Compound probiotic for lowering blood lipid and regulating constipation and application thereof in resistant starch fermented milk

Abstract

The application discloses a compound probiotic for lowering blood lipid and regulating constipation and application of the compound probiotic in resistant starch fermented milk, and belongs to the technical field of functional food. The compound probiotic is composed of Bifidobacterium animalis IU100 and Lacticaseibacillus paracasei ZYhyy-004, and has a synergistic effect. The compound probiotic is used for preparing resistant starch fermented milk, can significantly reduce the levels of serum total cholesterol, triglyceride and low-density lipoprotein cholesterol, increase the content of high-density lipoprotein cholesterol, enhance the intestinal propulsion rate and improve functional constipation. The fermented milk can regulate intestinal flora balance, promote the proliferation of beneficial bacteria and inhibit the growth of pathogenic bacteria, and realizes the dual effects of lowering blood lipid and improving intestinal function. The application provides a new way for developing multifunctional probiotic food.

Description

Technical Field

[0001] This invention relates to the fields of microorganisms and dairy processing, specifically to a compound probiotic that lowers blood lipids and regulates constipation, and its application in resistant starch fermented milk. Background Technology

[0002] Dyslipidemia and functional constipation have become two common and increasingly serious health problems. High-fat, high-sugar, and high-calorie diets have led to a continuous rise in the prevalence of dyslipidemia, especially high cholesterol and triglyceride intake, increasing the risk of cardiovascular disease. Meanwhile, functional constipation also plagues a large population, affecting their quality of life and digestive health. Studies have shown that probiotics play a significant role in regulating blood lipids and improving gut health. Probiotics such as lactobacilli and bifidobacteria can effectively lower total cholesterol and low-density lipoprotein (LDL) cholesterol in the blood by promoting bile acid excretion and inhibiting cholesterol absorption, thus helping to lower blood lipid levels. Furthermore, probiotics help maintain the balance of gut microbiota, reduce intestinal inflammation, and improve gut health, thereby indirectly regulating blood lipids while alleviating symptoms of functional constipation.

[0003] Fermented milk products incorporating type III resistant starch offer dual health benefits as an innovative probiotic food. Type III resistant starch, as an effective prebiotic, promotes the growth of beneficial bacteria in the gut, regulates gut microbiota, and improves gut microecological balance, thereby helping to alleviate functional constipation. Simultaneously, resistant starch lowers blood cholesterol and triglyceride levels by slowing carbohydrate digestion and absorption, further promoting cardiovascular health. Compared to traditional fermented milk products, fermented milk made with type III resistant starch fermented by probiotics such as Bifidobacterium not only increases the quantity and activity of probiotics, improving gut health, but also enhances its lipid-lowering function. Through this innovative combination of probiotics and resistant starch, this fermented milk product effectively addresses two major health issues: dyslipidemia and functional constipation, providing a healthier and more functional dairy product option that meets the needs of modern consumers for comprehensive health management. Summary of the Invention

[0004] To address the shortcomings and practical needs of existing resistant starch fermented milk, this invention provides a compound probiotic that lowers blood lipids and regulates constipation, and its application in resistant starch fermented milk. The study investigates the effects of Bifidobacterium animalis IU100 + Lactobacillus paracasei ZYhyy-004 resistant starch fermented milk on improving blood lipids in a hyperlipidemic mouse model and functional constipation in constipated mice. Animal experiments were conducted to detect serum cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglyceride levels. The effects of Bifidobacterium animalis IU100 and Lactobacillus paracasei ZYhyy-004 resistant starch fermented milk on improving blood lipids in hyperlipidemic mice were evaluated. The effect of Bifidobacterium animalis IU100 + Lactobacillus paracasei ZYhyy-004 resistant starch fermented milk on improving functional constipation in constipated mice was evaluated by the time to first black stool excretion, fecal volume within 6 hours, fecal water content, and intestinal propulsion rate.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] This invention provides a strain of Bifidobacterium animalis subsp. IU-100, which was deposited on September 5, 2016, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China. The strain is classified as Bifidobacterium animalis and its accession number is CGMCC No. 12942.

[0007] The present invention provides a method for culturing Bifidobacterium animalis IU-100, characterized in that Bifidobacterium animalis IU-100 is cultured in a modified MRS liquid medium at 37±1℃ and 0% oxygen concentration for 11h.

[0008] The present invention provides the above-mentioned improved MRS liquid culture medium suitable for Bifidobacterium animalis IU-100, the formula of which is: 5g anhydrous sodium acetate, 2g diammonium hydrogen citrate, 2g dipotassium hydrogen phosphate, 0.58g magnesium sulfate, 0.19g manganese sulfate tetrahydrate, 1mL Tween 80, 20g glucose, 10g peptone, 5g yeast powder, 5g beef meal, 0.5g cysteine ​​salt, and the pH is adjusted to 6.5.

