Lactobacillus helveticus with blood fat reducing effect as well as segmented fermentation preparation method and application of lactobacillus helveticus

Abstract

The invention belongs to the technical field of microorganisms, and in particular relates to lactobacillus helveticus IOB726 with an effect of reducing blood fat as well as a segmented fermentation preparation method and application of the lactobacillus helveticus IOB726. The invention discovers that the lactobacillus helveticus IOB726 has the effect of reducing blood fat for the first time. The lactobacillus helveticus IOB726 bacterial powder and the preparation thereof are prepared by adopting segmented liquid state fermentation for the first time, not only is the bacterial activity improved, but also an inulin component is added into a second-stage fermentation culture medium, so that a carbon source is provided for the growth of the lactobacillus helveticus IOB726, and the lactobacillus helveticus IOB726 is promoted to generate short-chain fatty acid in the fermentation metabolism process; the liver cholesterol synthesis can be inhibited, so that the hyperlipemia symptom can be improved. Besides, experimental results show that the lactobacillus helveticus IOB726 has an efficient blood fat reducing effect, has good acid resistance, bile salt resistance and gastrointestinal tract simulation tolerance, can play a role in human intestinal tracts, supplements strain resources of blood fat reducing probiotics, and shows a better blood fat reducing effect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a type of Lactobacillus helveticus with lipid-lowering effects. Lactobacillus helveticus IOB726 and its segmented fermentation preparation method and applications. Background Technology

[0002] Hyperlipidemia is a common metabolic disease, mainly caused by abnormally high levels of lipids such as cholesterol and triglycerides in the blood, which can lead to serious consequences such as arteriosclerosis, cardiovascular disease, and cerebrovascular disease. Hyperlipidemia is one of the important pathogenic factors of atherosclerotic cardiovascular and cerebrovascular diseases and has become a serious public health problem. Therefore, the prevention and treatment of hyperlipidemia are particularly important. With the improvement of people's living standards, the acceleration of the pace of life, the increase in life pressure, the reduction of physical labor and exercise, and the deterioration of the work and environment, the body's metabolic capacity is easily reduced and disordered, immunity is weakened, and the proportion of sub-healthy people is constantly rising. Especially, dietary changes have changed drastically, with high-fat and cholesterol-rich foods being ubiquitous in daily life. However, long-term and excessive consumption of such foods can promote the development of chronic diseases, including hyperlipidemia, leading to abnormal lipid metabolism, elevated serum cholesterol and / or triglycerides, seriously affecting normal life and endangering health.

[0003] Currently, most products used in clinical practice for regulating blood lipids are drugs, and most of them are Western medicines, such as fibrates, statins, and niacin. Although these are effective in lowering blood lipids, they often have the following disadvantages: (1) They have large side effects, which can easily cause abnormal liver enzymes, gastrointestinal discomfort, abdominal pain, diarrhea, and loss of appetite; (2) The effects are short-lasting. Usually, after taking the medication, it is necessary to continue taking it for several courses of treatment to maintain the effect, and the duration of the effect is poor; (3) The effects are unstable. For example, the components of compound Chinese medicines are complex and the treatment mechanism is not clear. Therefore, the treatment effect often varies from person to person and is not stable; (4) The cost is high. Long-term use of drugs will bring a lot of economic pressure to patients. With the development of science and technology and the improvement of people's living standards, more and more patients with hyperlipidemia hope to have more comprehensive, healthier, and safer products to improve hyperlipidemia, rather than just lowering blood lipids.

[0004] Probiotics are live microorganisms that can produce health benefits when ingested in sufficient quantities. Numerous studies both domestically and internationally have shown that some probiotics have significant effects on lowering serum lipids. Although the connection between the gut microbiota and specific human diseases, especially chronic diseases, is well understood, the availability of lactic acid bacteria for clinical use in improving hyperlipidemia remains very limited.

[0005] Therefore, finding a type of lactic acid bacteria that can lower blood cholesterol and triglycerides has become an important breakthrough direction in this field. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a *Lactobacillus helveticus* with lipid-lowering effects. Lactobacillus helveticus This invention relates to IOB726, its staged fermentation preparation method, and its applications. The *Lactobacillus helveticus* described in this invention can reduce blood cholesterol and triglyceride levels, and inhibit pancreatic lipase and cholesterol esterase activity.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In the first aspect, the present invention provides a *Lactobacillus helveticus* with lipid-lowering effects (…). Lactobacillus helveticus IOB726, the classification name of the Lactobacillus helveticus is Lactobacillus helveticus ( Lactobacillus helveticus The accession number is CGMCC No.28044, the accession date is July 28, 2023, the accession site is China General Microbiological Culture Collection Center (CGMCC), and the address of the accession institution is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0008] The Lactobacillus helveticus described in this invention ( Lactobacillus helveticus IOB726 was selected by the inventor from traditional yogurt products.

[0009] Alternatively, the *Lactobacillus helveticus* (Lactobacillus) described in this invention... Lactobacillus helveticus IOB726 lowers cholesterol and triglyceride levels and inhibits the activity of pancreatic lipase and cholesterol esterase.

[0010] Alternatively, the Lactobacillus helveticus (Lactobacillus schreiberensis) with lipid-lowering effects described in this invention (…) Lactobacillus helveticus IOB726 lowers blood lipids through the following synergistic mechanism: (a) In vitro cholesterol degradation rate: 30.12%; (b) In vitro triglyceride degradation rate: 59.63%; (c) Pancreatic lipase inhibition rate: 38.91%; (d) Cholesterol esterase inhibition rate: 32.36%; (e) Bile salt binding rate 21.39%; and the bacterial powder has a survival rate of ≥57.5% after 4 hours at pH 2.0 and a survival rate of ≥80.7% after 4 hours at 0.5% bile salt.

