lr-121, taxonomically named Lactobacillus rhamnosus, was deposited on September 30, 2016, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 13076.

[0063] Biological material: DH216, classified and named: Bifidobacterium animalis, was deposited on November 2, 2016, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC NO.13232.

[0064] Biological material: DH-Lp-22, classified and named Lactobacillus plantarum, was deposited on September 30, 2016, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC NO.13074. Attached Figure Description

[0065] Figure 1 The defecation status of mice under different drug doses is shown in A, which is the time of first defecation / min, B, the number of black stool particles / 6h / particle, and C, the weight of black stool / g. The 'a' above the bar chart indicates a significant difference compared with group Z2 (P < 0.05).

[0066] Figure 2 The results of small intestinal propulsion rate measurement in each group of mice are shown. Note: 'a' above the bar chart indicates a significant difference compared to group Z2 (P < 0.05). Detailed Implementation

[0067] This invention provides a compound probiotic composition, its application, and a compound probiotic powder. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0068] In this document, the terms "including", "comprising", and "having" describe both closed-loop technical solutions consisting of the listed features and open-loop technical solutions that include the listed features.

[0069] In this document, the term “and / or” as used includes any and all combinations of one or more of the related listed items.

[0070] In this document, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when a range refers to an integer, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0071] In this article, when referring to units for data ranges, if the unit is only followed by the right endpoint, it indicates that the units for the left and right endpoints are the same. For example, 3~15g / L means that the units for the left endpoint "3" and the right endpoint "15" are both g / L.

[0072] This invention targets a compound probiotic composed of *Lactobacillus rhamnosus* DH-Lr-121, *Bifidobacterium animalis* DH216, *Lactobacillus acidophilus* DH-La-166, and *Lactobacillus plantarum* DH-Lp-22. The culture medium formulation and fermentation process of this compound probiotic have been optimized, providing a culture medium combination and fermentation method that significantly improves the viable cell count and stability of the four strains. Experiments show that this system significantly increases the viable cell count of the four probiotics, enhances the activity of metabolites, and reduces production costs, meeting the demand for high-quality probiotic raw materials. It solves the problems of slow growth, low viable cell yield, and susceptibility to environmental factors leading to decreased activity in existing technologies, thus laying the foundation for the preparation of highly active compound probiotic powder and related laxative products.

[0073] The culture medium screening and optimization experiment provided by this invention includes the following steps:

[0074] (1) Activation of microbial strains

[0075] Lactobacillus rhamnosus DH-Lr-121, Bifidobacterium animalis DH216, Lactobacillus acidophilus DH-La-166, and Lactobacillus plantarum DH-Lp-22 were inoculated onto solid MRS agar plates and cultured at 37°C for 24 h. After single colonies formed, a single colony was picked and inoculated onto MRS liquid agar and cultured at 37°C until the seed culture cell concentration reached 1.0 × 10⁻⁶. 8 CFU / ml was used to activate the bacterial strain.

[0076] (2) Optimization of culture medium formulation

[0077] Based on MRS medium, its components were screened and optimized.

[0078] Screening media were prepared by adding 0.1-2% of carbon source, nitrogen source, trace element, inorganic salt, or growth factor to MRS medium that did not contain carbon source, nitrogen source, trace element, inorganic salt, or growth factor. Seed cultures of *Lactobacillus rhamnosus* DH-Lr-121, *Bifidobacterium animalis* DH216, *Lactobacillus acidophilus* DH-La-166, and *Lactobacillus plantarum* DH-Lp-22 were inoculated at 3% of each of these screening media and anaerobically cultured at 37°C for 24 hours, with a blank control included. The absorbance (A600) of the bacterial cultures of *Lactobacillus rhamnosus* DH-Lr-121, *Bifidobacterium animalis* DH216, *Lactobacillus acidophilus* DH-La-166, and *Lactobacillus plantarum* DH-Lp-22 was measured at 0 h, 12 h, and 24 h to determine the optimal carbon source, nitrogen source, trace element, inorganic salt, or growth factor.

[0079] (3) Comparative experiment on the culture effects of different culture medium formulations

[0080] Different culture media were prepared, and the pH was adjusted according to the requirements of each media. The media were sterilized at 121℃ for 15 min, and the seed culture was transferred at a 3% inoculum to 80 ml of each media. The media were then anaerobically incubated at 37℃ for 24 h, with a blank control included. The absorbance (A600) of *Lactobacillus rhamnosus* DH-Lr-121, *Bifidobacterium animalis* DH216, *Lactobacillus acidophilus* DH-La-166, and *Lactobacillus plantarum* DH-Lp-22 was measured at 0 h, 12 h, and 24 h to observe the growth of *Lactobacillus rhamnosus* and *Bifidobacterium animalis* in different media. After reaching the stationary phase, the selection media were counted, and the optimal culture medium formulation was determined by combining the measured indicators.

[0081] (4) Fermentation process optimization

[0082] Optimization of inoculation ratio: Through extensive experimentation, it was determined that the optimal seed-liquid volume ratio of Lactobacillus acidophilus DH-La-166, Bifidobacterium animalis DH216, Lactobacillus rhamnosus DH-Lr-121, and Lactobacillus plantarum DH-Lp-22 resulted in the best mixed fermentation effect. At this ratio, the four bacteria could fully exert their metabolic synergistic effects, achieving complementary advantages and improving fermentation efficiency and product quality.