[0009] This invention provides a strain of *Lactobacillus paracasei* ZYhyy-004, which was deposited on August 5, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China. The strain is classified as *Lactobacillus paracasei*, with accession number CGMCC No. 31554.

[0010] The present invention provides a method for culturing Lactobacillus paracasei ZYhyy-004, characterized in that Lactobacillus paracasei ZYhyy-004 is cultured in a modified MRS liquid medium at 37±1℃ for 10h.

[0011] This invention provides the above-mentioned improved MRS liquid culture medium suitable for Lactobacillus paracasei ZYhyy-004, the formulation of which is: 5g anhydrous sodium acetate, 2g diammonium hydrogen citrate, 2g dipotassium hydrogen phosphate, 0.58g magnesium sulfate, 0.19g manganese sulfate tetrahydrate, 1mL Tween 80, 20g glucose, 10g peptone, 5g yeast powder, 5g beef meal, and the pH is adjusted to 6.5.

[0012] On the other hand, the present invention provides a compound microbial agent comprising the above-mentioned Bifidobacterium animalis subsp. lactis IU100 and Lactobacillus paracasei ZYhyy-004, and the application of the compound microbial agent.

[0013] On the other hand, the present invention provides the application of *Bifidobacterium animalis* subsp. *lactospirum* IU100 and *Lactobacillus paracasei* ZYhyy-004 in the preparation of products. Preferably, the product is a food product, more preferably a dairy product, further preferably yogurt or fermented milk, and further preferably a fermented milk for lowering hyperlipidemia.

[0014] Preferably, the amount of Bifidobacterium animalis subsp. lactis IU100 added to the product is not less than 1×10⁻⁶. 6 The concentration of CFU / mL and the addition level of *Lactobacillus paracasei* ZYhyy-004 should not be less than 5 × 10⁻⁶ CFU / mL. 5 CFU / mL 1×10 7 CFU / mL; preferably, the addition amount of Bifidobacterium animalis subsp. lactis IU100 is 1×10⁻⁶. 6 ~10 9 The concentration of CFU / mL and the addition amount of *Lactobacillus paracasei* ZYhyy-004 was 5 × 10⁻⁶. 5 ~5×10 7 CFU / mL; preferably, the ratio of Bifidobacterium animalis subsp. lactis IU100 to Lactobacillus paracasei ZYhyy-004 is (1-2):(2-1), and more preferably (0.8-1.2):(0.4-0.6). Preferably, the amount of Bifidobacterium animalis subsp. lactis IU100 added is 5×10⁻⁶. 6 –2×10 7 CFU / mL; the bacterial count of *Lactobacillus paracasei* ZYhyy-004 was 1×10⁻⁶. 6 –1×10 7 CFU / mL.

[0015] On the other hand, this invention provides the application of the above-mentioned compound microbial agent in fermented milk products for regulating functional constipation. Preferably, the amount of Bifidobacterium animalis subsp. lactis IU100 added is 5 × 10⁻⁶. 6 –2×10 7 CFU / mL; the bacterial count of *Lactobacillus paracasei* ZYhyy-004 was 1×10⁻⁶. 6 –1×10 7 CFU / mL; preferably, the amount of Bifidobacterium animalis subsp. lactis IU100 added to the product is 1×10⁻⁶ CFU / mL. 7 The concentration of CFU / mL and the addition amount of *Lactobacillus paracasei* ZYhyy-004 was 5 × 10⁻⁶. 6 CFU / mL.

[0016] On the other hand, the present invention provides a resistant starch fermented milk and its application in lowering hyperlipidemia. Preferably, the fermented milk base contains 1%-2% pea RS3 resistant starch.

[0017] Additionally, a fermented milk base is provided, comprising the aforementioned compound microbial agent. The fermented milk base is a combination of pure milk and pea type III resistant starch. Preferably, the milk base is obtained by adding 1%-2% pea type III resistant starch to pure milk, dissolving and homogenizing it, then pasteurizing it at 95°C for 5 minutes, and cooling it to 43°C. The aforementioned milk base contains a starter culture, preferably comprising Bifidobacterium animalis IU100 and Lactobacillus paracasei ZYhyy-004 at the aforementioned concentrations or ratios, as well as a basic starter culture, which includes Streptococcus salivarius subsp. thermophilus and Lactobacillus bulgaricus.