[0011] In a second aspect, the present invention provides the *Lactobacillus helveticus* described in the first aspect above. Lactobacillus helveticus A method for preparing IOB726, the method comprising the following steps: (1) Activation of strain: Lactobacillus helveticus ( Lactobacillus helveticus IOB726 was taken out of the cryopreservation tube and inoculated into the activation medium at a volume ratio of 1:20-1:30 for activation culture. The culture temperature was 36±2℃, sealed, and the culture time was 24±2h. The activated strain is the primary seed culture. The primary seed culture is inoculated into the activation medium at a volume ratio of 1:30 and cultured at 36±2℃ for 22±2h to obtain the secondary seed culture. The activation medium is MRS medium, with the following formula: 8 g-10 g peptone, 3 g-4 g yeast extract, 15 g-20 g glucose, 1 g-2 g dipotassium hydrogen phosphate, 1 g-2 g triammonium citrate, 0.2 g-0.5 g MgSO4·7H2O, 0.05 g-0.1 g MnSO4, 8 g-10 g agar, 0.8 g-1 g Tween 80, pH=6.0±0.2, adjusted to 1 L, and sterilized at 121℃ for 15 min.

[0012] (2) Segmented fermentation: First stage fermentation: The secondary seed liquid was inoculated into the liquid fermentation medium at a volume ratio of 1:70-1:80, and cultured at a temperature of 36±2℃ and a rotation speed of 120 r / min-140 r / min for 18±2h to obtain the fermentation broth. The first stage of liquid fermentation medium is a modified MRS medium with the following formula: 40-50 g peptone, 15-20 g yeast extract, 60-80 g glucose, 5-10 g dipotassium hydrogen phosphate, 5-10 g triammonium citrate, 1-2 g MgSO4·7H2O, 0.25-0.5 g MnSO4, 4-5 g Tween 80, pH=6.0±0.2, adjusted to 5 L, and sterilized at 121℃ for 15 min.

[0013] Second stage fermentation: The first stage fermentation broth was concentrated by centrifugation at 4℃ and 6000 r / min for 20 min, with a concentration factor of 5-10 times. Then, the concentrated fermentation broth was inoculated into the second stage liquid fermentation medium at a volume ratio of 1:50-1:60. The temperature was 36±2℃ and the rotation speed was 120 r / min-140 r / min. The culture was carried out for 18±2 h to obtain the fermentation broth. The second stage of liquid fermentation medium is a modified MRS medium with the following formula: 40-50 g peptone, 15-20 g yeast extract, 30-50 g inulin, 5-10 g dipotassium hydrogen phosphate, 5-10 g triammonium citrate, 1-2 g MgSO4·7H2O, 0.25-0.5 g MnSO4, 4-5 g Tween 80, pH=6.0±0.2, adjusted to 5 L, and sterilized at 121℃ for 15 min.

[0014] (3) Preparation of bacterial powder: The fermentation broth obtained after the second stage of fermentation was centrifuged at 4℃ and 4000 r / min for 15 min to obtain the supernatant and bacterial sludge. The supernatant was concentrated, mixed with the obtained bacterial sludge, pre-cooled, and then freeze-dried under vacuum. The pre-cooling temperature was -40℃, the vacuum degree was ≤10 Pa, and the freeze-drying time was 48±2h to obtain Lactobacillus helveticus ( Lactobacillus helveticus IOB726 bacterial powder or its preparations.

[0015] In a third aspect, the present invention provides a product having a lipid-lowering effect, said product comprising the *Lactobacillus helveticus* described in the first aspect above. Lactobacillus helveticus Live and / or inactivated cells of IOB726.

[0016] In a fourth aspect, the present invention provides the *Lactobacillus helveticus* described in the first aspect above (… Lactobacillus helveticus IOB726. Lactobacillus helveticus prepared using the preparation method described in the second aspect above ( Lactobacillus helveticus The use of IOB726 bacterial powder or its preparations, or the product described in the third aspect above, in the preparation of products with lipid-lowering effects.

[0017] Alternatively, in the above-described uses, the product may be selected from pharmaceuticals, functional foods, or health products.

[0018] Alternatively, in the above-described uses, the product may be in the form of an oral dosage form.

[0019] Alternatively, in the above-described uses, the oral dosage form is a tablet, capsule, oral liquid, enteric-coated tablet, granule, syrup, or sublingual tablet.

[0020] Alternatively, in the above uses, the product may also include other medicines, functional foods, or health products for the prevention, improvement, or treatment of hyperlipidemia.

[0021] This invention is the first discovery of Lactobacillus helveticus ( Lactobacillus helveticusIOB726 has the effect of lowering blood lipids, reducing cholesterol and triglyceride levels, and inhibiting the activity of pancreatic lipase and cholesterol esterase. It can be used in products that lower high blood lipids.

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to discover Lactobacillus helveticus ( Lactobacillus helveticus IOB726 has the effect of lowering blood lipids.

[0023] 2. This invention is the first to use segmented liquid fermentation to prepare Lactobacillus helveticus (… Lactobacillus helveticus IOB726 bacterial powder and its preparations not only improved bacterial activity but also added inulin to the second-stage fermentation medium, thus enhancing the activity of *Lactobacillus helveticus* (Lactobacillus). Lactobacillus helveticus IOB726 provides a carbon source for growth and promotes the growth of Lactobacillus helveticus ( Lactobacillus helveticus IOB726 produces short-chain fatty acids during fermentation and metabolism, which help inhibit cholesterol synthesis in the liver, thereby improving symptoms of hyperlipidemia.

[0024] 3. While existing technologies have reported the lipid-lowering effects of inulin, such as a composition and preparation method for lowering blood lipids, blood pressure, and blood sugar (CN109757730B) and a lipid-lowering Enterococcus faecalis and inulin complex and its preparation method (CN108159084B), these methods involve combining fermented bacterial powder with inulin to form a composition that exerts the lipid-lowering effect. However, no studies have found that inulin is used as a culture medium component to promote bacterial proliferation, nor has the correlation between its inhibition of hepatic cholesterol synthesis and lipid-lowering function been verified. This invention is the first to demonstrate that inulin can enhance the lipid-lowering effect by promoting the production of short-chain fatty acids by Lactobacillus helveticus IOB726, with a unique mechanism.