[0083] Segmented temperature control: During the initial fermentation stage (0-3 hours), the temperature was maintained at 39℃. This temperature is conducive to the rapid initiation and growth of *Lactobacillus rhamnosus* DH-Lr-121 and *Lactobacillus plantarum* DH-Lp-22, which synthesize large amounts of extracellular enzymes such as proteases, decompose proteins in the culture medium, and provide sufficient amino acids and other nutrients for the subsequent growth of *Bifidobacterium animalis* DH216 and *Lactobacillus acidophilus* DH-La-166. After 3 hours, the temperature was lowered to 37℃, which is more suitable for bacterial growth and reproduction, maintaining their high activity and promoting an increase in bacterial biomass.

[0084] Precise pH control: An automatic control system monitors the pH of the fermentation broth in real time. When the pH falls below 5.5, a 20% (w / v) potassium carbonate solution is automatically added to stabilize the pH between 5.5 and 6.5. This stable pH environment helps maintain the cell membrane stability and enzyme activity of *Lactobacillus acidophilus* and *Lactobacillus plantarum*, ensuring the smooth progress of the fermentation process.

[0085] Feeding strategy optimization: After 4 hours of fermentation, a carbon and nitrogen source stream was started for feeding. The feed consisted of 5-20 g / L glucose and 2-10 g / L soybean peptone, maintaining the glucose concentration in the fermentation broth at 2-5 g / L and the amino acid concentration at ≥300 mg / L. Feeding provided additional nutrients to the cells, prolonging their growth time and further increasing the viable cell count.

[0086] The fermentation method provided by this invention includes the following steps:

[0087] Step (1): Inoculate various different seed bacteria into modified MRS medium and culture for 14-21 h to obtain various different seed bacterial solutions;

[0088] Preferably, the modified MRS liquid culture medium comprises (but is not limited to): 3-15 g / L peptone, 3-15 g / L beef extract, 2-10 g / L yeast extract, 0.5-5 g / L diammonium hydrogen citrate, 5-25 g / L glucose, 0.5-3 g / L Tween-80, 2-10 g / L sodium acetate, 1-5 g / L dipotassium hydrogen phosphate, 0.1-1.5 g / L magnesium sulfate, 0.1-1 g / L manganese sulfate, 0.1-2 g / L cysteine, and water;

[0089] Preferably, the modified MRS liquid culture medium comprises (but is not limited to): 3-15 g / L peptone, 3-15 g / L beef extract, 2-10 g / L yeast extract, 0.5-5 g / L diammonium citrate, 5-25 g / L glucose, 0.5-3 g / L Tween-80, 2-10 g / L sodium acetate, 1-5 g / L dipotassium hydrogen phosphate, 0.1-1.5 g / L magnesium sulfate, 0.1-1 g / L manganese sulfate, 0.1-2 g / L tomato extract, and water;

[0090] Preferably, the modified MRS liquid culture medium comprises (but is not limited to): 3-15 g / L tryptone, 3-15 g / L beef extract, 2-10 g / L yeast extract, 0.5-5 g / L diammonium hydrogen citrate, 5-25 g / L glucose, 0.5-3 g / L Tween-80, 2-10 g / L sodium acetate, 1-5 g / L dipotassium hydrogen phosphate, 0.1-1.5 g / L magnesium sulfate, 0.1-1 g / L manganese sulfate, and water;

[0091] Preferably, the modified MRS liquid culture medium comprises (but is not limited to): 3-15 g / L soybean peptone, 3-15 g / L beef extract, 2-10 g / L yeast extract, 0.5-5 g / L diammonium hydrogen citrate, 5-25 g / L glucose, 0.5-3 g / L Tween-80, 2-10 g / L sodium acetate, 1-5 g / L dipotassium hydrogen phosphate, 0.1-1.5 g / L magnesium sulfate, 0.1-1 g / L manganese sulfate, and water.

[0092] Step (2) involves adding the various seed culture solutions to a fermentation medium for fermentation. The fermentation medium comprises water and the following components:

[0093] Peptone 15-40 g / L, beef extract 10-30 g / L, yeast extract 5-30 g / L, glucose 5-30 g / L, sucrose 10-30 g / L, fructooligosaccharides 1-10 g / L, Tween-80 0.5-3 g / L, sodium acetate 2-10 g / L, dipotassium hydrogen phosphate 1-5 g / L, magnesium sulfate 0.1-1.5 g / L, manganese sulfate 0.1-1 g / L, calcium carbonate 5-15 g / L, cysteine ​​0.5-5 g / L, and tomato extract 0.5-5 g / L.

[0094] In a preferred embodiment, during the fermentation process in step (2), the pH of the fermentation broth is controlled at 5.0 to 8.0, preferably 5.0 to 7.0, and more preferably 5.5 to 6.5.

[0095] In another preferred embodiment, during the fermentation process in step (2), the sugar concentration in the fermentation broth is controlled to be 0.05~10 g / L by adding a carbon source, preferably 0.1~8 g / L, and more preferably 0.5~5 g / L.

[0096] In another preferred embodiment, during the fermentation process in step (2), the concentration of amino acids in the fermentation broth is controlled to be 0.05~5 g / L, preferably 0.1~3 g / L, and most preferably 0.5~2 g / L by adding a nitrogen source.

[0097] In another preferred embodiment, the carbon source is a 100-600 g / L glucose aqueous solution, wherein the concentration of the glucose aqueous solution is preferably 200-400 g / L, more preferably 250-350 g / L. The feeding rate of the glucose aqueous solution is controlled at 0.2-3 L / h, preferably 0.5-5 L / h.

[0098] In this invention, the seed culture of the four strains is added to the fermentation medium simultaneously or in stages, and this invention does not impose any special restrictions on this.