[0018] On the other hand, this invention provides the application of compound microbial agents in preparing resistant starch fermented milk, and the application of resistant starch fermented milk prepared with compound microbial agents in fermented milk products that reduce hyperlipidemia and regulate functional constipation. Preferably, the product is either fermented milk or yogurt.

[0019] Preferably, the fermented milk uses a basic starter culture, which is a compound microbial agent containing the above-mentioned microbial agent. The amount of basic starter culture added is 0.005‰-0.04‰ (weight percentage) of the milk base, preferably 0.01‰-0.03‰. The amount of single or multiple microbial agents composed of Bifidobacterium animalis IU100 and / or Lactobacillus paracasei ZYhyy-004 added is 0.005‰-0.04‰ (weight percentage) of the milk base, preferably 0.01‰-0.03‰, more preferably 0.015‰-0.025‰.

[0020] In another aspect of the present invention, the activation conditions for Bifidobacterium animalis IU100 and Lactobacillus paracasei ZYhyy-004 are as follows: Bifidobacterium animalis subsp. lactis IU100 or Lactobacillus paracasei ZYhyy-004 stored at -80°C are cultured in a modified MRS liquid medium at 37±1°C and 0% oxygen concentration for 9-11 hours to activate the strains.

[0021] The compound microbial agent is fermented on a milk base containing resistant starch for 5-6 hours.

[0022] Fermented milk or resistant starch fermented milk prepared with the above-mentioned compound microbial agent has the effect of reducing blood lipids in hyperlipidemic mouse models / patients; fermented milk or resistant starch fermented milk prepared with the above-mentioned compound microbial agent has the effect of regulating functional constipation in constipated mouse models / patients.

[0023] In another aspect of the present invention, the compound microbial agent or the fermented milk prepared therefrom simultaneously has the application of lowering blood lipids and regulating functional constipation. Further, lowering blood lipids refers to lowering serum cholesterol, low-density lipoprotein cholesterol, and triglycerides. Further, it includes increasing high-density lipoprotein cholesterol.

[0024] Furthermore, the study modulated the time to first black stool, stool volume within 6 hours, stool water content, and intestinal propulsion rate in a mouse model of functional constipation. This also included the intestinal propulsion rate in patients.

[0025] In another aspect, the present invention provides the use of the aforementioned *Bifidobacterium animalis* IU100 and *Lactobacillus paracasei* ZYhyy-004 in the preparation of resistant starch fermented milk. Preferably, the fermented milk is yogurt or fermented milk. The advantages of the present invention compared to the prior art are:

[0026] (1) This invention provides a strain of Bifidobacterium animalis subsp. lactis IU100, with accession number CGMCC No.12942; and a strain of Lactobacillus paracasei ZYhyy-004, with accession number CGMCC No.31554.

[0027] Fermented milk prepared from Bifidobacterium animalis subsp. lactis IU100 and Lactobacillus paracasei ZYhyy-004 reduced serum cholesterol, triglycerides, and low-density lipoprotein cholesterol by 20.3%, 26.3%, and 34.2% in hyperlipidemic mice, while increasing high-density lipoprotein cholesterol by 23.2%.

[0028] Bifidobacterium animalis subsp. lactis IU100 and Lactobacillus paracasei ZYhyy-004 reduced the time to first black stool excretion by 73 min in constipated mice, increased fecal volume by 18 stools within 6 hours, increased fecal water content by 17.3%, and increased intestinal propulsion rate by 28.3%.

[0029] (2) The present invention provides a basic fermented milk that can reduce the time of first black excretion of constipated mice by 58.5 min, increase the amount of feces by 7 within 6 hours, increase the water content of feces by 9.3%, and increase the intestinal propulsion rate by 15.4%.

[0030] (3) This invention provides a resistant starch fermented milk, specifically a fermented milk containing 1%-2% pea RS3 resistant starch. It can reduce serum cholesterol, triglycerides, and low-density lipoprotein cholesterol in hyperlipidemic mice by 22.1%, 27.6%, and 30.7%, respectively, while increasing high-density lipoprotein cholesterol by 28.1%. It can also reduce the time to first black stool excretion in constipated mice by 61 minutes, increase fecal volume by 9 stools within 6 hours, increase fecal water content by 10%, and increase intestinal propulsion rate by 17.7%.

