Compound probiotics capable of reducing blood fat and regulating constipation and application of compound probiotics in resistant starch fermented milk

Abstract

The invention discloses compound probiotics for reducing blood fat and regulating constipation and application of the compound probiotics in resistant starch fermented milk, and belongs to the technical field of functional food. The compound bacterium is prepared from bifidobacterium animalis IU100 and lactobacillus paracasei ZYhyy-004, and the compound bacterium has a synergistic interaction effect, and the compound bacterium can be used for preparing the compound bacterium. The resistant starch is used for preparing the resistant starch fermented milk, the levels of serum total cholesterol, triglyceride and low-density lipoprotein cholesterol can be remarkably reduced, the content of high-density lipoprotein cholesterol is increased, meanwhile, the intestinal tract propulsion rate is increased, and functional constipation is improved. The fermented milk realizes dual effects of reducing blood fat and improving intestinal functions by regulating intestinal flora balance, promoting proliferation of beneficial bacteria and inhibiting growth of pathogenic bacteria. The invention provides a new way for developing multifunctional probiotic foods.

Description

Technical Field

[0001] The present invention relates to the fields of microorganisms and dairy product processing, and particularly relates to a compound probiotic for reducing blood lipid and regulating constipation and its application in resistant starch fermented milk. Background Art

[0002] Dyslipidemia and functional constipation have become two common and increasingly serious health problems. High-fat, high-sugar and high-calorie eating habits have led to an increasing prevalence of dyslipidemia. In particular, the intake of high cholesterol and high triglycerides increases the risk of cardiovascular diseases. At the same time, the problem of functional constipation also plagues a large number of people, affecting people's quality of life and digestive health. Research shows that probiotics play a significant role in regulating blood lipid and improving intestinal health. Probiotics such as lactic acid bacteria and bifidobacteria can effectively reduce the total cholesterol and low-density lipoprotein (LDL) cholesterol in the blood, and help reduce blood lipid levels by promoting bile acid excretion and inhibiting cholesterol absorption. In addition, probiotics contribute to maintaining the balance of the intestinal flora, reducing intestinal inflammatory responses, improving intestinal health, and thus alleviating the symptoms of functional constipation while indirectly regulating blood lipid.

[0003] Fermented milk products combined with type III resistant starch, as an innovative compound probiotic food, have dual health benefits. As an effective prebiotic, type III resistant starch can promote the growth of probiotics in the intestine, regulate the intestinal flora, improve the intestinal microecological balance, and thus help alleviate functional constipation. At the same time, resistant starch promotes cardiovascular health by delaying the digestion and absorption of carbohydrates and reducing the cholesterol and triglyceride levels in the blood. Compared with traditional fermented dairy products, type III resistant starch fermented milk fermented by probiotics such as bifidobacteria not only increases the number and activity of probiotics, improves intestinal health, but also enhances its blood lipid-lowering function. Through the combination of this innovative compound probiotic and resistant starch, this fermented milk product can effectively address the two major health problems of dyslipidemia and functional constipation, providing a healthier and more functional dairy product option to meet the needs of modern consumers for comprehensive health management. Summary of the Invention

[0004] In view of the deficiencies and actual needs of existing resistant starch fermented milk, the present invention provides a compound probiotic for reducing blood lipid and regulating constipation and its application in resistant starch fermented milk, and studies the effects of Bifidobacterium animalis subsp. lactis IU100 + Lacticaseibacillus paracasei ZYhyy-004 resistant starch fermented milk on blood lipid in a hyperlipidemic mouse model and on functional constipation in constipated mice. Through animal experiments, the contents of serum cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglyceride were detected to evaluate the improvement effect of Bifidobacterium animalis subsp. lactis IU100 and Lacticaseibacillus paracasei ZYhyy-004 resistant starch fermented milk on blood lipid in hyperlipidemic mice; the improvement effect of Bifidobacterium animalis subsp. lactis IU100 + Lacticaseibacillus paracasei ZYhyy-004 resistant starch fermented milk on functional constipation in constipated mice was evaluated by the first black feces excretion time, fecal volume within 6 hours, fecal water content, and intestinal propulsion rate.