[0025] 4. Lactobacillus helveticus ( Lactobacillus helveticus IOB726 improves symptoms of hyperlipidemia through at least one of the following (1) to (5) or through a combination of the following: (1) Lowering cholesterol levels in vitro; (2) Reduces triglyceride levels in vitro; (3) Inhibits pancreatic lipase activity; (4) Inhibits cholesterol esterase activity; (5) Ability to bind bile salts.

[0026] 5. Among the lactic acid bacteria disclosed in the prior art, such as *Lactobacillus plantarum* HYC (patent CN121160567A) The in vitro cholesterol-lowering rate of the fermentation broth can reach 89.4%, and the *Lactobacillus plantarum* SMU, patented in CN120966705A, is also mentioned. KDLP2312 showed an in vitro cholesterol-lowering rate of 40.51% (patent CN105969680A). Lactobacillus pentosus GUFHSL-69 showed an in vitro cholesterol removal rate of 25.66%. Most published data on in vitro lipid-lowering effects of lactic acid bacteria only validated the effect from a single dimension: cholesterol-lowering rate. This invention, however, validated multiple dimensions—cholesterol degradation rate (30.41%), triglyceride degradation rate (60.03%), and pancreatic lipase inhibition rate (41.77%)—demonstrating that Lactobacillus helveticus IOB726 has a highly efficient lipid-lowering effect. Furthermore, it exhibits good acid and bile salt resistance and simulated gastrointestinal tolerance, allowing it to function effectively in the human gut. This supplements the resources of lipid-lowering probiotics, demonstrating a superior lipid-lowering effect. Attached Figure Description

[0027] Figure 1 Cholesterol standard curve.

[0028] Figure 2 Lactobacillus helveticus ( Lactobacillus helveticus IOB726's cholesterol degradation rate.

[0029] Figure 3 Lactobacillus helveticus ( Lactobacillus helveticus The degradation rate of triglycerides by IOB726.

[0030] Figure 4 Lactobacillus helveticus ( Lactobacillus helveticus The inhibition rate of IOB726 on pancreatic lipase.

[0031] Figure 5 Lactobacillus helveticus ( Lactobacillus helveticus The inhibition rate of IOB726 on cholesterol esterase.

[0032] Figure 6 : Bile salt standard curve.

[0033] Figure 7 Lactobacillus helveticus ( Lactobacillus helveticus IOB726's ability to bind bile salts. Detailed Implementation

[0034] The present invention will be further described in detail below through specific embodiments. The following embodiments are merely descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0035] The following content refers to Lactobacillus helveticus ( Lactobacillus helveticus strain IOB726, classified as Lactobacillus helveticus. Lactobacillus helveticusThe deposit date is July 28, 2023, the accession number is CGMCC No.28044, the depositary institution is the China General Microbiological Culture Collection Center (CGMCC), and the address is No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0036] Example 1: Lactobacillus helveticus ( Lactobacillus helveticus Preparation method of IOB726 bacterial powder 1 A type of Lactobacillus helveticus with lipid-lowering effects ( Lactobacillus helveticus The preparation method of IOB726 bacterial powder includes the following steps: (1) Activation of strain: Lactobacillus helveticus ( Lactobacillus helveticus IOB726 was taken out of the cryopreservation tube and inoculated into the activation medium at a volume ratio of 1:30 for activation culture. The culture temperature was 36±2℃, sealed, and the static culture time was 24±2h. The activated strain is the seed liquid.

[0037] The activation medium is MRS medium, with the following formula: 10 g peptone, 4 g yeast extract, 20 g glucose, 2 g dipotassium hydrogen phosphate, 2 g triammonium citrate, 5 g MgSO4·7H2O, 0.05 g MnSO4, 10 g agar, 0.8 g Tween 80, pH=6.0±0.2, adjusted to 1 L, sterilized at 121℃ for 15 min.

[0038] (2) Fermentation: The activated seed liquid was inoculated into the liquid fermentation medium at a volume ratio of 1:80. The temperature was 36±2℃, the rotation speed was 120 r / min-140 r / min, and the fermentation liquid was obtained after 18±2 h. The liquid fermentation medium is a modified MRS medium with the following formula: 50 g peptone, 20 g yeast extract, 80 g glucose, 10 g dipotassium hydrogen phosphate, 10 g triammonium citrate, 2 g MgSO4·7H2O, 0.25 g MnSO4, 4 g Tween 80, pH=6.0±0.2, adjusted to 5 L, and sterilized at 121℃ for 15 min.

[0039] (3) Preparation of bacterial powder: The fermentation broth obtained after fermentation is centrifuged at 4℃ and 4000 r / min for 15 min to obtain the supernatant and bacterial sludge. The bacterial sludge is pre-cooled and then freeze-dried under vacuum to obtain Lactobacillus helveticus (Lactobacillus). Lactobacillus helveticus IOB726 bacterial powder.

[0040] Example 2 Lactobacillus helveticus ( Lactobacillus helveticus Preparation method of IOB726 bacterial powder 2 A type of Lactobacillus helveticus with lipid-lowering effects ( Lactobacillus helveticus The preparation method of IOB726 bacterial powder includes the following steps: (1) Activation of strain: Lactobacillus helveticus ( Lactobacillus helveticus IOB726 was taken out of the cryopreservation tube and inoculated into the activation medium at a volume ratio of 1:30 for activation culture. The culture temperature was 36±2℃, sealed, and the static culture time was 24±2h. The activated strain is the seed liquid.

[0041] The activation medium is MRS medium, with the following formula: 10 g peptone, 4 g yeast extract, 20 g glucose, 2 g dipotassium hydrogen phosphate, 2 g triammonium citrate, 5 g MgSO4·7H2O, 0.05 g MnSO4, 10 g agar, 0.8 g Tween 80, pH=6.0±0.2, adjusted to 1 L, sterilized at 121℃ for 15 min.