[0099] In another preferred embodiment, the seed cultures of the four strains are added to the fermentation medium in steps, specifically including: first adding the seed cultures of Lactobacillus acidophilus and Lactobacillus rhamnosus to the fermentation medium, fermenting for a period of time (preferably fermenting for 2-4 hours or when the seed cultures of Lactobacillus acidophilus and Lactobacillus rhamnosus are in the early stage of logarithmic growth in the fermentation medium), and then adding the seed cultures of Bifidobacterium animalis and Lactobacillus plantarum to the fermentation medium.

[0100] Preferably, the ratio of viable bacteria counts in the seed culture of *Lactobacillus acidophilus* DH-La-166, *Bifidobacterium animalis* DH216, *Lactobacillus rhamnosus* DH-Lr-121, and *Lactobacillus plantarum* DH-Lp-22 is (1~4):(1~4):(1~4):(1~4), more preferably (1~3):(1~3):(1~3):(1~3.2). Specifically, the viable bacteria count ratio is 1:1:3:3, 1:1.3:2.9:3.2, 1:1:1:1, 3:3:3:2, 2:2:1:1, or 3:3:2:1.

[0101] Preferably, the fermentation includes an early fermentation stage and a middle and late fermentation stage. The fermentation temperature is controlled in stages: during the early fermentation stage (0-3 hours), the temperature is controlled at 38-40℃; during the middle and late fermentation stage (3 hours to the end of fermentation), the temperature is controlled at 35-37℃.

[0102] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0103] In 2022, the National Health Commission of China updated the "List of Microbial Strains that Can Be Used in Food" and the "List of Microbial Strains that Can Be Used in Infant Food," which included changes to the names of some strains. Specifically, *Lactobacillus rhamnosus* was renamed *Lactaseibacillus rhamnosus*, *Bifidobacterium animalis* was renamed *Bifidobacterium animalis subsp. lactis*, and *Lactobacillus plantarum* was renamed *Lactiplantibacillus plantarum*. In this invention, the *Lactobacillus rhamnosus*, *Bifidobacterium animalis*, and *Lactobacillus plantarum* mentioned can also use their renamed strain names, i.e., *Lactobacillus rhamnosus*, *Bifidobacterium animalis subsp. lactis*, and *Lactobacillus plantarum*, and the scope of protection involved is not affected by the renaming.

[0104] Among them, *Lactobacillus acidophilus* DH213, *Lactobacillus rhamnosus* DH-Lr-121, *Bifidobacterium animalis* DH216, and *Lactobacillus plantarum* DH-Lp-22 have been deposited at the China General Microbiological Culture Collection Center, and the deposit information is as follows:

[0105] Biological material: DH213, classified and named Lactobacillus acidophilus, was deposited on August 11, 2015, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC NO.11228.

[0106] Biological material: DH-Lr-121, taxonomically named Lactobacillus rhamnosus, was deposited on September 30, 2016, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 13076.

[0107] Biological material: DH216, classified and named: Bifidobacterium animalis, was deposited on November 2, 2016, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC NO.13232.

[0108] Biological material: DH-Lp-22, classified and named Lactobacillus plantarum, was deposited on September 30, 2016, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC NO.13074.

[0109] The present invention will be further illustrated below with reference to the embodiments:

[0110] Example 1: Preparation of different Lactobacillus glycerol tube strains

[0111] Lactobacillus rhamnosus DH-Lr-121, Bifidobacterium animalis DH216, Lactobacillus acidophilus DH-La-166, and Lactobacillus plantarum DH-Lp-22 were inoculated onto solid MRS agar plates and cultured at 37°C for 24 h. After single colonies formed, a single colony was picked and inoculated onto MRS liquid medium and cultured at 37°C until the seed culture cell concentration reached above 1.0 × 10^8 cfu / ml, thus completing the strain activation. The seed culture was mixed thoroughly, and 1 mL was taken and mixed evenly with 1 mL of 60% glycerol solution to prepare batches of glycerol tubes, which were then frozen at -20°C for direct use in subsequent fermentation process optimization.

[0112] Example 2: Seed culture medium optimization and seed liquid preparation experiment

[0113] MRS liquid culture medium incubation:

[0114] Different Lactobacillus glycerol tube strains prepared in Example 1 were taken from a -20℃ freezer and inoculated (with the same inoculation amount) into 300mL shake flasks. MRS liquid medium was used to culture *Lactobacillus rhamnosus* DH-Lr-121, *Bifidobacterium animalis* DH216, *Lactobacillus acidophilus* DH-La-166, and *Lactobacillus plantarum* DH-Lp-22, respectively. The cultures were then anaerobic and statically incubated at 37℃ for 24 hours. The viable cell counts were then determined, as shown in Table 1 below.

[0115] Table 1. Viable cell counts of different lactic acid bacteria cultured in MRS liquid medium.

[0116]

[0117] Culture in modified MRS liquid medium:

[0118] Different Lactobacillus glycerol tube strains prepared in Example 1 were taken out from a -20℃ freezer and inoculated (with the same inoculation amount) into 300mL shake flasks. They were cultured in modified MRS liquid medium as Lactobacillus rhamnosus DH-Lr-121, Bifidobacterium animalis DH216, Lactobacillus acidophilus DH-La-166 and Lactobacillus plantarum DH-Lp-22. They were anaerobic and statically cultured at 37℃ for 24h. The number of viable bacteria was detected and shown in Table 2 below.

[0119] Table 2. Viable cell counts of different lactic acid bacteria cultured in modified MRS liquid medium.