[0031] (4) The present invention provides a resistant starch-fermented milk containing *Bifidobacterium animalis* subsp. *lactotrium* IU100 and *Lactobacillus paracasei* ZYhyy-004, which can reduce serum cholesterol, triglycerides, and low-density lipoprotein cholesterol by 32.4%, 35.9%, and 47.4%, respectively, while increasing high-density lipoprotein cholesterol by 42.7% in hyperlipidemic mice. It can also reduce the time to first black stool excretion in constipated mice by 77.5 min, increase fecal volume by 19 stools within 6 hours, increase fecal water content by 18.3%, and increase intestinal propulsion rate by 32%. Furthermore, the resistant starch-fermented milk containing *Bifidobacterium animalis* subsp. *lactotrium* IU100 and *Lactobacillus paracasei* ZYhyy-004 contributes to the balance of intestinal microbial ecology and avoids environmental pollution caused by antibiotic abuse, showing broad application prospects. Attached Figure Description

[0032] Figure 1 Comparison of body weight changes in mouse models. w represents weeks.

[0033] Figure 2 Effects of each intervention group on serum total cholesterol and triglycerides in mouse models.

[0034] Figure 3 Effects of each intervention group on low-density lipoprotein cholesterol and high-density lipoprotein cholesterol in mouse models.

[0035] Figure 4 The effects of each intervention group on the time of first black pellet excretion and the number of fecal pellets at 6 hours in mouse models.

[0036] Figure 5 The effects of each intervention group on fecal water content and intestinal propulsion rate in mouse models. Detailed Implementation

[0037] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0038] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0039] Bifidobacterium animalis subsp. lactis IU100 was isolated from samples from a Shandong dairy company, while Lactobacillus paracasei ZYhyy-004 was isolated from samples from Henan.

[0040] Example 1: Activation of bacterial strains

[0041] Preparation of Bifidobacterium animalis subsp. lactis IU100 cells according to the present invention: After two subcultures in modified MRS liquid medium, Bifidobacterium animalis subsp. lactis IU100 was inoculated into modified MRS liquid medium at an inoculum of 2% (v / v) and cultured at 37±1℃ and 0% oxygen for 11 h. The supernatant was removed by centrifugation (4000 rpm, 10 min), and the cells were washed twice with PBS. Before gavage, the cells were resuspended in 0.85% physiological saline and the bacterial suspension concentration was adjusted to 1×10⁻⁶. 10 CFU / mL.

[0042] Preparation of *Lactobacillus paracasei* ZYhyy-004 cells according to the present invention: *Lactobacillus paracasei* ZYhyy-004 was passaged twice in modified MRS liquid medium, and then inoculated into modified MRS liquid medium at an inoculum of 1% (v / v). The culture was carried out at 37±1℃ and 0% oxygen for 10 h. The supernatant was removed by centrifugation (5000 rpm, 8 min), and the cells were washed twice with PBS. Before gavage, the cells were resuspended in 0.85% physiological saline, and the bacterial suspension concentration was adjusted to 1×10⁻⁶. 10 CFU / mL.

[0043] Example 2: Preparation of resistant starch fermented milk

[0044] Preparation of fermented milk base: 1%-2% resistant starch is added to pure milk at a mass ratio, and stirred at 500 r / min for 30-50 min at 50℃-55℃ to obtain a mixed emulsion; the mixed emulsion is homogenized at 15-20 MPa at 50℃-55℃ to obtain a homogeneous mixed emulsion; the homogeneous mixed emulsion is sterilized at 95℃ for 5 min and cooled to 43℃ to obtain the fermented milk base.

[0045] The basic starter culture consists of Streptococcus thermophilus and Lactobacillus bulgaricus, which is added to the fermented milk base at a ratio of 25g / t for fermentation.

[0046] The preparation methods for each intervention group are as follows:

[0047] Basic fermented milk group: The basic starter culture is added to the fermented milk base at a ratio of 25g / t for fermentation.

[0048] Resistant starch fermented milk group: 1%-2% pea type III resistant starch was added to the fermented milk base, and the rest was the same as the basic fermented milk group.

[0049] Bifidobacterium animalis subsp. lactis IU100 group (IU100 fermented milk group): Bifidobacterium animalis subsp. lactis IU100 was added to the fermented milk base, with a final addition amount of 1×10⁻⁶. 7 CFU / g milk base. Other parameters are the same as those in the basic fermented milk group.

[0050] Compound microbial agent group (IU100+ZYhyy-004): After culturing Lactobacillus paracasei ZYhyy-004 and Bifidobacterium animalis subsp. lactis IU100 separately, a compound microbial agent was prepared at a bacterial volume ratio of 1:2.