[0042] (2) Fermentation: The activated seed liquid was inoculated into the liquid fermentation medium at a volume ratio of 1:80. The temperature was 36±2℃, the rotation speed was 120 r / min-140 r / min, and the fermentation liquid was obtained after 18±2h. The liquid fermentation medium is a modified MRS medium with the following formula: 50 g peptone, 20 g yeast extract, 35 g inulin, 10 g dipotassium hydrogen phosphate, 10 g triammonium citrate, 2 g MgSO4·7H2O, 0.25 g MnSO4, 4 g Tween 80, pH=6.0±0.2, adjusted to 5 L, and sterilized at 121℃ for 15 min.

[0043] (3) Preparation of bacterial powder: The fermentation broth obtained after fermentation is centrifuged at 4℃ and 4000 r / min for 15 min to obtain the supernatant and bacterial sludge. The bacterial sludge is pre-cooled and then freeze-dried under vacuum to obtain Lactobacillus helveticus (Lactobacillus). Lactobacillus helveticus IOB726 bacterial powder.

[0044] Example 3 Lactobacillus helveticus ( Lactobacillus helveticus A segmented fermentation method for preparing IOB726 bacterial powder A type of Lactobacillus helveticus with in vitro lipid-lowering properties ( Lactobacillus helveticus The preparation method of IOB726 bacterial powder includes the following steps: (1) Activation of strain: Lactobacillus helveticus ( Lactobacillus helveticusIOB726 was taken out of the cryopreservation tube and inoculated into the activation medium at a volume ratio of 1:30 for activation culture. The culture temperature was 36±2℃, sealed, and the culture time was 24±2h. The activated strain is the primary seed culture. The primary seed culture is inoculated into the activation medium at a volume ratio of 1:25 and cultured at 36±2℃ for 22±2h to obtain the secondary seed culture. The activation medium is MRS medium, with the following formula: 10 g peptone, 4 g yeast extract, 20 g glucose, 2 g dipotassium hydrogen phosphate, 2 g triammonium citrate, 0.5 g MgSO4·7H2O, 0.05 g MnSO4, 10 g agar, 0.8 g Tween 80, pH=6.0±0.2, adjusted to 1 L, sterilized at 121℃ for 15 min.

[0045] (2) Segmented fermentation: First stage fermentation: The secondary seed liquid was inoculated into the liquid fermentation medium at a volume ratio of 1:80, and cultured at a temperature of 36±2℃ and a rotation speed of 120 r / min-140 r / min for 18±2h to obtain the fermentation broth; The first stage of liquid fermentation medium is a modified MRS medium with the following formula: 50 g peptone, 20 g yeast extract, 80 g glucose, 10 g dipotassium hydrogen phosphate, 10 g triammonium citrate, 2 g MgSO4·7H2O, 0.25 g MnSO4, 4 g Tween 80, pH=6.0±0.2, adjusted to 5 L, and sterilized at 121℃ for 15 min.

[0046] Second stage fermentation: After concentrating the first stage fermentation broth, it is inoculated into liquid fermentation medium, and cultured at 36±2℃ and 120 r / min-140 r / min for 18±2h to obtain fermentation broth. The second stage of liquid fermentation medium is a modified MRS medium with the following formula: 50 g peptone, 20 g yeast extract, 35 g inulin, 10 g dipotassium hydrogen phosphate, 10 g triammonium citrate, 2 g MgSO4·7H2O, 0.25 g MnSO4, 4 g Tween 80, pH=6.0±0.2, adjusted to 5 L, and sterilized at 121℃ for 15 min.

[0047] (3) Preparation of bacterial powder: The fermentation broth obtained after the second stage of fermentation was centrifuged at 4℃ and 4000 r / min for 15 min to obtain supernatant and bacterial sludge. The supernatant was concentrated, mixed with the obtained bacterial sludge, pre-cooled, and then freeze-dried under vacuum. The pre-cooling temperature was -40℃, the vacuum degree was ≤10 Pa, and the freeze-drying time was 48±2h to obtain Lactobacillus helveticus ( Lactobacillus helveticusIOB726 bacterial powder and its preparations.

[0048] The *Lactobacillus helveticus* obtained in Examples 1-3 ( Lactobacillus helveticus The activity of IOB726 bacterial powder was tested according to GB 4789.35. 2016 National Food Safety Standard, Microbiological Examination of Food, Lactic Acid Bacteria Examination. The test results are shown below: Table 1 Lactobacillus helveticus ( Lactobacillus helveticus IOB726 bacterial culture activity test results (CFU / ml) Experimental Example 4: Lactobacillus helveticus ( Lactobacillus helveticus IOB726 Bioactivity Assay Experimental methods: 1. Lactobacillus helveticus ( Lactobacillus helveticus Biliary salt tolerance and acid resistance test of IOB726 Lactobacillus helveticus (passed three times) Lactobacillus helveticus IOB726 bacterial suspension was inoculated at 10% in blank control and MRS medium with different bile salt concentrations and pH values, and cultured at 37 ℃ for 18 h. A series of dilutions were performed, and 1000 μL of each appropriate dilution was used for viable cell counting, with each dilution repeated three times. After static incubation at 37 ℃ for 36–48 h, the viable cell count was determined. The viable cell counts under different treatment conditions are represented by N; the viable cell counts at different time points in the blank control experiment are represented by N0.

[0049] 2. In vitro cholesterol lowering (1) Drawing the cholesterol standard curve Prepare a cholesterol standard solution. Accurately weigh 50 mg of cholesterol and dissolve it in n-hexane, then dilute to 50 mL to obtain a concentration of 1 mg / mL. Use a 5 mL pipette to transfer 5 mL of this solution to a 50 mL volumetric flask and dilute to 50 mL to obtain a concentration of 0.1 mg / mL (100 μg / mL).

[0050] Pipette 0 mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, and 0.5 mL of cholesterol standard solution into separate 10 mL test tubes. After evaporating the solvent in a 60℃ drying oven, add 2 mL of colorimetric reagent, let stand at room temperature for 10 min, then slowly add 1 mL of concentrated sulfuric acid, mix well, and measure the OD value at 550 nm using a T6-NewJoy visible spectrophotometer within 10–90 min. Plot a standard curve with OD value on the x-axis and cholesterol standard concentration on the y-axis. Colorimetric reagent: 0.5 mg of o-phthalaldehyde dissolved in 1 mL of glacial acetic acid.