[0120]

[0121] As can be seen from Tables 1 and 2 above, the improved MRS medium formula increases the number of viable Lactobacillus and Bifidobacterium to varying degrees, especially the increase in Bifidobacterium animalis and Lactobacillus acidophilus. Therefore, the improved MRS liquid medium can significantly increase the number of viable Lactobacillus species.

[0122] Seed liquid preparation method:

[0123] Lactobacillus rhamnosus DH-Lr-121: Prepare 100 mL of modified MRS medium containing 0.5% tryptone, inoculate Lactobacillus rhamnosus DH-Lr-121 into the medium, and incubate at 37°C for 18 h. After culture, perform subculturing twice.

[0124] Bifidobacterium animalis DH216: Prepare 100 mL of modified MRS medium containing 0.1% cysteine, inoculate with Bifidobacterium animalis DH216, and then place in an anaerobic incubator and incubate at 37°C for 20 h.

[0125] Lactobacillus acidophilus DH-La-166: Take 100 mL of modified MRS medium containing 0.1% tomato extract, inoculate with Lactobacillus acidophilus DH-La-166, and incubate statically at 37℃ for 21 h.

[0126] Lactobacillus plantarum DH-Lp-22: Prepare 100 mL of modified MRS medium containing 0.5% soybean peptone, inoculate with Lactobacillus plantarum DH-Lp-22, and incubate at 37℃ for 14 h.

[0127] Example 3: Comparative Experiment of Cultivation Effects of Different Culture Medium Formulations

[0128] Five different culture media were prepared. The pH was adjusted according to the requirements of each culture medium, and the cultures were sterilized at 121℃ for 15 min. Seed cultures of the four strains prepared in Example 2 were transferred to 300 ml of each culture medium at an inoculation rate of 3% (for each strain). The cultures were anaerobic and statically incubated at 37℃ for 24 h, with a blank control included. The absorbance (A600) of *Lactobacillus rhamnosus* DH-Lr-121, *Bifidobacterium animalis* DH216, *Lactobacillus acidophilus* DH-La-166, and *Lactobacillus plantarum* DH-Lp-22 was measured at 0 h, 12 h, and 24 h to observe the growth of *Lactobacillus rhamnosus* and *Bifidobacterium animalis* in different culture media. After reaching the stationary phase, the selection solutions were counted, and the optimal culture medium formulation was determined by comprehensively measuring the indicators. The viable cell counts in different culture media are shown in Table 3 below.

[0129] The five culture media consist of water and the following components in weight percentages:

[0130] Modified MRS broth medium: 3.0% peptone, 1.0% beef extract, 1.5% yeast extract, 1.0% glucose, 15% sucrose, 0.5% fructooligosaccharides, 0.1% Tween-80, 0.5% sodium acetate, 0.2% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.01% manganese sulfate, 0.3% cysteine, 0.3% tomato extract, 1.0% calcium carbonate, pH adjusted to 6.8.

[0131] MRS broth medium: peptone 1.0%, beef extract 0.8%, yeast extract 0.4%, glucose 2.0%, dipotassium hydrogen phosphate 0.2%, diammonium hydrogen citrate 0.2%, sodium acetate 0.5%, magnesium sulfate 0.02%, manganese sulfate 0.004%, Tween-80 0.1%, pH adjusted to 6.8.

[0132] BBL liquid medium: peptone 1.5%, yeast extract 0.2%, glucose 2.0%, soluble starch 0.05%, sodium chloride 0.5%, L-cysteine ​​0.05%, tomato extract 0.5%, liver extract 0.2%, Tween-80 0.1%, pH adjusted to 6.8.

[0133] TPY liquid culture medium: glucose 0.5%, hydrolyzed casein 1.0%, soybean peptone 0.5%, yeast extract 0.2%, dipotassium hydrogen phosphate 0.02%, magnesium chloride 0.05%, zinc sulfate 0.025%, calcium chloride 0.015%, Tween-80 0.1%, magnesium sulfate heptahydrate 0.01%, L-cysteine ​​hydrochloride 0.05%, pH adjusted to 6.2-6.4.

[0134] PTYG liquid medium: 0.5% peptone, 0.5% tryptone, 1.0% yeast extract, 1.0% glucose, 0.02% calcium chloride, 0.048% magnesium sulfate heptahydrate, 0.1% potassium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate, 1.0% sodium bicarbonate, 0.2% sodium chloride, pH adjusted to 6.2–6.4.

[0135] Table 3. Number of viable lactic acid bacteria cultured in different culture media

[0136]

[0137] As can be seen from Table 3 above, the modified MRS broth medium formula has the best culture effect on lactobacillus and bifidobacteria. In other media, the co-culture effect of Lactobacillus rhamnosus DH-Lr-121, Bifidobacterium animalis DH216, Lactobacillus acidophilus DH-La-166 and Lactobacillus plantarum DH-Lp-22 is poor or cannot be co-cultured.

[0138] Example 4: Investigation of high-density fermentation of different lactic acid bacteria individually

[0139] Industrial-scale high-density fermentation of Lactobacillus rhamnosus DH-Lr-121

[0140] Seed culture preparation: Take the Lactobacillus rhamnosus DH-Lr-121 glycerol tube culture prepared in Example 1 and store it in a -20℃ refrigerator, and prepare Lactobacillus rhamnosus DH-Lr-121 seed culture according to Example 2, with an inoculation amount of 3%.