[0051] IU100+ZYhyy-004 resistant starch fermented milk group: 1%-2% pea type III resistant starch was added to the fermented milk base. *Lactobacillus paracasei* ZYhyy-004 and *Bifidobacterium animalis* subsp. lactis IU100 were cultured separately and then a compound bacterial agent was prepared at a 1:2 ratio. The final addition of *Bifidobacterium animalis* subsp. lactis IU100 to the milk base was 1×10⁻⁶. 7 CFU / g milk base, the final addition amount of Lactobacillus paracasei ZYhyy-004 is 5×10⁻⁶. 6 CFU / g milk base. Other parameters are the same as those in the basic fermented milk group.

[0052] After the above intervention groups were properly prepared, except for the compound microbial agent group which did not require fermentation, the other four groups were fermented at 42±1℃ for 5-6 hours to obtain fermented milk for each group.

[0053] During gavage, each group received 200 μL of the solution.

[0054] Example 3: Animal Model of Hyperlipidemia

[0055] The experimental animals were SPF-grade BALB / c (6 weeks old, female) mice purchased from Beijing Huafukang Biotechnology Co., Ltd. The animal experiments were divided into 10 groups: blank group, model group, drug group, basic fermented milk group, resistant starch fermented milk group, IU100 fermented milk group, and Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy-004 (1×10⁻⁶). 9 The CFU / mL group and the Bifidobacterium animalis subsp. lactis IU100+ Lactobacillus paracasei ZYhyy-004 resistant starch fermented milk group each consisted of 10 mice, for a total of 70 mice.

[0056] Mice were housed at 24℃ with a controlled 12-hour light and 12-hour dark cycle, and their bedding was changed every three days. After a week of acclimatization, they were fed a high-fat diet daily (66.5 kg basal diet, 20 kg sucrose, 1 kg sodium cholate, 10 kg lard, and 2.5 kg cholesterol), and weighed weekly. Daily food intake was monitored, and weight changes were tracked weekly. The control group was administered 0.9% saline via gavage until the end of the experiment. After the last gavage, mice were fasted for 12 hours but allowed free access to water. Blood was collected from the orbital veins of the mice, and successful modeling was indicated by total cholesterol (TC) and triglycerides (TG) levels being 3-4 times higher than in the control group.

[0057] Model group and intervention groups: Initially fed a high-fat diet daily until successful model establishment (4-8 weeks). After successful model establishment, the model group and intervention groups were administered 1×10⁻⁶ mg / L via gavage every other day. 9 CFU / g probiotics (Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy-004 prepared at a 1:2 ratio, also prepared as 200ul) or 200ul fermented milk until the end of the experiment; at the same time, the drug group was given 5mg / kg lovastatin by gavage for 4-8 weeks.

[0058] Example 4: Detection of serum cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides

[0059] During the experiment, mice were allowed free access to food and water. They were weighed every 7 days. After the last gavage, mice were fasted for 12 hours but allowed free access to water. Blood was then collected from the orbital veins of the mice using heparin anticoagulant tubes. The blood was centrifuged at 4000 r / min for 3 min at 4°C. Serum was collected and total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured using an animal-specific fully automated biochemical analyzer.

[0060] Weight changes such as Figure 1As shown, in the 7 groups of mice, the body weight of the mice increased with the increase of the cycle. The body weight of the model group mice reached 37.71g in the sixth week, which was significantly higher than that of the control group (p<0.01), indicating that the high-fat diet can significantly increase the body weight of mice and the experimental model was successful.

[0061] Compared with the model group, the weight of mice in the statin group at week 6 was 34.37 g, which was significantly lower than that of the model group (p<0.05); there was no significant difference in weight between the basal fermented milk group and the resistant starch fermented milk group; the weights of mice in the resistant starch fermented milk group and the IU100 fermented milk group at week 6 were 34.72 g and 34.80 g, respectively, showing significant differences (p<0.05); the weight of mice in the Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy-004 group at week 6 was 34.72 g, showing a significant difference (p<0.05); the weight of mice in the Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy-004 resistant starch fermented milk group at week 6 was 33.66 g, showing a significant difference (p<0.01).

[0062] The results showed that statins, resistant starch fermented milk, IU100 fermented milk group, compound microbial agent group (IU100+ZYhyy-004), and Bifidobacterium animalis subsp. lactis IU100+Lactobacillus paracasei ZYhyy-004 resistant starch fermented milk could all effectively inhibit the increase of mouse body weight.

[0063] Serum total cholesterol results as follows Figure 2 As shown in Figure A, among the seven groups of mice, the serum total cholesterol in the model group was 3.49 mmol / L, which was significantly higher than that in the control group (p<0.01). Compared with the model group, the serum total cholesterol in the statin group was 2.31 mmol / L, which was significantly lower than that in the model group (p<0.01). The serum total cholesterol in the Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy-004 resistant starch fermented milk group was 2.36 mmol / L, which was significantly lower than that in the model group (p<0.01), achieving almost the same total cholesterol-lowering function as the drug group. There were no significant differences among the basic fermented milk group, the resistant starch fermented milk group, the IU100 fermented milk group, and the compound probiotic group.