[0051] (2) Cholesterol content determination Lactobacillus helveticus (Lactobacillus) was obtained from Examples 1-3. Lactobacillus helveticus IOB726 strain and commercially available Lactobacillus helveticus strain were each inoculated into 10 mL of MRS medium and incubated at 37 ℃ for 20 ± 0.5 h. The culture was then inoculated into 100 mL of the above medium and kept for 17 h.

[0052] 1 mg / mL cholesterol solution: Dilute 0.1 g cholesterol to 100 mL volumetric flask with anhydrous ethanol, filter sterilize through a 0.22 μm filter membrane, and store at 4 °C for later use.

[0053] A standard curve for cholesterol was plotted, with the linear equation y = 0.0039x - 0.015, R0. 2 =0.909, indicating a good linear relationship.

[0054] Lactobacillus helveticus (Lactobacillus) was obtained from Examples 1-3. Lactobacillus helveticus Strain IOB726 and commercially available Lactobacillus helveticus strain were inoculated into MRS-CHOL medium, with uninoculated MRS-CHOL medium serving as a control. After incubation at 37 ℃ for 24 h, centrifugation (6000×g, 5 ℃, 10 min) was performed, and the supernatant was collected for analysis using the OPA method. The MRS-CHOL medium formulation was as follows: 1 mg / mL cholesterol solution (sterilized by 0.22 μm filtration) was added to basal MRS medium (same as activation medium), resulting in a final cholesterol concentration of 100 μg / mL and a pH of 6.0 ± 0.2.

[0055] Take 4.0 mL of the supernatant, add 2.0 mL of 50% KOH, mix well, then add 3.0 mL of 95% ethanol, vortex, place the reactants at 60 °C for 15 min to saponify, add 3.0 mL of n-hexane, mix well, add 2.0 mL of distilled water, vortex for 1 min, and let stand for 15 min.

[0056] Take 2.0 mL of the n-hexane layer solution into a clean centrifuge tube, evaporate it under nitrogen at 60 °C, redissolve it with 2.0 mL of phthalaldehyde solution, add 1.0 mL of concentrated sulfuric acid, vortex, and measure the OD. 550nm .

[0057] The cholesterol content of the sample was determined according to the standard curve. Cholesterol degradation rate / % = In the formula: C0 — Measured cholesterol concentration (μg / mL) in uninoculated supernatant; C – Measured concentration of cholesterol (μg / mL) in the supernatant of the fermentation broth after inoculation.

[0058] 3. In vitro triglyceride-lowering effect (1) Lactobacillus helveticus ( Lactobacillus helveticus Activation of IOB726 and commercially available Lactobacillus helveticus strains Lactobacillus helveticus (Lactobacillus) was obtained from Examples 1-3. Lactobacillus helveticus The IOB726 strain and the commercially available Lactobacillus helveticus strain were each inoculated into 5 mL of culture medium and cultured at 37 °C for 20 h. 5 mL of each culture was then transferred to 100 mL of culture medium and cultured at a suitable temperature for another 18 h. Finally, the Lactobacillus helveticus obtained in Examples 1-3 was... (Lactobacillus helveticus IOB726 and commercially available Lactobacillus helveticus culture were inoculated at a 3% inoculum into 100 mL of suitable triglyceride medium and uninoculated suitable triglyceride medium, respectively, and cultured at 37 °C for 24 h, pending further experiments. The suitable triglyceride medium formulation was: 10 g peptone, 5 g yeast extract, 15 g triglycerides, 2 g dipotassium hydrogen phosphate, 2 g triammonium citrate, and MgSO4. 0.5 g of 7H2O, 0.1 g of MnSO4, 1 g of Tween 80, pH=6.5±0.2, bring to a final volume of 1 L, and sterilize at 121℃ for 15 min.

[0059] (2) Determination of triglyceride content The triglyceride removal rate was determined by inoculating the activated *Lactobacillus helveticus* (obtained in Examples 1-3) at a 10% (V / V) inoculum. Lactobacillus helveticus IOB726 strain and commercially available Lactobacillus helveticus strain were inoculated into triglyceride liquid medium and incubated at 37°C for 24 h. 1 mL of fermentation broth was centrifuged at 6000 r / min for 10 min, and the supernatant was collected. The triglyceride content in the supernatant was determined according to the triglyceride kit instructions. The triglyceride removal rate was calculated using the following formula.

[0060] In the formula: A0 is the triglyceride content of the uninoculated supernatant, and A1 is the triglyceride content (mmol / L) of the fermentation supernatant of the strain.

[0061] 4. Lactobacillus helveticus ( Lactobacillus helveticus The inhibitory effect of IOB726 on the activity of lipid metabolism-related enzymes (1) Preparation of bacterial suspension The activated *Lactobacillus helveticus* obtained in Examples 1-3 (…) Lactobacillus helveticus IOB726 and commercially available Lactobacillus helveticus bacterial suspensions were centrifuged at 10,000 r / min for 10 min at 4°C, the supernatant was discarded, and the bacterial cells were washed three times with sterile water, then resuspended in sterile water and the OD was adjusted. 600nm The bacterial suspension was obtained by adjusting the solution to 2.0.

[0062] (2) Determination of pancreatic lipase activity Mix 200 μL of sample with 200 μL of 2 U / mL pancreatic lipase solution thoroughly and incubate at 37℃ for 15 min. Immediately add 400 μL of 10 mmol / L pNPP solution and mix well. Incubate again at 37℃ for 15 min. After the reaction is complete, quickly place the mixture in a 100℃ water bath for 5 min to terminate the reaction. Centrifuge the terminated reaction solution at 6000 r / min for 5 min, collect the supernatant, and measure the absorbance at 405 nm. Use 10 μg / mL orlistat as a positive control. The enzyme activity inhibition rate is calculated using the following formula.