[0141] Culture medium preparation: Weigh out the following per liter of culture medium: 30 g / L peptone, 10 g / L beef extract, 15 g / L yeast extract, 10 g / L glucose, 15 g / L sucrose, 5 g / L fructooligosaccharides, 1 g / L Tween-80, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, 0.1 g / L manganese sulfate, 3 g / L cysteine, 3 g / L tomato extract, 10 g / L calcium carbonate, and water. Add the above ingredients sequentially to an appropriate amount of deionized water, stir thoroughly to dissolve, and then adjust the pH to 6.8 with dilute hydrochloric acid or sodium hydroxide solution. Transfer the prepared culture medium to a 5 L fermenter, autoclave at 121 °C for 30 min, and allow to cool to a suitable temperature before use.

[0142] Inoculation and Fermentation Control: Early Fermentation Stage (0-3h): Set the fermenter temperature to 39℃ and the stirring speed to 150rpm to ensure an anaerobic environment, promoting the rapid growth of *Lactobacillus acidophilus* DH-La-166, *Bifidobacterium animalis* DH216, *Lactobacillus rhamnosus* DH-Lr-121, and *Lactobacillus plantarum* DH-Lp-22, and the synthesis of extracellular enzymes such as proteases. Mid-Fermentation Stage (3-9h): Adjust the temperature to 37℃ and the stirring speed to 150rpm to ensure an anaerobic environment. Simultaneously, add 20% potassium carbonate solution and 30% carbon and nitrogen source feed solution via a peristaltic pump, monitoring and maintaining the pH of the fermentation broth at approximately 5.5, the glucose concentration at 2-5g / L, and the amino acid concentration at ≥300mg / L. Late Fermentation Stage (9h to end of fermentation): Continue to maintain the temperature at 37℃ and the stirring speed at 150rpm to ensure an anaerobic environment. Meanwhile, the addition of carbon and nitrogen sources was stopped, but 20% potassium carbonate solution was continued to be added via a peristaltic pump. The pH of the fermentation broth was monitored and maintained at around 5.5 in real time, and fermentation was continued for 12 hours before being terminated.

[0143] Sampling and testing: During fermentation, 10 mL of fermentation broth was taken from the fermenter every 2 hours. A portion of the fermentation broth was used to determine the viable cell count using qPCR, with counting performed in primer systems for the corresponding bacterial species; another portion was used to determine the pH using a precision pH meter; and a third portion was used to determine the OD600, with absorbance measured at 600 nm using a spectrophotometer.

[0144] The industrial-scale high-density fermentation of other lactic acid bacteria was carried out using the same fermentation method as described above. Bifidobacterium animalis DH216, Lactobacillus acidophilus DH-La-166, and Lactobacillus plantarum DH-Lp-22 were fermented separately. The viable cell counts after fermentation are shown in Table 4 below.

[0145] Table 4. Viable bacterial counts of different lactobacilli after individual fermentation using the fermentation method described in Example 3.

[0146]

[0147] Example 5: Investigation of high-density fermentation of mixed *Lactobacillus rhamnosus*, *Bifidobacterium animalis*, *Lactobacillus acidophilus*, and *Lactobacillus plantarum*.

[0148] Seed culture preparation: Take the Lactobacillus rhamnosus DH-Lr-121, Bifidobacterium animalis DH216, Lactobacillus acidophilus DH-La-166 and Lactobacillus plantarum DH-Lp-22 glycerol tube strains prepared in Example 1 and store them in a -20℃ refrigerator. Prepare seed cultures of the four strains according to Example 2, and inoculate them into the fermentation medium at a total inoculation amount of 3% and an inoculation ratio of Lactobacillus acidophilus: Bifidobacterium animalis: Lactobacillus rhamnosus: Lactobacillus plantarum 3:3:2:1 (ratio of viable cells).

[0149] Preparation of fermentation medium: Weigh out the following per liter of medium: 30 g / L peptone, 10 g / L beef extract, 15 g / L yeast extract, 10 g / L glucose, 15 g / L sucrose, 5 g / L fructooligosaccharides, 1 g / L Tween-80, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, 0.1 g / L manganese sulfate, 3 g / L cysteine, 3 g / L tomato extract, 10 g / L calcium carbonate, and water. Add the above ingredients sequentially to an appropriate amount of deionized water, stir thoroughly to dissolve, and then adjust the pH to 6.8 with dilute hydrochloric acid or sodium hydroxide solution. Transfer the prepared medium to a 5 L fermenter, autoclave at 121 °C for 30 min, and allow to cool to a suitable temperature before use.

[0150] Inoculation and fermentation control:

[0151] Early stage of fermentation (0~3h): Set the fermenter temperature to 39℃ and the stirring speed to 150rpm to ensure an anaerobic environment, promote the rapid start-up growth of Lactobacillus acidophilus DH-La-166, Bifidobacterium animalis DH216, Lactobacillus rhamnosus DH-Lr-121 and Lactobacillus plantarum DH-Lp-22 and synthesize a large amount of extracellular enzymes such as proteases.

[0152] Mid-fermentation stage (3-9 hours): Adjust the temperature to 37℃ and the stirring speed to 150 rpm to ensure an anaerobic environment. Simultaneously, add 20% potassium carbonate solution and 30% carbon and nitrogen source feed solution via a peristaltic pump, monitor and maintain the pH of the fermentation broth at around 5.5 in real time, and maintain the glucose concentration in the fermentation broth at 2-5 g / L and the amino acid concentration at ≥300 mg / L.

[0153] Late stage of fermentation (9 hours to end of fermentation): The temperature is maintained at 37℃, and the stirring speed is set to 150 rpm to ensure an anaerobic environment. At the same time, the addition of carbon and nitrogen sources is stopped, but 20% potassium carbonate solution is continued to be added through a peristaltic pump. The pH of the fermentation broth is monitored and maintained at around 5.5 in real time, and fermentation is continued until 12 hours are completed and then terminated.