[0064] Triglyceride results as follows Figure 2As shown in Figure B, the triglyceride level in the model group mice was 1.56 mmol / L, which was significantly higher than that in the control group (p<0.01). Compared with the model group mice, the triglyceride level in the statin drug group mice was 1.04 mmol / L, which was significantly lower than that in the model group (p<0.01). The triglyceride level in the Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy-004 resistant starch fermented milk group mice was 1.00 mmol / L, which was significantly lower than that in the model group (p<0.01). There were no significant differences between the basal fermented milk group, the resistant starch fermented milk group, the IU100 fermented milk group, and the compound probiotic group.

[0065] The basic fermented milk group can reduce serum total cholesterol levels to a certain extent. Adding resistant starch (resistant starch fermented milk group) can slightly enhance the effect, while using IU100 fermented milk has no significant effect. Adding only a combination of microorganisms has a similar effect to the basic fermented milk group. However, the fermented milk group prepared by fermenting resistant starch with only two microorganisms can achieve a lipid-lowering function similar to that of drugs.

[0066] Low-density lipoprotein cholesterol results as follows Figure 3 As shown in Figure A, among the seven groups of mice, the LDL cholesterol level in the model group was 1.14 mmol / L, which was significantly higher than that in the control group (p<0.01). Compared with the model group, the LDL cholesterol level in the statin group was 0.63 mmol / L, which was significantly lower than that in the model group (p<0.01). The LDL cholesterol levels in the resistant starch fermented milk group and the control group were 0.79 mmol / L and 0.76 mmol / L, respectively. The LDL cholesterol content in mice in the *Bifidobacterium animalis* subsp. *milk* IU100 + *Lactobacillus paracasei* ZYhyy-004 group was 0.75 mmol / L, which was significantly lower than that in the model group (p<0.05). The LDL cholesterol content in mice in the *Bifidobacterium animalis* subsp. *milk* IU100 + *Lactobacillus paracasei* ZYhyy-004 resistant starch fermented milk group was 0.6 mmol / L, which was extremely significantly lower than that in the model group (p<0.01).

[0067] High-density lipoprotein cholesterol results as follows Figure 3As shown in B, among the seven groups of mice, the high-density lipoprotein cholesterol (HDL-C) level in the model group was 0.82 mmol / L, which was significantly lower than that in the control group (p<0.01). Compared with the model group, the HDL-C level in the statin group was 1.13 mmol / L, which was significantly higher than that in the model group (p<0.05). The HDL-C levels in the resistant starch fermented milk and IU100 fermented milk groups were 1.05 mmol / L and 1.03 mmol / L, respectively, which were significantly higher than those in the model group (p<0.05). The HDL-C level in the Bifidobacterium animalis subsp. lactis IU100 group was 1.01 mmol / L, which was significantly higher than that in the model group (p<0.05). The HDL-C level in the Bifidobacterium animalis subsp. lactis IU100 resistant starch fermented milk group was 1.17 mmol / L, which was significantly higher than that in the model group (p<0.05).

[0068] Example 5: Functional constipation animal model

[0069] The experimental animals were SPF-grade BALB / c (6 weeks old, male) mice, purchased from Spiford (Beijing) Biotechnology Co., Ltd. The animal experiments were divided into 10 groups: blank group, model group, drug group, basal fermented milk group, resistant starch fermented milk group, *Bifidobacterium animalis* subsp. *lactotrichum* IU100 and *Lactobacillus paracasei* ZYhyy-004 (1×10⁻⁶). 9 The mice were divided into three groups: a CFU / mL group, a Bifidobacterium animalis subsp. lactis IU100 group, and a Lactobacillus paracasei ZYhyy-004 resistant starch fermented milk group, with 10 mice in each group, for a total of 70 mice.

[0070] Mice were housed at a controlled temperature of 23±2℃ and humidity of 60±5%, with bedding changed every three days. After one week of acclimatization, except for the control group, mice in other groups were treated with loperamide at 15 mg / kg / d for 14 days to induce constipation. The gavage dose was 0.1 mL / 20 g. The establishment of the constipation model was determined by the time of the first black vomiting, the number of fecal particles at 6 hours, and the fecal water content. A successful constipation model in BALB / c male mice was indicated when the time of the first black vomiting was twice or more than that of the control group, the decrease in fecal water content was >15%, and the decrease in the number of fecal particles at 6 hours was more than 15.