[0063] In the formula: As is the absorbance of the sample group; An is the absorbance of the sample control group; Ac is the absorbance of the blank group; A0 is the absorbance of the blank control group.

[0064] (3) Determination of cholesterol esterase activity Add 50 μL of sample and 50 μL of cholesterol esterase solution (2 U / mL) to 1 mL of phosphate buffer (0.1 mol / L, pH 7.0, containing 0.1 mol / L NaCl and 5.16 mmol / L sodium taurocholate), mix well, and incubate at 37°C for 15 min. Immediately after incubation, add 100 μL of 4 mmol / L PNPB solution, mix well, and incubate again at 37°C for 15 min. After the reaction, centrifuge at 6000 r / min for 5 min, collect the supernatant, and measure the absorbance at 405 nm. The enzyme activity inhibition rate is calculated using the following formula.

[0065] In the formula: As is the absorbance of the sample group; An is the absorbance of the sample control group; Ac is the absorbance of the blank group; A0 is the absorbance of the blank control group.

[0066] 5. Lactobacillus helveticus ( Lactobacillus helveticus IOB726 combined with bile salt analysis The 0.01 mol / L HCl solution and the *Lactobacillus helveticus* obtained in Examples 1-3 were mixed. Lactobacillus helveticus 1 mL each of IOB726 and commercially available Lactobacillus helveticus bacterial cultures were incubated at 37°C for 1 h. The pH was adjusted to 6.3, and 5 mL of 1 mmol / L sodium taurocholate (STC) standard solution and 5 mL of 0.1 mol / L phosphate buffer (pH 7.0) were added. The mixture was incubated at 37°C for 1 h. After centrifugation, 2 mL of the supernatant was added to 6 mL of 60% sulfuric acid solution and incubated in a 70°C water bath for 30 min, followed by cooling. The absorbance of each group was measured at 387 nm. The absorbance of sodium taurocholate solution at different concentrations (0, 0.1, 0.2, 0.4, 0.5, 0.8 mmol / L) was measured to establish a taurocholate standard curve. The linear equation was y = 0.1464x - 0.0123, R0. 2 =0.9583. Calculate and determine EC using the following formula. 50 : In the formula: A is the amount of bile salt added, μmol; B is the amount of remaining bile salt, μmol.

[0067] Experimental results: 1. Lactobacillus helveticus ( Lactobacillus helveticus Results of bile salt tolerance and acid resistance tests on IOB726 Gastric acid and bile salts have antibacterial properties; only a very small number of lactic acid bacteria with strong acid resistance can survive in large numbers in the stomach and reach the intestines to exert their probiotic effects. This experiment used a pH of 2.0 to investigate the effects of *Lactobacillus helveticus* (…). Lactobacillus helveticus The survival rate of IOB726 was tested, and the results are shown in Table 2.

[0068] Table 2 Lactobacillus helveticus ( Lactobacillus helveticus IOB726's acid tolerance Literature review and analysis showed that the survival rate of lactic acid bacteria stored at pH 2.0 for 2 hours was approximately 50%. As shown in Table 2, the *Lactobacillus helveticus* (Lactobacillus) prepared by this method exhibits in vitro lipid-lowering properties. Lactobacillus helveticusIOB726, when stored at pH 2.0 for 2 hours, showed a survival rate of 80.7%, and after 4 hours, the survival rate reached 57.5%. Therefore, the *Lactobacillus helveticus* strain prepared by this method exhibits in vitro lipid-lowering properties. Lactobacillus helveticus IOB726 has good acid resistance.

[0069] Lactic acid bacteria strains that survive gastric juice will encounter bile salts in the upper small intestine. Bile salt tolerance is an important indicator of their ability to grow, survive, and exert beneficial effects in the intestines. This experiment selected a 4-hour survival test under a bile salt concentration of 0.5%.

[0070] Table 3 Lactobacillus helveticus ( Lactobacillus helveticus IOB726 tolerance to bile salts As shown in Table 3, the *Lactobacillus helveticus* (L. helveticus) prepared by this method has the effect of lowering blood lipids in vitro. Lactobacillus helveticus When IOB726 is stored at a bile salt concentration of 0.5%, the survival rate reaches 86.7% after 2 hours and 80.7% after 4 hours. As shown in Tables 2 and 3, the *Lactobacillus helveticus* (L. helveticus) prepared by this method has the effect of lowering blood lipids in vitro. Lactobacillus helveticus IOB726 has a strong ability to withstand acids and bile salts, a strong ability to pass through the gastrointestinal tract, and maintains a high survival rate.

[0071] 2. Lactobacillus helveticus ( Lactobacillus helveticus Results of in vitro cholesterol-lowering experiments using IOB726 cholesterol standard curve as follows Figure 1 As shown, the linear equation is y = 0.0039x - 0.015, R0 2 =0.909, indicating a good linear relationship, and is used to calculate cholesterol concentration.

[0072] Lactobacillus helveticus ( Lactobacillus helveticus The cholesterol degradation rates of IOB726 and commercially available Lactobacillus helveticus are as follows: Figure 2 As shown. Compared with the control group, the *Lactobacillus helveticus* (Lactobacillus) obtained in Examples 1-3... Lactobacillus helveticus The cholesterol degradation rates of IOB726 were 25.01%, 27.46%, and 30.12%, respectively, while the cholesterol degradation rate of commercially available Lactobacillus helveticus was 18.68%. Its degradation mechanism may be achieved through adsorption or co-precipitation. This indicates that Lactobacillus helveticus (… Lactobacillus helveticus IOB726 has an in vitro cholesterol-lowering effect, and Lactobacillus helveticus (Lactobacillus) prepared through segmented fermentation... Lactobacillus helveticus IOB726 has a better effect on cholesterol degradation.