[0005] The object of the present invention is achieved by the following technical solutions:

[0006] The present invention provides a Bifidobacterium animalis subsp. lactis IU-100, which was deposited on September 5, 2016 at the China General Microbiological Culture Collection Center (CGMCC), Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode 100101, and was classified and named as Bifidobacterium animalis, with the deposit number of CGMCC No. 12942.

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

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

[0009] The present invention provides a Lacticaseibacillus paracasei ZYhyy-004, which was deposited on August 5, 2024 at the China General Microbiological Culture Collection Center (CGMCC), Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode 100101, and was classified and named as Lacticaseibacillus paracasei, with the deposit number of CGMCC No. 31554.

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

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

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

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

[0014] Preferably, the addition amount of Bifidobacterium animalis subsp. lactis IU100 in the product is not less than 1×10 6 CFU / mL, and the addition amount of Lactobacillus paracasei ZYhyy-004 is not less than 5×10 5 CFU / mL1×10 7 CFU / mL; preferably, the addition amount of Bifidobacterium animalis subsp. lactis IU100 is 1×10 6 ~10 9 CFU / mL, and the addition amount of Lactobacillus paracasei ZYhyy-004 is 5×10 5 ~5×10 7 CFU / mL; preferably, the addition amount ratio of Bifidobacterium animalis subsp. lactis IU100 and Lactobacillus paracasei ZYhyy-004 is (1-2):(2-1), and the preferred ratio is (0.8-1.2):(0.4-0.6). Preferably, the addition amount of Bifidobacterium animalis subsp. lactis IU100 is 5×10 6 –2×10 7 CFU / mL; the number of Lactobacillus paracasei ZYhyy-004 is 1×10 6 –1×10 7 CFU / mL.

[0015] On the other hand, the present invention provides the application of the above-mentioned compound bacterium agent in fermented milk products for regulating functional constipation. Preferably, the addition amount of Bifidobacterium animalis subsp. lactis IU100 is 5×10 6 –2×10 7 CFU / mL; the number of Lactobacillus paracasei ZYhyy-004 is 1×10 6 –1×10 7 CFU / mL; preferably, the addition amount of Bifidobacterium animalis subsp. lactis IU100 in the product is 1×10 7 CFU / mL, and the addition amount of Lactobacillus paracasei ZYhyy-004 is 5×10 6 CFU / mL.

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

[0017] In addition, a fermented milk base is provided, which contains the above-mentioned compound bacterium agent, and the fermented milk base is a combination of pure milk and pea type III resistant starch. Preferably, the milk base is prepared by adding 1%-2% of pea type III resistant starch to pure milk, dissolving and homogenizing, and then performing pasteurization at 95°C for 5 minutes and cooling to 43°C to obtain the fermented milk base. The above milk base contains a starter, and the preferred starter is a combination of Bifidobacterium animalis IU100 and Lactobacillus paracasei ZYhyy-004 at the above-mentioned concentration or ratio, and a basic starter, and the basic starter includes Streptococcus salivarius subsp. thermophilus and Lactobacillus bulgaricus.

[0018] On the other hand, the present invention provides the application of the compound bacterium agent in preparing resistant starch fermented milk, and the application of the resistant starch fermented milk prepared by the compound bacterium agent in fermented milk products for reducing hyperlipidemia and regulating functional constipation. Preferably, the product is any one of fermented milk and yogurt.

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

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

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

[0022] The fermented milk or resistant starch fermented milk prepared from the above compound bacterium agent has the effect of reducing blood lipids in hyperlipidemic mouse models / patients; the fermented milk or resistant starch fermented milk prepared from the above compound bacterium agent has the effect of regulating functional constipation in constipated mouse models / patients.

[0023] On the other hand of the present invention, the compound bacterium agent or the fermented milk prepared therefrom has the application of reducing blood lipids and regulating functional constipation at the same time. Further, reducing blood lipids means reducing serum cholesterol, low-density lipoprotein cholesterol, and triglycerides. Further, it includes increasing high-density lipoprotein cholesterol.

[0024] Further, regulating functional constipation refers to the first black feces excretion time, fecal volume within 6 hours, fecal water content, and intestinal propulsion rate in the mouse model. It also includes the intestinal propulsion rate of patients.