[0071] Model group and intervention groups: After successful modeling, the model group and intervention groups were administered 1×10⁻⁶ gavage every other day. 9CFU / g probiotics (Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy-004 prepared at a 1:2 ratio, which can also be prepared into a 200ul bacterial solution) or 200ul fermented milk until the end of the experiment.

[0072] Example 6: Detection of first black stool time, fecal volume within 6 hours, fecal water content, and intestinal propulsion rate in a functional constipation animal model.

[0073] After 14 days of intervention, mice were fasted for 12 hours, then administered 15% carmine by gavage. The number of fecal pellets and the time of the first black pellet excreted within six hours were recorded. Finally, feces collected over six hours were weighed ("A"), dried, and weighed again ("B"). The fecal moisture content percentage (%) was calculated as (AB) / A × 100%. Mice were sacrificed 30 minutes after gavage with 15% carmine. The entire small intestine was then removed and stretched into a straight line; its length was measured ("a"), and the travel distance of activated charcoal was measured ("b"). The gastrointestinal transit ratio (%) was calculated as b / a × 100%.

[0074] The results of the first black spot removal time are as follows Figure 4 As shown in Figure A, among the seven groups of mice, the time to first blackening of the pellet in the model group was 148 min, which was significantly higher than that in the control group (p<0.05). Compared with the model group, the time to first blackening of the pellet in the loperamide group was 71 min, which was significantly lower than that in the model group (p<0.05). The time to first blackening of the pellet in the basal fermented milk group was 89.5 min, which was significantly lower than that in the model group (p<0.05). The time to first blackening of the pellet in the resistant starch fermented milk group and the 0.79 mmol / L group was 8... At 7 min and 89 min, the time to first black pellet expulsion was significantly shorter than that in the model group (p<0.05). In the group with *Bifidobacterium animalis subsp. lactis* IU100 + *Lactobacillus paracasei* ZYhyy-004, the time to first black pellet expulsion was 75 min, significantly shorter than that in the model group (p<0.05). In the group with *Bifidobacterium animalis subsp. lactis* IU100 + *Lactobacillus paracasei* ZYhyy-004 resistant starch fermented milk, the time to first black pellet expulsion was 70.5 min, significantly shorter than that in the model group (p<0.05). All four intervention groups significantly reduced the time to first black pellet expulsion in constipated mice. The group with *Bifidobacterium animalis subsp. lactis* IU100 + *Lactobacillus paracasei* ZYhyy-004 resistant starch fermented milk showed the most significant effect, with the time to first black pellet expulsion significantly shorter than that in the basal fermented milk and resistant starch fermented milk groups.

[0075] Results of stool volume over 6 hours Figure 4As shown in Figure B, among the seven groups of mice, the model group had 48 fecal pellets within 6 hours, significantly lower than the control group (p<0.05). Compared with the model group, the loperamide group had 65 fecal pellets within 6 hours, significantly higher than the model group (p<0.05). The basal fermented milk group had 55 fecal pellets within 6 hours, with no significant difference from the model group (p>0.05). The resistant starch fermented milk group and the IU100 fermented milk group had the following fecal pellet counts within 6 hours: The number of feces in mice in the groups with 57 and 55 feces within 6 hours was not significantly different from that in the model group (p>0.05); the number of feces in mice in the group with Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy-004 within 6 hours was 64 feces, which was significantly higher than that in the model group (p<0.05); the number of feces in mice in the group with Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy-004 resistant starch fermented milk within 6 hours was 67 feces, which was significantly higher than that in the model group (p<0.05). Among the four intervention groups, the Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy-004 and the Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy-004 resistant starch fermented milk group significantly reduced fecal volume in constipated mice within 6 hours, with the Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy-004 resistant starch fermented milk group showing the most significant effect.

[0076] The results of fecal water content are as follows Figure 5 As shown in Figure A, among the seven groups of mice, the fecal water content of the model group was 47.7%, significantly lower than that of the control group (p<0.05); compared with the model group, the fecal water content of the loperamide group was 65.7%, significantly higher than that of the model group (p<0.05); the fecal water content of the basal fermented milk group was 57%, significantly higher than that of the model group (p<0.05); the fecal water content of the resistant starch fermented milk group and the IU100 fermented milk group were 57% and 57%, respectively. The fecal water content in the *Bifidobacterium animalis* subsp. lactis IU100 + *Lactobacillus paracasei* ZYhyy-004 group was 65%, significantly higher than that in the model group (p<0.05); the fecal water content in the *Bifidobacterium animalis* subsp. lactis IU100 + *Lactobacillus paracasei* ZYhyy-004 resistant starch fermented milk group was 66%, significantly higher than that in the model group (p<0.05). All four intervention groups significantly increased the fecal water content in constipated mice, with the *Bifidobacterium animalis* subsp. lactis IU100 + *Lactobacillus paracasei* ZYhyy-004 resistant starch fermented milk group showing the most significant effect.

