Strain ZW081, composition, preparation method and application thereof

Abstract

The present invention provides a strain ZW081, a composition, a preparation method and an application thereof, belonging to the field of microbial technology, specifically a strain of Heindrixella coagulans ( Heyndrickxia coagulans ) ZW081, deposited in the China Center for Type Culture Collection on October 21, 2024, with the deposit address being Wuhan University, Wuhan, China, with the deposit number being CCTCC NO: M 20242271; a microbial composition comprising strain ZW081, Lactobacillus kefir ZJ13, Acetobacter ZJ10, Lactobacillus paracasei ZJ22, Lactobacillus rhamnosus ZJ18, Leuconostoc mesenteroides ZJ09, Lactobacillus fermentans ZJ20 and Kluyveromyces ZJ19; a method for preparing the microbial composition; and use of the strain and composition in the preparation of lipid-lowering products, wherein the strain and composition have the function of lowering lipids and losing weight.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and specifically relates to strain ZW081, a composition, and a preparation method and application thereof. Background Art

[0002] The prevalence of obesity continues to increase worldwide, with an imbalance between energy intake and expenditure being the primary driver of obesity. The gut microbiome is an environmental factor implicated in obesity and metabolic disorders, with obese animals and humans exhibiting altered gut microbiome composition and structure. Studies have shown that probiotics can effectively alleviate obesity caused by a high-fat diet.

[0003] Kefir is a traditional fermented dairy product made from cow's or goat's milk. Its unique bacterial structure and the complex interactions between the bacterial communities allow the kefir grains in the milk to degrade protein, fat and lactose during the fermentation process, forming a variety of amino acids, short-chain fatty acids, ethanol, vitamins and some biologically active substances, which exert functions such as angiotensin-converting enzyme (ACE) inhibition, antibacterial, immune regulation, mineral binding, and antioxidant.

[0004] Bacillus coagulans ( Bacillus coagulans ) will be analyzed in 2020 based on whole genome sequence B. coagulans The classification changed to Weizmannia coagulans ( Weizmannia ), based on whole genome sequence analysis in 2023, its classification was changed to Hendrixella coagulans at the genus level ( Heyndrickxia coagulans ). That is, Bacillus coagulans is Bacillus coagulans.

[0005] Bacillus coagulans is widely used for its lactic acid production and ability to form spores with excellent stress resistance. However, unlike other probiotics, Bacillus coagulans has a weak adhesion to intestinal epithelial cells, making it difficult to persist in the intestines under natural conditions. Therefore, as an intestinal "migrant," Bacillus coagulans can only stay there for a short time. After a single oral dose of Bacillus coagulans, the bacteria in the intestines will disappear through defecation after approximately 3-5 days. Therefore, only continuous use of Bacillus coagulans inoculation can fully realize its probiotic effects in the intestine.

[0006] Therefore, the market is in urgent need of screening a Bacillus coagulans with lipid-lowering and weight-loss effects, and developing a product with strong stress resistance and the ability to prolong the colonization time of Bacillus coagulans in the intestines, thereby promoting its lipid-lowering and weight-loss effects. Summary of the Invention

[0007] In response to the deficiencies in the prior art, the present invention provides strain ZW081, a composition, and a preparation method and application thereof.

[0008] The present invention provides a strain of Hendrixella coagulans ( Heyndrickxia coagulans ) ZW081, the strain ZW081 has the function of alleviating fatty liver symptoms, lowering lipids and losing weight.

[0009] The present invention discovers for the first time that a microbial composition obtained by combining kefir Lactobacillus ZJ13, Acetobacter ZJ10, Lactobacillus paracasei ZJ22, Lactobacillus rhamnosus ZJ18, Leuconostoc mesenteroides ZJ09, Lactobacillus mucilaginosus ZJ20 and Kluyveromyces ZJ19 isolated from kefir grains with strain ZW081 has good stability, can improve the colonization ability of strain ZW081 in the intestine, and the components exert a synergistic weight-loss effect through multi-target intervention, and have obvious lipid-lowering and weight-loss effects.

[0010] Heindrixella coagulans ( Heyndrickxia coagulans ) ZW081, referred to as strain ZW081.

[0011] The technical solutions of the present invention are as follows:

[0012] A strain of Hendrixella coagulans ( Heyndrickxia coagulans ) ZW081, deposited in the China Center for Type Culture Collection on October 21, 2024, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCCNO: M 20242271.

[0013] A microbial composition comprising the following strains:

[0014] The above-mentioned strains are ZW081, Lactobacillus kefir ZJ13, Acetobacter ZJ10, Lactobacillus paracasei ZJ22, Lactobacillus rhamnosus ZJ18, Leuconostoc mesenteroides ZJ09, Lactobacillus fermentans ZJ20 and Kluyveromyces ZJ19.