[0025] On the other hand of the present invention, there is provided the use of the above-mentioned Bifidobacterium animalis subsp. lactis IU100 and Lactobacillus paracasei ZYhyy-004 for preparing resistant starch fermented milk. Preferably, the fermented milk is yogurt or fermented milk. The beneficial effects of the present invention compared with the prior art are as follows:

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

[0027] The fermented milk prepared from Bifidobacterium animalis subsp. lactis IU100 and Lactobacillus paracasei ZYhyy-004 can reduce serum cholesterol, triglycerides, and low-density lipoprotein cholesterol in hyperlipidemic mice by 20.3%, 26.3%, and 34.2% respectively, and increase high-density lipoprotein cholesterol by 23.2%.

[0028] Bifidobacterium animalis subsp. lactis IU100 and Lactobacillus paracasei ZYhyy-004 can reduce the first black feces excretion time in constipated mice by 73 minutes, increase the fecal volume within 6 hours by 18, increase the fecal water content by 17.3%, and increase the intestinal propulsion rate by 28.3%.

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

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

[0031] (4) The resistant starch fermented milk of Bifidobacterium animalis subsp. lactis IU100 and Lactobacillus paracasei ZYhyy-004 of the present invention can reduce the serum cholesterol, triglyceride, and low-density lipoprotein cholesterol of hyperlipidemic mice by 32.4%, 35.9%, and 47.4% respectively, and increase the high-density lipoprotein cholesterol by 42.7%. It can reduce the first black stool excretion time of constipated mice by 77.5 min, increase the fecal volume within 6 hours by 19 grains, increase the fecal water content by 18.3%, and increase the intestinal propulsion rate by 32%. At the same time, the resistant starch fermented milk of Bifidobacterium animalis subsp. lactis IU100 and Lactobacillus paracasei ZYhyy-004 helps to maintain the balance of the intestinal microbial ecosystem and can avoid environmental pollution caused by the abuse of antibiotics, with broad application prospects. Description of the Drawings

[0032] Figure 1 Comparison of the body weight changes of the mouse model. w represents week.

[0033] Figure 2 Effects of each intervention group on the total serum cholesterol and triglyceride of the mouse model.

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

[0035] Figure 4 Effects of each intervention group on the first black stool excretion time and the number of feces within 6 hours of the mouse model.

[0036] Figure 5 Effects of each intervention group on the fecal water content and intestinal propulsion rate of the mouse model. Detailed Embodiments

[0037] The endpoints and any values within the ranges disclosed in this document are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this document.

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

[0039] Bifidobacterium animalis subsp. lactis IU100 was obtained from the isolation of samples from Shandong Dairy Company, and Lactobacillus paracasei ZYhyy - 004 was obtained from the isolation of samples from Henan.

[0040] Example 1 Strain Activation

[0041] Preparation of Bifidobacterium animalis subsp. lactis IU100 cells of the present invention: After Bifidobacterium animalis subsp. lactis IU100 was passaged twice in modified MRS liquid medium, it was inoculated into the modified MRS liquid medium at an inoculation amount of 2% (v / v) and cultured at 37 ± 1 °C and 0% oxygen concentration for 11 h. The supernatant was removed by centrifugation (4000 rpm, 10 min). After the cells were washed twice with PBS, when used for gavage, they were resuspended with 0.85% physiological saline and the concentration of the cell suspension was adjusted to 1×10 10 CFU / mL.

[0042] Preparation of Lactobacillus paracasei ZYhyy - 004 cells of the present invention: After Lactobacillus paracasei ZYhyy - 004 was passaged twice in modified MRS liquid medium, it was inoculated into the modified MRS liquid medium at an inoculation amount of 1% (v / v) and cultured at 37 ± 1 °C and 0% oxygen concentration for 10 h. The supernatant was removed by centrifugation (5000 rpm, 8 min). After the cells were washed twice with PBS, when used for gavage, they were resuspended with 0.85% physiological saline and the concentration of the cell suspension was adjusted to 1×10 10 CFU / mL.

[0043] Example 2 Preparation of Resistant Starch Fermented Milk

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

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

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

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

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

[0049] Bifidobacterium animalis subsp. lactis IU100 group (IU100 fermented milk group): Bifidobacterium animalis subsp. lactis IU100 is added to the fermented milk base, and the final addition amount of IU100 is 1×10 7 CFU / g milk base. The others are the same as the basic fermented milk group.