[0154] Sampling and testing: During fermentation, 10 mL of fermentation broth was taken from the fermenter every 2 hours. A portion of the fermentation broth was used to determine the viable cell count using qPCR, with counting performed in primer systems for the corresponding bacterial species; another portion was used to determine the pH using a precision pH meter; and a third portion was used to determine the OD600, with absorbance measured at 600 nm using a spectrophotometer.

[0155] Table 5. Viable cell counts of *Lactobacillus rhamnosus*, *Bifidobacterium animalis*, *Lactobacillus acidophilus*, and *Lactobacillus plantarum* after mixed fermentation using the fermentation method described in Example 4.

[0156]

[0157] 4.2 Preparation of Compound Microbial Powder

[0158] Centrifugation: After fermentation, transfer the fermentation broth to a centrifuge and centrifuge at 8000 rpm for 15 minutes at a low temperature of 4℃. After centrifugation, carefully discard the supernatant and collect the bottom sludge. The wet weight of the sludge is approximately 40 g / L.

[0159] Freeze-drying protection: Prepare a preservative consisting of 10 parts skim milk powder, 5 parts trehalose, 0.5 parts ascorbic acid, and 84.5 parts purified water, totaling 100 parts. Thoroughly mix the collected bacterial sludge with the preservative at a 1:1 mass ratio. Then transfer the mixture to a freeze-drying container and pre-freeze at -80℃ for 12 hours to ensure complete freezing. Next, place the freeze-drying container in a vacuum freeze dryer and dry at -55℃ and 5Pa for 48 hours to remove moisture through sublimation. After drying, remove the freeze-dried material, pulverize it, and pass it through an 80-mesh sieve to obtain the final compound bacterial powder.

[0160] Table 6. Viable bacterial counts of *Lactobacillus rhamnosus*, *Bifidobacterium animalis*, *Lactobacillus acidophilus*, and *Lactobacillus plantarum* after treatment using the method described in the preparation of the compound bacterial powder.

[0161]

[0162] 4.3 Bowel movement test of probiotic composition

[0163] 4.3.1 Experimental Objective

[0164] The above-screened compound probiotic combination was used to establish a constipation mouse model to simulate human constipation. The product intervention was conducted on the mice, and the laxative effect of the composition of the present invention was confirmed by the time of the first black stool excretion, the number of stools excreted within 5 hours, and the weight of the stool.

[0165] 4.3.2 Experimental Animals

[0166] Adult male mice weighing 18-22g were selected, with 10-15 mice per group.

[0167] 4.3.3 Test strains

[0168] Lactobacillus rhamnosus DH-Lr-121, Bifidobacterium animalis DH216, Lactobacillus acidophilus DH-La-166, Lactobacillus plantarum DH-Lp-22

[0169] 4.3.4 Preparation of Probiotic Suspension

[0170] According to the different probiotic powder combinations and their addition ratios in Table 7, they were prepared into bacterial suspensions of 1×10^9 CFU / mL.

[0171] Table 7 Different probiotic powder combinations

[0172]

[0173] Note: Microbial powder combination 6 is a compound of microbial powders obtained from the individual fermentation of each microbial species; microbial powder combination 5 is a microbial powder obtained from the mixed culture of Bifidobacterium animalis, Lactobacillus rhamnosus, and Lactobacillus plantarum; microbial powder combinations 7 and 8 are microbial powders obtained from the mixed culture of Lactobacillus acidophilus, Bifidobacterium animalis, Lactobacillus rhamnosus, and Lactobacillus plantarum.

[0174] 4.3.3 Animal Grouping

[0175] After one week of acclimatization feeding, the mice were randomly divided into 9 groups of 20 mice each: blank group (K), model group (M), bacterial powder combination group 1 (Z1), bacterial powder combination group 2 (Z2), bacterial powder combination group 3 (Z3), bacterial powder combination group 4 (Z4), bacterial powder combination group 5 (Z5), bacterial powder combination group 6 (Z6), bacterial powder combination group 7 (Z7), and bacterial powder combination group 8 (Z8). Modeling and subsequent drug administration began.

[0176] 4.3.4 Experimental Procedure

[0177] 4.3.4.2 Experiment on the determination of defecation time, number of black stool particles, and weight of black stool

[0178] 4.3.4.1 Administration method

[0179] Modeling: After the adaptive feeding period, mice in the blank group (K), model group (M), bacterial powder combination group 1 (Z1), bacterial powder combination group 2 (Z2), bacterial powder combination group 3 (Z3), bacterial powder combination group 4 (Z4), bacterial powder combination group 5 (Z5), bacterial powder combination group 6 (Z6), bacterial powder combination group 7 (Z7), and bacterial powder combination group 8 (Z8) were administered the corresponding formula of the test sample by gavage at a dose of 0.2 mL of bacterial suspension (2.0×10^8 CFU) per day. The blank group (K) and model group (M) were given the same dose of physiological saline. The gavage was performed once a day for 7 consecutive days.

[0180] Preparation of loperamide solution: Take 3.6 ml of loperamide solution with a concentration of 0.7 mg / ml, add 0.6 ml of distilled water, mix well to obtain a loperamide solution with a concentration of 0.6 mg / ml.