[0015] The preparation method of the above-mentioned microbial composition comprises the following steps:

[0016] (1) Preparation of seed solution: Lactobacillus kefir ZJ13, Acetobacter aceticus ZJ10, Lactobacillus paracasei ZJ22, Lactobacillus rhamnosus ZJ18, Leuconostoc mesenteroides ZJ09, Lactobacillus fermentans ZJ20, and Kluyveromyces ZJ19 were inoculated into sterilized milk, and cultured at 28-42°C for 48-96 hours to obtain seed solution of each strain;

[0017] (2) adding the Kluyveromyces ZJ19 seed solution and the Leuconostoc mesenteroides ZJ09 seed solution prepared in step (1) to the sterilized milk at a volume ratio of 5-10%, mixing, and culturing at 28-37° C. for 24-36 hours to obtain a culture;

[0018] (3) Adding the Acetobacter ZJ10 seed solution prepared in step (1) to the culture obtained in step (2) at a volume ratio of 10-20%, mixing, and culturing at 35-40° C. for 16-24 hours to obtain a culture;

[0019] (4) Add strain ZW081 to the culture obtained in step (3) until the concentration of strain ZW081 is 10 8 ~10 10 cuf / mL, mix well, and culture at 30-37°C for 24-96 hours to obtain the culture;

[0020] (5) To the culture obtained in step (4), whole milk powder was added in a weight-to-volume ratio of (1-2): 100 g / mL, and at the same time, the fermented Lactobacillus mucilaginosus ZJ20 seed liquid, the kefir Lactobacillus ZJ13 seed liquid, the Lactobacillus paracasei ZJ22 seed liquid and the Lactobacillus rhamnosus ZJ18 seed liquid prepared in step (1) were added in a volume ratio of 5-10%, the mixture was mixed, and the mixture was statically cultured at 40-45°C for 24-96 hours to obtain a microbial composition.

[0021] Preferably, according to the present invention, in step (1) and step (2), the sterilized milk is pasteurized fresh milk.

[0022] According to the preferred embodiment of the present invention, in step (1), the culture is kept at 28-42°C for 48-96 hours, and the sterilized milk is diluted to produce a bacterial count of 10 6 ~10 8 cfu / mL of seed solution.

[0023] Preferably, according to the present invention, in step (4), the spore rate of strain ZW081 is above 98%.

[0024] Further preferably, in step (4), the strain ZW081 is cultured in MRS medium, statically cultured at 35-40° C. until the spore rate is above 98%, and then solid-liquid separation is performed to obtain the strain ZW081.

[0025] According to a preferred embodiment of the present invention, the method for preparing the microbial composition comprises the following steps:

[0026] ① Preparation of seed solution: Lactobacillus kefir ZJ13, Acetobacter ZJ10, Lactobacillus paracasei ZJ22, Lactobacillus rhamnosus ZJ18, Leuconostoc mesenteroides ZJ09, Lactobacillus fermentans ZJ20 and Kluyveromyces ZJ19 were inoculated into pasteurized fresh milk, incubated at 28°C for 72 hours, and diluted with pasteurized fresh milk to make a bacterial count of 10 6 cfu / mL of seed solution;

[0027] ② Add the Kluyveromyces ZJ19 seed solution and the Leuconostoc mesenteroides ZJ09 seed solution prepared in step ① to pasteurized fresh milk at a volume ratio of 8%, mix well, and incubate at 30° C. for 24 hours to obtain a culture;

[0028] ③ Add the Acetobacter ZJ10 seed solution prepared in step ① to the culture obtained in step ② at a volume ratio of 15%, mix well, and incubate at 37° C. for 24 hours to obtain a culture;

[0029] ④ Add strain ZW081 to the culture obtained in step ③ until the concentration of strain ZW081 is 10 8 cuf / mL, mix well, and culture at 37°C for 24 hours to obtain the culture;

[0030] ⑤ To the culture obtained in step ④, whole milk powder was added at a weight-to-volume ratio of 1.5:100 g / mL, and then the fermented Lactobacillus mucilaginosus ZJ20 seed solution, kefir Lactobacillus ZJ13 seed solution, Lactobacillus paracasei ZJ22 seed solution, and Lactobacillus rhamnosus ZJ18 seed solution prepared in step ① were added at a volume ratio of 8%, mixed, and statically cultured at 42°C for 96 hours to obtain a microbial composition;

[0031] The strain ZW081 in step ④ is cultured in MRS medium, statically cultured at 35-40° C. until the spore rate reaches 99.5%, solid-liquid separation is performed, and the solid is collected to obtain the strain ZW081.