[0050] Compound bacterium agent group (IU100+ZYhyy-004): After separately culturing Lactobacillus paracasei ZYhyy-004 and Bifidobacterium animalis subsp. lactis IU100, a compound bacterium agent is prepared according to the bacterial mass ratio of 1:2.

[0051] IU100+ZYhyy-004 resistant starch fermented milk group: 1%-2% pea type III resistant starch is added to the fermented milk base. After separately culturing Lactobacillus paracasei ZYhyy-004 and Bifidobacterium animalis subsp. lactis IU100, a compound bacterium agent is prepared according to the bacterial mass ratio of 1:2. An additional 1×10 7 CFU / g milk base of Bifidobacterium animalis subsp. lactis IU100 and 5×10 6 CFU / g milk base of Lactobacillus paracasei ZYhyy-004 are finally added to the milk base. The others are the same as the basic fermented milk group.

[0052] After the above-mentioned intervention groups are prepared, except that the compound bacterium agent group does not need fermentation, the other four groups are fermented at 42±1°C for 5-6 hours to obtain fermented milk of each group.

[0053] During gavage, 200 μL is taken from each group for gavage.

[0054] Example 3 Hyperlipidemia animal model

[0055] SPF-grade BALB / c (6-week-old, female) mice were selected as experimental animals and purchased from Beijing Huafukang Biotechnology Co., Ltd. The animal experiment was divided into 10 groups, namely the blank group, the model group, the drug group, the basic fermented milk group, the resistant starch fermented milk group, the IU100 fermented milk group, the Lactobacillus animalis subsp. IU100 + Lactobacillus paracasei ZYhyy-004 (1×10 9 CFU / mL) group, and the Lactobacillus animalis subsp. IU100 + Lactobacillus paracasei ZYhyy-004 resistant starch fermented milk group. There were 10 mice in each group, with a total of 70 mice.

[0056] Mouse breeding conditions: temperature 24°C, controlled light for 12 h and darkness for 12 h, and the bedding was changed every three days. After one week of adaptive breeding, the mice were fed a high-fat diet daily (66.5 kg of basal diet, 20 kg of sucrose, 1 kg of sodium cholate, 10 kg of lard, 2.5 kg of cholesterol), and weighed once a week. The food intake was monitored daily, and the body weight change was detected weekly. The blank group was continuously gavaged with 0.9% normal saline until the end of the experiment; after 12 h of fasting but not water deprivation after the last gavage, blood was collected from the orbital vein of the mice, and the total cholesterol (TC) and triglyceride (TG) of the mice after modeling were detected to be 3-4 times higher than those of the blank group, indicating successful modeling.

[0057] Model group and each intervention group: First, the mice were fed a high-fat diet daily until the model was successfully established (4-8 weeks). After the model was successfully established, the model group and the intervention group were gavaged with 1×10 9 CFU / g probiotics (Lactobacillus animalis subsp. IU100 + Lactobacillus paracasei ZYhyy-004 was prepared according to the bacterial cell mass ratio of 1:2 and also formulated into 200 μL) or 200 μL of fermented milk until the end of the experiment; at the same time, the drug group was gavaged with 5 mg / kg of lovastatin for 4-8 weeks.

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

[0059] During the experiment, the mice had free access to food and water, and their body weights were measured every 7 days. After 12 h of fasting but not water deprivation after the last gavage, blood was collected from the orbital vein of the mice, collected with a heparin anticoagulant tube, centrifuged at 4000 r / min for 3 min at 4°C, and the serum was collected and the total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) indexes were measured using a fully automatic biochemical analyzer for animals.

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

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

[0062] The results showed that statin drugs, resistant starch fermented milk, IU100 fermented milk group, compound bacterium 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 the body weight of the mice.

[0063] The results of serum total cholesterol were as Figure 2 shown in Figure A. Among the 7 groups of mice, the serum total cholesterol of the mice in the model group was 3.49 mmol / L, and the content of serum total cholesterol was significantly higher than that of the control group (p < 0.01); compared with the mice in the model group, the serum total cholesterol of the mice in the statin drug group was 2.31 mmol / L, and the content of serum total cholesterol was significantly lower than that of the model group (p < 0.01); the serum total cholesterol of the mice in the Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy - 004 resistant starch fermented milk group was 2.36 mmol / L, and the content of serum total cholesterol was significantly lower than that of the model group (p < 0.01), and it could nearly achieve the same function of reducing total cholesterol as the drug group. There was no significant difference among the basic fermented milk group, the resistant starch fermented milk group, the IU100 fermented milk group, and the compound bacterium agent group.