[0181] Preparation of the ink: Prepare according to the method for "testing methods that help with bowel movements" in the "Methods for Functional Testing and Evaluation of Health Foods (2023 Edition)". Accurately weigh 10g of gum arabic, add 80mL of water, and boil until the solution is clear. Weigh 5g of activated charcoal (powder) and add it to the above solution, boiling three times. After the solution cools, add water to bring the volume to 100mL. Store in a refrigerator at 4℃ and shake well before use.

[0182] After the drug administration was completed, 10 mice from each group were used to measure defecation time, number of fecal pellets, fecal weight, and water content. Mice in each group were fasted but allowed free access to water. After 16 hours of fasting, except for the control group, the other groups were administered loperamide (6 mg / kg BW) by gavage. The control group received the same dose of physiological saline. 0.5 hours after loperamide administration, each mouse was given 0.2 mL of ink by gavage, and timing began. Each mouse was placed individually in a cage with free access to food and water. The time of the first black feces excreted by each mouse was recorded, and the number and weight of black feces excreted by the mice within 6 hours were counted.

[0183] 4.3.4.3 Small bowel motility test

[0184] Preparation of loperamide solution: Take 2.4 ml of loperamide solution with a concentration of 0.7 mg / ml, add 1.8 ml of distilled water, mix well to obtain a loperamide solution with a concentration of 0.4 mg / ml.

[0185] Preparation of the ink: Prepare according to the method for "testing methods that help with bowel movements" in the "Methods for Functional Testing and Evaluation of Health Foods (2023 Edition)". Accurately weigh 10g of gum arabic, add 80mL of water, and boil until the solution is clear. Weigh 5g of activated charcoal (powder) and add it to the above solution, boiling three times. After the solution cools, add water to bring the volume to 100mL. Store in a refrigerator at 4℃ and shake well before use.

[0186] After the drug administration was completed, another 10 mice from each group were used for the small bowel motility test. After fasting for 16 hours with free access to water, all mice except the control group were administered loperamide (4 mg / kg BW) by gavage. The control group received the same volume of physiological saline. 0.5 hours after gavage, all mice were administered 0.2 mL of ink by gavage, and timing began. The mice were euthanized by cervical dislocation immediately after 25 minutes. The abdominal cavity was opened, the mesentery was separated, and a section of the intestine from the pylorus to the ileocecal junction was cut and placed on a clean experimental table. The small intestine was gently stretched into a straight line, and the length of the intestinal section was measured as the "total small bowel length." The distance from the pylorus to the leading edge of the ink was measured as the "ink propulsion length."

[0187] Calculate the ink propulsion rate using the following formula:

[0188] Ink propulsion rate (%) = N / n × 100%

[0189] In the formula: N is the length of ink propulsion, and n is the total length of the small intestine, in cm.

[0190] 4.3.5 Experimental Results and Analysis

[0191] 4.3.5.1 Results of the experiment on defecation in each group of mice

[0192] The results are shown in Table 8 and Figure 1 .

[0193] Table 8. Defecation patterns of mice in each group

[0194]

[0195] Note: # indicates a significant difference compared to the blank group (P < 0.05), * indicates a significant difference compared to the model group (P < 0.05), and ** indicates a highly significant difference compared to the model group (P < 0.01).

[0196] 4.3.5.2 Results of the small intestinal propulsion test in each group of mice

[0197] The results are shown in Table 9 and Figure 2 .

[0198] Table 9 Results of small intestinal propulsion rate measurement in each group of mice

[0199]

[0200] Note: # indicates a significant difference compared to the blank group (P < 0.05), * indicates a significant difference compared to the model group (P < 0.05), and ** indicates a highly significant difference compared to the model group (P < 0.01).

[0201] 4.3.5.3 Results Analysis

[0202] Tables 8 and 9 show that the small intestinal propulsion rate, time to first black stool expulsion, number of black stool particles at 6 hours, and weight of black stool in the model group mice were significantly different from those in the control group (P < 0.05), indicating that the mouse constipation model induced by loperamide was successfully established. The time to first black stool expulsion in each bacterial powder combination group was significantly reduced compared to the constipation model group (P < 0.05), while the weight and number of black stool particles at 6 hours were increased compared to the model group. Furthermore, the small intestinal propulsion test results showed that each bacterial powder combination group was improved compared to the model group, indicating that the intervention with the bacterial powder of this invention can alleviate the constipation symptoms induced by loperamide in constipated mice.

[0203] Depend on Figure 1 , 2 It can be seen that the bacterial powder combination 8 has the strongest effect on improving various indicators of constipated mice. Its effect on improving various indicators of constipated mice is significantly better than that of bacterial powder combinations 1-7, and there is a significant or extremely significant difference compared with the mouse constipation model group. This indicates that the bacterial powder obtained by fermentation of a single bacterial species, or the compound bacterial powder obtained by fermentation of a single bacterial species, or the bacterial powder obtained by fermentation of less than four bacterial species, or the bacterial powder obtained by fermentation of four other bacterial species, is far less effective in relieving constipation than the compound bacterial powder obtained by fermentation of the combination of bacterial species. It also shows that the bacterial species in the compound probiotics of this invention can better exert a synergistic effect, thereby jointly promoting intestinal peristalsis and improving the constipation of patients.

[0204] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Lactobacillus acidophilus DH-La-166, characterized in that, Its accession number is CGMCC No. 13077.

2. A compound probiotic, characterized in that, Including Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium animalis, and Lactobacillus plantarum; The Lactobacillus acidophilus includes Lactobacillus acidophilus DH-La-166 as described in claim 1 and / or Lactobacillus acidophilus DH213 with accession number CGMCCNO.11228.