[0032] Use of the strain ZW081, the microbial composition, or the microbial composition prepared by the method described above in any of the following:

[0033] a. Application in the preparation of weight loss products;

[0034] b. Application in the preparation of lipid-lowering products;

[0035] c. Application in the preparation of drugs for preventing or treating fatty liver.

[0036] Preferably according to the present invention, the lipid-lowering product and weight-loss product are health foods or pharmaceutical preparations.

[0037] The beneficial effects of the present invention include at least the following:

[0038] 1. The present invention provides a strain of Bacillus coagulans ( Heyndrickxia coagulans ) ZW081 can reduce abdominal fat index, reduce fatty liver level, reduce the levels of serum liver function related indicators ALT, AST and ALP, alleviate liver function damage, and has the function of relieving fatty liver symptoms, lowering blood lipids and losing weight.

[0039] 2. The kefir Lactobacillus ZJ13, Acetobacter ZJ10, Lactobacillus paracasei ZJ22, Lactobacillus rhamnosus ZJ18, Leuconostoc mesenteroides ZJ09, Lactobacillus fermentans ZJ20 and Kluyveromyces ZJ19 in the present invention are all derived from the same natural kefir grains and have inherently good symbiotic properties. Therefore, they can quickly form a stable symbiotic community during the fermentation process.

[0040] 3. The microbial composition provided by the present invention, strain ZW081 is combined with the above-mentioned strains. Strain ZW081 forms a symbiotic system with other microorganisms in the microcommunity. Under the action of the biofilm formed by extracellular polysaccharides, etc., it integrates with intestinal microorganisms such as Lactobacillus paracasei ZJ22, Lactobacillus rhamnosus ZJ18, Leuconostoc mesenteroides ZJ09, and Lactobacillus mucilaginosus ZJ20. With the help of the intestinal colonization of the symbiotic microorganisms, strain ZW081 can remain in the intestine for a long time.

[0041] 4. The microbial composition provided by the present invention, strain ZW081, when combined with the above strains, can improve the intestinal microecological environment, improve the body's ability to decompose and metabolize, reduce fat accumulation, and achieve the effect of lowering lipids and losing weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Figure 3 shows the body weight of mice in different groups.

[0043] Figure 2 Figure 3 shows the abdominal fat index of mice in different groups.

[0044] Figure 3 Figure 2 shows the liver function indicators of mice in different groups.

[0045] Figure 4 Figures are paraffin sections of the livers of mice in different groups. DETAILED DESCRIPTION

[0046] The present invention is described in detail below with reference to the examples. It should be understood that the examples are only used to further illustrate and explain the present invention and are not intended to limit the present invention.

[0047] The contents not described in detail in the examples are based on conventional techniques in the art; the experimental materials and reagents not described in detail are common commercial products.

[0048] Lactobacillus kefir ZJ13, Acetobacter aceticus ZJ10, Lactobacillus paracasei ZJ22, Lactobacillus rhamnosus ZJ18, Leuconostoc mesenteroides ZJ09, Lactobacillus fermentans ZJ20, and Kluyveromyces ZJ19 strains were purchased from Jinan Jingyan Biotechnology Co., Ltd.

[0049] Strain ZW081 was obtained from the intestine of dairy cows and was identified as Heindrixella coagulans ( Heyndrickxia coagulans), has been preserved, the specific preservation information is as follows:

[0050] A strain of Hendrixella coagulans ( Heyndrickxia coagulans ) ZW081, deposited in the China Center for Type Culture Collection on October 21, 2024, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCCNO: M 20242271.

[0051] Example 1

[0052] The strain ZW081 was cultured in MRS medium (Qingdao Haibo Biotechnology Co., Ltd.) at 35-40°C until the spore rate reached 99.5%. The culture was then centrifuged at 6000 rpm for 10 minutes, and the precipitate was collected to obtain the strain ZW081.

[0053] Example 2

[0054] A method for preparing a microbial composition comprises the following steps:

[0055] (1) Preparation of seed liquid: Lactobacillus kefir ZJ13, Acetobacter ZJ10, Lactobacillus paracasei ZJ22, Lactobacillus rhamnosus ZJ18, Leuconostoc mesenteroides ZJ09, Lactobacillus fermentans ZJ20 and Kluyveromyces ZJ19 were inoculated into pasteurized fresh milk, and cultured at 28°C for 72 hours. The seed liquid was diluted with pasteurized fresh milk to prepare 10 6 cfu / mL of seed solution;

[0056] (2) adding the Kluyveromyces ZJ19 seed solution and the Leuconostoc mesenteroides ZJ09 seed solution prepared in step (1) to pasteurized fresh milk at a ratio of 8% (v / v), mixing, and culturing at 30° C. for 24 hours to obtain a culture;