[0064] The results of triglyceride were as Figure 2As shown in Figure B, the triglyceride level of the mice in the model group was 1.56 mmol / L, and the triglyceride content was extremely significantly higher than that of the control group (p < 0.01); compared with the mice in the model group, the triglyceride level of the mice in the statin drug group was 1.04 mmol / L, and the triglyceride content was extremely significantly lower than that of the model group (p < 0.01); the triglyceride level of the mice in the Lactobacillus animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy - 004 resistant starch fermented milk group was 1.00 mmol / L, and the triglyceride content was extremely significantly lower than that of the model group (p < 0.01). There was no significant difference among the basic fermented milk group, the resistant starch fermented milk group, the IU100 fermented milk group and the compound bacterium agent group.

[0065] The basic fermented milk group could reduce the serum total cholesterol content to a certain extent. Adding resistant starch (resistant starch fermented milk group) could slightly enhance the effect, while using IU100 fermented milk had no significant effect; only adding the bacterial combination had a function similar to that of the basic fermented milk group. Only the fermented milk group prepared by co - fermenting resistant starch with two kinds of bacteria could achieve a lipid - lowering function similar to that of drugs.

[0066] The results of low - density lipoprotein cholesterol are as Figure 3 As shown in Figure A, among the 7 groups of mice, the low - density lipoprotein cholesterol of the mice in the model group was 1.14 mmol / L, and the low - density lipoprotein cholesterol content was extremely significantly higher than that of the control group (p < 0.01); compared with the mice in the model group, the low - density lipoprotein cholesterol of the mice in the statin drug group was 0.63 mmol / L, and the low - density lipoprotein cholesterol content was extremely significantly lower than that of the model group (p < 0.01); the low - density lipoprotein cholesterol of the resistant starch fermented milk group and mmol / L was 0.79 mmol / L and 0.76 mmol / L respectively, and the low - density lipoprotein cholesterol content was significantly lower than that of the model group (p < 0.05); the low - density lipoprotein cholesterol of the Lactobacillus animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy - 004 group of mice was 0.75 mmol / L, and the low - density lipoprotein cholesterol content was significantly lower than that of the model group (p < 0.05); the low - density lipoprotein cholesterol of the Lactobacillus animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy - 004 resistant starch fermented milk group of mice was 0.6 mmol / L, and the low - density lipoprotein cholesterol content was extremely significantly lower than that of the model group (p < 0.01).

[0067] The results of high - density lipoprotein cholesterol are as Figure 3As shown in B, among the 7 groups of mice, the HDL cholesterol of the mice in the model group was 0.82mmol / L, which was significantly lower than that in the control group (p<0.01); compared with the mice in the model group, the HDL cholesterol of the mice in the statin group was 1.13mmol / L, which was significantly higher than that in the model group (p<0.05); the HDL cholesterol of the mice in the resistant starch fermented milk and IU100 fermented milk groups were 1.05mmol / L and 1.03mmol / L, respectively, which were significantly higher than those in the model group (p<0.05); the HDL cholesterol of the mice in the animal Bifidobacterium lactis subsp. IU100 group was 1.01mmol / L, which was significantly higher than that in the model group (p<0.05); the HDL cholesterol of the mice in the animal Bifidobacterium lactis subsp. IU100 resistant starch fermented milk group was 1.17mmol / L, which was significantly higher than that in the model group (p<0.05).

[0068] Example 5 Functional constipation animal model

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

[0070] Mouse housing conditions: controlled temperature of 23±2℃ and humidity of 60±5%, with bedding changed every three days. After one week of adaptive breeding, except for the control group, the other groups treated mice with 15mg / kg / d of loperamide for 14 days to induce constipation in mice, and the oral dose for mice was 0.1mL / 20g. The first black discharge time, the number of fecal particles in 6 hours and the water content of feces were used to determine whether the mouse constipation model was successfully established. When the first black discharge time of the mouse was twice or more than that of the control group, the degree of reduction in fecal water content was >15%, and the number of fecal particles in six hours decreased by more than 15 particles, it indicated that the loperamide-induced constipation model of BALB / c male mice was successfully established.