3. The compound probiotic according to claim 2, characterized in that, The ratio of viable counts of Lactobacillus acidophilus, Bifidobacterium animalis, Lactobacillus rhamnosus and Lactobacillus plantarum is (1~4):(1~4):(1~4):(1~4). Preferably, the compound probiotics include: Lactobacillus acidophilus DH-La-166, Bifidobacterium animalis DH216, Lactobacillus rhamnosus DH-Lr-121 and Lactobacillus plantarum DH-Lp-22; Or include: Lactobacillus acidophilus DH213, Bifidobacterium animalis DH216, Lactobacillus rhamnosus DH-Lr-121 and Lactobacillus plantarum DH-Lp-22; The preservation number of the animal Bifidobacterium DH216 is CGMCC NO.13232; The preservation number of *Lactobacillus rhamnosus* DH-Lr-121 is CGMCC NO. 13076; The preservation number of the *Lactobacillus plantarum* DH-Lp-22 is CGMCC NO.13074.

4. A culture medium combination for culturing Lactobacillus acidophilus as described in claim 1 or the compound probiotics as described in any one of claims 2-3, characterized in that, Including seed culture media and / or fermentation culture media; The seed culture medium is selected from at least one of the following: Peptone 3~15g / L, beef extract 3~15g / L, yeast extract 2~10g / L, diammonium hydrogen citrate 0.5~5g / L, glucose 5~25g / L, Tween-80 0.5~3g / L, sodium acetate 2~10g / L, dipotassium hydrogen phosphate 1~5g / L, magnesium sulfate 0.1~1.5g / L, manganese sulfate 0.1~1g / L, cysteine ​​0.1~2g / L, and water; Ingredients: 3-15 g / L peptone, 3-15 g / L beef extract, 2-10 g / L yeast extract, 0.5-5 g / L diammonium citrate, 5-25 g / L glucose, 0.5-3 g / L Tween-80, 2-10 g / L sodium acetate, 1-5 g / L dipotassium hydrogen phosphate, 0.1-1.5 g / L magnesium sulfate, 0.1-1 g / L manganese sulfate, 0.1-2 g / L tomato extract, and water; Tryptone 3~15g / L, beef extract 3~15g / L, yeast extract 2~10g / L, diammonium hydrogen citrate 0.5~5g / L, glucose 5~25g / L, Tween-80 0.5~3g / L, sodium acetate 2~10g / L, dipotassium hydrogen phosphate 1~5g / L, magnesium sulfate 0.1~1.5g / L, manganese sulfate 0.1~1g / L, and water; Soy protein peptone 3~15g / L, beef extract 3~15g / L, yeast extract 2~10g / L, diammonium hydrogen citrate 0.5~5g / L, glucose 5~25g / L, Tween-80 0.5~3g / L, sodium acetate 2~10g / L, dipotassium hydrogen phosphate 1~5g / L, magnesium sulfate 0.1~1.5g / L, manganese sulfate 0.1~1g / L, and water; The fermentation medium comprises water and the following components: Peptone 15-40 g / L, beef extract 10-30 g / L, yeast extract 5-30 g / L, glucose 5-30 g / L, sucrose 10-30 g / L, fructooligosaccharides 1-10 g / L, Tween-80 0.5-3 g / L, sodium acetate 2-10 g / L, dipotassium hydrogen phosphate 1-5 g / L, magnesium sulfate 0.1-1.5 g / L, manganese sulfate 0.1-1 g / L, calcium carbonate 5-15 g / L, cysteine ​​0.5-5 g / L, and tomato extract 0.5-5 g / L.

5. A fermentation method for Lactobacillus acidophilus, characterized in that, include: The Lactobacillus acidophilus of claim 1 or the compound probiotics of any one of claims 2 to 3 are inoculated into the seed culture medium of the culture medium combination of claim 4 and cultured to obtain seed liquid; The seed culture is inoculated into the fermentation medium for fermentation.

6. The fermentation method according to claim 5, characterized in that, The fermentation includes an early fermentation stage and a middle and late fermentation stage; the early fermentation stage is from 0 to 3 hours of fermentation, with a temperature of 38 to 40°C; the middle and late fermentation stage is from 3 hours of fermentation to the end of fermentation, with a temperature of 35 to 37°C. The pH of the fermentation is 5.0~8.0; Preferably, the fermentation process further includes a step of adding a carbon source and / or a nitrogen source; The amount of carbon source added is such that the sugar concentration in the fermentation broth is 0.05~10 g / L. The amount of nitrogen source added is such that the concentration of amino acids in the fermentation broth is 0.05~5 g / L.

7. The fermentation product obtained by the fermentation method according to any one of claims 5 to 6.

8. The use of Lactobacillus acidophilus as described in claim 1, the compound probiotic as described in any one of claims 2 to 3, or the fermentation product as described in claim 7 in the preparation of products that help with bowel movements.

9. A product that helps with bowel movements, characterized in that: It includes Lactobacillus acidophilus as described in claim 1, the compound probiotic as described in any one of claims 2 to 3, or the fermentation product as described in claim 7, and acceptable adjuvants.

10. A compound probiotic powder, characterized in that, It is produced by freeze-drying water and the following raw materials: The compound probiotics according to any one of claims 2 to 3 or the fermentation product, skim milk powder, trehalose and ascorbic acid according to claim 7; Preferably, the compound probiotic powder is prepared by freeze-drying the following raw materials in parts by weight: 30 to 100 parts of the compound probiotics as described in any one of claims 2 to 3 or the fermentation product as described in claim 7, 10 parts of skim milk powder, 5 parts of trehalose, 0.5 parts of ascorbic acid and 84.5 parts of water.

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