[0073] 3. Lactobacillus helveticus ( Lactobacillus helveticus Results of in vitro triglyceride (TG) lowering assay using IOB726 Lactobacillus helveticus ( Lactobacillus helveticus The degradation rates of triglycerides by IOB726 and commercially available Lactobacillus helveticus are as follows: Figure 3 As shown, compared with the blank group, the *Lactobacillus helveticus* (Lactobacillus) obtained in Examples 1-3 (…) Lactobacillus helveticus The degradation rates of triglycerides by IOB726 were 40.39%, 44.01%, and 59.63%, respectively, while the degradation rate of triglycerides by commercially available *Lactobacillus helveticus* was 37.53%. This indicates that *Lactobacillus helveticus* (… Lactobacillus helveticus IOB726 has the effect of lowering triglycerides in vitro, and Lactobacillus helveticus (Lactobacillus) prepared through segmented fermentation... Lactobacillus helveticus IOB726 has a good degradation effect on triglycerides.

[0074] 4. Lactobacillus helveticus ( Lactobacillus helveticus Experimental results on the inhibitory effect of IOB726 on the activity of lipid metabolism-related enzymes Pancreatic lipase, secreted by the acinar cells of the pancreas, is a key enzyme in the hydrolysis of fats and plays a crucial role in the digestion of fats in the human body. It hydrolyzes triglycerides into monoglycerides and fatty acids, which are then reabsorbed and ultimately synthesized into the fats needed by the body. Therefore, inhibiting pancreatic lipase activity is an important strategy for the prevention and treatment of hyperlipidemia and obesity.

[0075] The inhibition rates of *Lactobacillus helveticus* IOB726 and commercially available *Lactobacillus helveticus* on pancreatic lipase are as follows: Figure 4 As shown, compared with the blank group, the positive drug group had an inhibition rate of 41.06% against pancreatic lipase. Examples 1-3: Lactobacillus helveticus ( Lactobacillus helveticus The inhibition rates of IOB726 on pancreatic lipase activity were 30.18%, 35.99%, and 38.91%, respectively, while the inhibition rate of commercially available Lactobacillus helveticus on pancreatic lipase activity was 25.37%. This indicates that Lactobacillus helveticus (… Lactobacillus helveticus IOB726 exhibits high inhibitory activity against pancreatic lipase, and Lactobacillus helveticus (Lactobacillus) prepared through fractional fermentation... Lactobacillus helveticus IOB726 has a strong inhibitory effect on pancreatic lipase activity.

[0076] Cholesterol esterase can catalyze the breakdown of cholesterol esters into cholesterol and corresponding fatty acids. Therefore, inhibiting the activity of cholesterol esterase can reduce the absorption of cholesterol and fatty acids in the human body to a certain extent, thereby lowering serum cholesterol levels and regulating blood lipid levels.

[0077] Lactobacillus helveticus ( Lactobacillus helveticusThe inhibition rates of IOB726 and commercially available Lactobacillus helveticus on cholesterol esterase are as follows: Figure 5 As shown, compared with the blank group, the *Lactobacillus helveticus* (Lactobacillus) obtained in Examples 1-3 (…) Lactobacillus helveticus The inhibition rates of IOB726 on cholesterol esterase activity were 21.52%, 26.52%, and 32.36%, respectively, while the inhibition rate of commercially available Lactobacillus helveticus on cholesterol esterase activity was 18.77%. This indicates that Lactobacillus helveticus (… Lactobacillus helveticus IOB726 exhibits high inhibitory activity against cholesterol esterase activity, and Lactobacillus helveticus (Lactobacillus) prepared through segmented fermentation... Lactobacillus helveticus IOB726 has a strong inhibitory effect on cholesterol esterase activity.

[0078] 5. Lactobacillus helveticus ( Lactobacillus helveticus IOB726 combined with bile salt analysis results Bile salts are synthesized into cholesterol in the liver via a multi-enzyme pathway, and then secreted into the intervascular space and between hepatocytes. When substances bind to bile salts, the body's bile salt content decreases, prompting the liver to break down cholesterol to replenish the reduced bile salts, thus maintaining bile acid balance and achieving a lipid-lowering effect. The bile salt standard curve is shown below. Figure 6 As shown, the linear equation is y = 0.1464x - 0.0123, R0 2 =0.9583, indicating a good linear relationship.

[0079] Lactobacillus helveticus ( Lactobacillus helveticus IOB726 and commercially available Lactobacillus helveticus have similar bile salt binding capabilities. Figure 7 As shown, compared with the blank group, the *Lactobacillus helveticus* (Lactobacillus) obtained in Examples 1-3 (…) Lactobacillus helveticus The binding rates of IOB726 to sodium taurocholate were 10.24%, 17.33%, and 21.39%, respectively, while the binding rate of commercially available Lactobacillus helveticus to sodium taurocholate was 10.24%. This indicates that Lactobacillus helveticus (… Lactobacillus helveticus IOB726 has a strong ability to bind bile salts, which helps lower blood cholesterol levels, thus playing a role in lowering blood lipids. Furthermore, Lactobacillus helveticus (Lactobacillus) prepared through segmented fermentation... Lactobacillus helveticus IOB726 has a strong ability to bind bile salts.

[0080] 6. Lactobacillus helveticus ( Lactobacillus helveticus IOB726 bacterial powder storage stability test Lactobacillus helveticus (prepared in Example 4) Lactobacillus helveticus IOB726 was stored at 4℃ and 25℃ for 30 days, and the changes in viable bacterial count were detected: After 30 days of storage at 4℃, the viable bacterial count was 3.82 ± 0.15 × 10⁻⁶. 9CFU / ml (initial 4.05×10⁻⁶) 9 CFU / ml, viability 94.3%); stored at 25℃ for 30 days: viable count 2.98±0.12×10⁻⁶. 9 CFU / ml (survival rate 73.6%). The results indicate that the bacterial powder has good stability under normal storage conditions.