[0071] Model group and intervention groups: After successful modeling, the model group and intervention group were gavaged with 1×10 9CFU / g probiotics (Lactobacillus animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy-004 formulated according to the bacterial mass ratio of 1:2, and can also be formulated into a 200 μl bacterial liquid) or 200 μl fermented milk until the end of the experiment.

[0072] Example 6 Detection of the 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, the mice were fasted for 12 hours, and then gavaged with 15% carmine to record the number of fecal pellets and the first black stool time within six hours. Finally, the feces within 6 hours were collected, weighed as "A", and weighed again after drying, recorded as "B". The percentage of fecal water content (%) = (A - B) / A × 100%. The mice after 14 days of intervention were sacrificed 30 min after gavaging with 15% carmine. Then the entire small intestine was taken out and straightened, and the length was measured as "a", while the travel distance of activated carbon was measured as "b". The gastrointestinal transit ratio (%) = b / a × 100%.

[0074] The results of the first black stool time are as Figure 4 shown in Figure A. Among the 7 groups of mice, the first black stool time of the model group mice was 148 min, which was significantly higher than that of the blank group (p < 0.05); compared with the model group mice, the first black stool time of the loperamide drug group mice was 71 min, which was significantly lower than that of the model group (p < 0.05); the first black stool time of the basic fermented milk group was 89.5 min, which was significantly lower than that of the model group (p < 0.05); the first black stool times of the resistant starch fermented milk group and the 0.79 mmol / L group were 87 min and 89 min respectively, which were significantly lower than that of the model group (p < 0.05); the first black stool time of the Lactobacillus animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy-004 group mice was 75 min, which was significantly lower than that of the model group (p < 0.05); the first black stool time of the Lactobacillus animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy-004 resistant starch fermented milk group mice was 70.5 min, which was significantly lower than that of the model group (p < 0.05). The four intervention groups could significantly reduce the first black stool time of constipated mice. Among them, the first black stool time of the Lactobacillus animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy-004 resistant starch fermented milk group was significantly lower than that of the basic fermented milk and resistant starch fermented milk groups, and the effect was the most significant.

[0075] The results of the fecal volume within 6 hours are as Figure 4As shown in Figure B, among the 7 groups of mice, the number of feces of the model group mice within 6 hours was 48, which was significantly lower than that of the blank group (p < 0.05); compared with the model group mice, the number of feces of the loperamide drug group mice within 6 hours was 65, which was significantly higher than that of the model group (p < 0.05); the number of feces of the basic fermented milk group within 6 hours was 55, and there was no significant difference in the number of feces within 6 hours compared with the model group (p > 0.05); the number of feces of the resistant starch fermented milk group and the IU100 fermented milk group within 6 hours were 57 and 55 respectively, and there was no significant difference in the number of feces within 6 hours compared with the model group (p > 0.05); the number of feces of the Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy - 004 group mice within 6 hours was 64, which was significantly higher than that of the model group (p < 0.05); the number of feces of the Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy - 004 resistant starch fermented milk group mice within 6 hours was 67, which was significantly higher than that of the model group (p < 0.05). Among the four intervention groups, 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 could significantly increase the number of feces of constipated mice within 6 hours, and the Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy - 004 resistant starch fermented milk group had the most significant effect.

[0076] The results of fecal water content are as Figure 5 As shown in Figure A, among the 7 groups of mice, the fecal water content of the model group mice was 47.7%, which was significantly lower than that of the blank group (p < 0.05); compared with the model group mice, the fecal water content of the loperamide drug group mice was 65.7%, which was significantly higher than that of the model group (p < 0.05); the fecal water content of the basic fermented milk group was 57%, which was 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 57.7% and 60.1% respectively, which were significantly higher than that of the model group (p < 0.05); the fecal water content of the Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy - 004 group mice was 65%, which was significantly higher than that of the model group (p < 0.05); the fecal water content of the Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy - 004 resistant starch fermented milk group mice was 66%, which was significantly higher than that of the model group (p < 0.05). All four intervention groups could significantly increase the fecal water content of constipated mice, and the Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy - 004 resistant starch fermented milk group had the most significant effect.