[0077] Results of intestinal propulsion rate as follows Figure 5 As shown in Figure B, among the seven groups of mice, the intestinal propulsion rate of the model group was 53.3%, significantly lower than that of the control group (p<0.05); compared with the model group, the intestinal propulsion rate of the loperamide group was 93.5%, significantly higher than that of the model group (p<0.05); the intestinal propulsion rate of the basal fermented milk group was 68.7%, significantly higher than that of the model group (p<0.05); the intestinal propulsion rates of the resistant starch fermented milk group and the IU100 fermented milk group were 7... The intestinal propulsion rate in the *Bifidobacterium animalis* subsp. lactis IU100 + *Lactobacillus paracasei* ZYhyy-004 group was 81.6%, significantly higher than the model group (p<0.05). The intestinal propulsion rate in the *Bifidobacterium animalis* subsp. lactis IU100 + *Lactobacillus paracasei* ZYhyy-004 resistant starch fermented milk group was 85.3%, significantly higher than the model group (p<0.05). All four intervention groups significantly increased the intestinal propulsion rate in constipated mice, with the *Bifidobacterium animalis* subsp. lactis IU100 + *Lactobacillus paracasei* ZYhyy-004 resistant starch fermented milk group showing the most significant effect.

[0078] In summary, *Bifidobacterium animalis* subsp. lactis IU100 and *Lactobacillus paracasei* ZYhyy-004 exhibit synergistic fermentation capabilities in resistant starch-based fermented milk. The fermented milk prepared through this synergistic fermentation demonstrates superior lipid-lowering and functional constipation-relieving effects compared to single-strain fermentation, bacterial cell addition, or resistant starch addition. Therefore, *Bifidobacterium animalis* subsp. lactis IU100 and *Lactobacillus paracasei* ZYhyy-004 exhibit a significant synergistic effect in the fermentation of resistant starch-based fermented milk. Their mutual cooperation and promotion not only enhance fermentation efficiency but also significantly improve the functionality of the fermented milk. The resulting resistant starch-based fermented milk not only excels in lowering blood lipids and relieving functional constipation but also demonstrates excellent potential health benefits such as regulating intestinal microecological balance and enhancing immunity. This compound microbial agent provides a foundation for the development and innovation of functional foods.

[0079] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A compound microbial agent, characterized in that, It contains Bifidobacterium animalis IU100 and Lactobacillus paracasei ZYhyy-004; The Bifidobacterium animalis IU100, classified and named Bifidobacterium animalis, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 12942 and deposit date of September 5, 2016. The Lactobacillus paracasei ZYhyy-004, classified and named Lactobacillus paracasei, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31554 and deposit date of August 5, 2024.

2. A fermentation composition, characterized in that, It is obtained by fermentation with the compound microbial agent of claim 1.

3. A fermented milk base, characterized in that, The compound microbial agent comprising the one described in claim 1, wherein the fermented milk base is a combination of pure milk and pea type III resistant starch.

4. The method for preparing fermented milk base as described in claim 3, characterized in that, Add 1% - 2% pea type III resistant starch to pure milk, dissolve and homogenize it, pasteurize it at 95℃ for 5 minutes, and cool it to 43℃ to obtain fermented milk base.

5. A fermenting agent, characterized in that, It includes Bifidobacterium animalis IU100 and Lactobacillus paracasei ZYhyy-004 as described in claim 1, as well as a basic starter culture, which includes Streptococcus salivarius subsp. thermophilus and Lactobacillus bulgaricus.

6. The fermenting agent as described in claim 5, characterized in that, The count of the animal Bifidobacterium IU100 was 5 × 10⁻⁶. 6 –2×10 7 CFU / mL; the bacterial count of the *Lactobacillus paracasei* ZYhyy-004 was 1 × 10⁻⁶. 6 – 1×10 7 CFU / mL.

7. The application of the compound microbial agent as described in claim 1, the fermentation composition as described in claim 2, the fermented milk base as described in claim 3, the fermented milk base prepared by the preparation method as described in claim 4, and the starter culture as described in claim 5 or 6 in the preparation of fermented milk.

8. The application of the compound microbial agent as described in claim 1, the fermentation composition as described in claim 2, the fermented milk base as described in claim 3, the fermented milk base prepared by the preparation method as described in claim 4, and the starter culture as described in claim 5 or 6 in the preparation of yogurt.

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