[0081] In summary, this invention studies *Lactobacillus helveticus* (… Lactobacillus helveticus The effect of IOB726 bacterial powder on blood lipids. Results showed that *Lactobacillus helveticus* (…) Lactobacillus helveticus IOB726 bacterial powder exhibits strong resistance to acid and bile salts, demonstrates excellent gastrointestinal tract passage, and maintains a high survival rate. It effectively promotes cholesterol and triglyceride degradation, inhibits the activity of pancreatic lipase and cholesterol esterase, reduces the absorption of cholesterol and fatty acids in the body, and enhances the ability to bind bile salts, thereby lowering blood lipids. This provides a certain pharmacological theoretical basis for the development of drugs and health products for the adjuvant prevention and treatment of hyperlipidemia, and also lays the foundation for the development of Lactobacillus helveticus (…). Lactobacillus helveticus The IOB726 bacterial powder and its formulations provide some innovative ideas.

[0082] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A type of Lactobacillus helveticus with lipid-lowering effects ( Lactobacillus helveticus IOB726, characterized in that: The classification name of the *Lactobacillus helveticus* is *Lactobacillus helveticus* (… Lactobacillus helveticus The accession number is CGMCC No.28044, the accession date is July 28, 2023, the accession site is China General Microbiological Culture Collection Center (CGMCC), and the address of the accession institution is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

2. The *Lactobacillus helveticus* according to claim 1 (… Lactobacillus helveticus IOB726, characterized in that: The Lactobacillus helveticus ( Lactobacillus helveticus IOB726 improves symptoms of hyperlipidemia through at least one of the following (1) to (5) or through a combination of the following: (1) Lowering cholesterol levels in vitro; (2) Reduces triglyceride levels in vitro; (3) Inhibits pancreatic lipase activity; (4) Inhibits cholesterol esterase activity; (5) Ability to bind bile salts.

3. The *Lactobacillus helveticus* as described in claim 1 or claim 2 (… Lactobacillus helveticus The preparation method of IOB726 is characterized by: The preparation method includes the following steps: (1) Activation of strain: Lactobacillus helveticus ( Lactobacillus helveticus IOB726 was taken out of the cryopreservation tube and inoculated into the activation medium at a volume ratio of 1:20-1:30 for activation culture. The culture temperature was 36±2℃, sealed, and the culture time was 24±2h. The activated strain is the primary seed culture. The primary seed culture is inoculated into the activation medium at a volume ratio of 1:30 and cultured at 36±2℃ for 22±2h to obtain the secondary seed culture. (2) Segmented fermentation: First stage fermentation: The secondary seed liquid was inoculated into the liquid fermentation medium at a volume ratio of 1:70-1:80, and cultured at a temperature of 36±2℃ and a rotation speed of 120 r / min-140 r / min for 18±2h to obtain the fermentation broth. Second stage fermentation: The first stage fermentation broth was concentrated by centrifugation at 4℃ and 6000 r / min for 20 min, with a concentration factor of 5-10 times. Then, the concentrated fermentation broth was inoculated into the second stage liquid fermentation medium at a volume ratio of 1:50-1:

60. The temperature was 36±2℃ and the rotation speed was 120 r / min-140 r / min. The culture was carried out for 18±2 h to obtain the fermentation broth. (3) Preparation of bacterial powder: After the second stage of fermentation, the fermentation broth obtained is centrifuged at 4℃ and 4000 r / min for 15 min to obtain the supernatant and bacterial sludge. The supernatant is concentrated, mixed with the obtained bacterial sludge, pre-cooled, and then freeze-dried under vacuum. The pre-cooling temperature is -40℃, the vacuum degree is ≤10 Pa, and the freeze-drying time is 48±2 h to obtain Lactobacillus helveticus ( Lactobacillus helveticus IOB726 bacterial powder or its preparations.

4. The preparation method according to claim 3, characterized in that: The activation medium is MRS medium, with the following formula: 8 g-10 g peptone, 3 g-4 g yeast extract, 15 g-20 g glucose, 1 g-2 g dipotassium hydrogen phosphate, 1 g-2 g triammonium citrate, 0.2 g-0.5 g MgSO4·7H2O, 0.05 g-0.1 g MnSO4, 8 g-10 g agar, 0.8 g-1 g Tween 80, pH=6.0±0.2, adjusted to 1 L, and sterilized at 121℃ for 15 min; The first stage of liquid fermentation medium is a modified MRS medium with the following formula: 40 g-50 g peptone, 15 g-20 g yeast extract, 60 g-80 g glucose, 5 g-10 g dipotassium hydrogen phosphate, 5 g-10 g triammonium citrate, 1 g-2 g MgSO4·7H2O, 0.25 g-0.5 g MnSO4, 4 g-5 g Tween 80, pH=6.0±0.2, adjusted to 5 L, and sterilized at 121℃ for 15 min. The second stage of liquid fermentation medium is a modified MRS medium with the following formula: 40-50 g peptone, 15-20 g yeast extract, 30-50 g inulin, 5-10 g dipotassium hydrogen phosphate, 5-10 g triammonium citrate, 1-2 g MgSO4·7H2O, 0.25-0.5 g MnSO4, 4-5 g Tween 80, pH=6.0±0.2, adjusted to 5 L, and sterilized at 121℃ for 15 min.

5. A product with lipid-lowering effect, characterized in that: The product contains *Lactobacillus helveticus* as described in claim 1 or claim 2. Lactobacillus helveticus Live and / or inactivated cells of IOB726.

6. The *Lactobacillus helveticus* as described in claim 1 or claim 2 (… Lactobacillus helveticus IOB726, Lactobacillus helveticus prepared by the preparation method described in claim 3 or claim 4 ( Lactobacillus helveticus The use of IOB726 bacterial powder or its preparations, or the product of claim 5, in the preparation of products with lipid-lowering effects.

7. The use according to claim 6, characterized in that: The products are selected from pharmaceuticals, functional foods, or health supplements.

8. The use according to claim 6, characterized in that: The product is an oral dosage form.

9. The use according to claim 8, characterized in that: The oral dosage form is a tablet, capsule, oral liquid, enteric-coated tablet, granule, syrup, or sublingual tablet.

10. The use according to claim 6, characterized in that: The products also include other drugs, functional foods, or health products used to prevent, improve, or treat hyperlipidemia.

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