[0077] The results of intestinal propulsion rate are asFigure 5 As shown in Figure B, among the 7 groups of mice, the intestinal propulsion rate of the mice in the model group was 53.3%, which was significantly lower than that of the blank group (p < 0.05); compared with the mice in the model group, the intestinal propulsion rate of the mice in the loperamide drug group was 93.5%, which was significantly higher than that of the model group (p < 0.05); the intestinal propulsion rate of the basic fermented milk group was 68.7%, which was 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 71% and 73% respectively, which were significantly higher than that of the model group (p < 0.05); the intestinal propulsion rate of the mice in the Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy-004 group was 81.6%, which was significantly higher than that of the model group (p < 0.05); the intestinal propulsion rate of the mice in the Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy-004 resistant starch fermented milk group was 85.3%, which was significantly higher than that of the model group (p < 0.05). The four intervention groups could significantly increase the intestinal propulsion rate of constipated mice, and the Bifidobacterium animalis subsp. lactis IU100 + Lactobacillus paracasei ZYhyy-004 resistant starch fermented milk group had the most significant effect.

[0078] In summary, Bifidobacterium animalis subsp. lactis IU100 and Lactobacillus paracasei ZYhyy-004 have the function of co-fermenting resistant starch fermented milk. Through the co-fermentation of the two strains, the obtained fermented milk has better effects on reducing blood lipids and relieving functional constipation than single-strain fermentation, bacterial cell addition, resistant starch addition and other treatments. Therefore, Bifidobacterium animalis subsp. lactis IU100 and Lactobacillus paracasei ZYhyy-004 have obvious synergistic effects in the fermentation process of resistant starch fermented milk base. The two cooperate and promote each other, and the synergistic effect is not only reflected in the improvement of fermentation efficiency, but more significantly in the improvement of the functionality of fermented milk. The prepared resistant starch fermented milk not only shows outstanding performance in reducing blood lipids and relieving functional constipation, but also has excellent performance in potential health benefits such as regulating intestinal microecological balance and enhancing body immunity. This compound bacterium agent provides a basis for the development and innovation of functional foods.

[0079] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Bifidobacterium animalis IU100, characterized in that, The Bifidobacterium animalis is classified and named as Bifidobacterium animalis, and is deposited in the China General Microbiological Culture Collection Center (CGMCC) with the deposit number CGMCC No. 12942 and the deposit date of September 5, 2016.

2. A Lactobacillus paracasei ZYhyy-004, characterized in that, The Lactiplantibacillus paracasei is classified and named as Lactiplantibacillus paracasei, and is deposited in the China General Microbiological Culture Collection Center (CGMCC) with the deposit number CGMCC No. 31554 and the deposit date of August 5, 2024.

3. A composite microbial agent, characterized in that, It contains the Bifidobacterium animalis IU100 described in claim 1 and the Lactiplantibacillus paracasei ZYhyy-004 described in claim 2.

4. A fermentation composition, characterized in that, It is obtained by fermenting the compound bacterium agent of claim 3.

5. A fermented milk base, characterized in that, It contains the compound bacterium agent described in claim 3, and the fermented milk base is a combination of pure milk and pea type III resistant starch.

6. The preparation method of the fermented milk base according to claim 5, characterized in that, 1%-2% of pea type III resistant starch is added to pure milk, dissolved and homogenized, and then pasteurized at 95 °C for 5 min and cooled to 43 °C to obtain the fermented milk base.

7. A starter culture, characterized in that, It includes the Bifidobacterium animalis IU100 and the Lactiplantibacillus paracasei ZYhyy-004 in claim 3, and a starter culture, and the starter culture includes Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

8. The starter according to claim 7, characterized in that, The number of bacteria of Bifidobacterium animalis IU100 is 5×10 6 –2×10 7 CFU / mL; the number of bacteria of Lactobacillus paracasei ZYhyy-004 is 1×10 6 –1×10 7 CFU / mL.

9. The application of the Bifidobacterium animalis of claim 1 or the Lactiplantibacillus paracasei of claim 2, the compound bacterium agent of claim 3, the fermented composition of claim 4, the fermented milk base of claim 5, the fermented milk base prepared by the preparation method of claim 6, and the starter culture of claim 7 or 8 in the preparation of products.

10. The application according to claim 9, wherein The product is yogurt or fermented milk.

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