[0057] (3) Adding the Acetobacter ZJ10 seed solution prepared in step (1) to the culture obtained in step (2) at a ratio of 15% (v / v), mixing well, and culturing at 37°C for 24 hours to obtain a culture;

[0058] (4) Add the strain ZW081 prepared in Example 1 to the culture obtained in step (3) until the concentration of strain ZW081 is 10 8 cuf / mL, mix well, and culture at 37°C for 24 hours to obtain the culture;

[0059] (5) To the culture obtained in step (4), whole milk powder was added at a weight-to-volume ratio of 1.5:100 g / mL, and then the fermented Lactobacillus mucilaginosus ZJ20 seed liquid, kefir Lactobacillus ZJ13 seed liquid, Lactobacillus paracasei ZJ22 seed liquid and Lactobacillus rhamnosus ZJ18 seed liquid prepared in step (1) were added at a ratio of 8% (v / v), mixed, and cultured at 42°C for 96 hours to obtain a microbial composition.

[0060] The colony counts were performed after the cultures were boiled in water for 10 minutes at the beginning of step (4) when the strain ZW081 prepared in Example 1 was added, at the end of step (4), and at the end of step (5). The spore count of strain ZW081 at the beginning of step (4) was 9.1×10 7 cuf / mL, and the spore count of strain ZW081 at the end of step (4) was 9.0×10 7 cuf / mL, indicating that after 24 hours of static culture at 37°C in step (4), the spore survival rate was 98.9%, and the spores had basically not germinated; the spore count of strain ZW081 at the end of step (5) was 8.7×10 7 cuf / mL, and the spore survival rate of strain ZW081 was 95.6%.

[0061] Comparative Example 1

[0062] A method for preparing a microbial composition comprises the following steps:

[0063] The difference from Example 2 is that strain ZW081 is not added, specifically as follows:

[0064] (1) Same as step (1) in Example 2;

[0065] (2) Same as step (2) in Example 2;

[0066] (3) Adding the Acetobacter ZJ10 seed solution prepared in step (1) to the culture obtained in step (2) at a ratio of 15% (v / v), mixing well, and culturing at 37°C for 24 hours to obtain a culture;

[0067] (4) To the culture obtained in step (3), whole milk powder was added at a weight-to-volume ratio of 1.5:100 g / mL, and then seed liquid of Lactobacillus mucilaginosus ZJ20, seed liquid of Lactobacillus kefir ZJ13, seed liquid of Lactobacillus paracasei ZJ22, and seed liquid of Lactobacillus rhamnosus ZJ18 were added at a ratio of 8% (v / v). The mixture was mixed and incubated at 42°C for 96 hours to obtain a microbial composition.

[0068] Comparative Example 2

[0069] A method for preparing a microbial composition comprises the following steps:

[0070] The difference from Example 2 is that strain ZW081 is first added to pasteurized fresh milk, and then other strains are added, as follows:

[0071] (1) Same as step (1) in Example 2;

[0072] (2) The strain ZW081 prepared in Example 1 was added to pasteurized fresh milk until the bacterial concentration reached 10 8 cuf / ml, mixed well, and cultured at 37°C for 24 h to obtain the culture;

[0073] (3) adding Kluyveromyces ZJ19 seed solution and Leuconostoc mesenteroides ZJ09 seed solution to the culture obtained in step (2) at a ratio of 8% (v / v), mixing, and culturing at 30° C. for 24 hours to obtain a culture;

[0074] (4) Adding 15% (v / v) Acetobacter ZJ10 seed solution to the culture obtained in step (3), mixing well, and culturing at 37°C for 24 hours to obtain a culture;

[0075] (5) To the culture obtained in step (4), whole milk powder was added at a weight-to-volume ratio of 1.5:100 g / mL, and then seed liquid of Lactobacillus mucilaginosus ZJ20, seed liquid of Lactobacillus kefir ZJ13, seed liquid of Lactobacillus paracasei ZJ22, and seed liquid of Lactobacillus rhamnosus ZJ18 were added at a ratio of 8% (v / v). The mixture was mixed and incubated at 42°C for 96 hours to obtain a microbial composition.

[0076] The obtained microbial composition was placed in a boiling water bath for 10 minutes and then colony counted. The survival rate of strain ZW081 was calculated to be 61.8%.

[0077] Comparative Example 3

[0078] A method for preparing a microbial composition comprises the following steps:

[0079] The difference from Example 2 is that strain ZW081 is added at the end, as follows:

[0080] (1) Same as step (1) in Example 2;

[0081] (2) Same as step (2) in Example 2;

[0082] (3) Same as step (3) in Example 2;

[0083] (4) Adding whole milk powder to the culture obtained in step (3) at a weight-to-volume ratio of 1.5:100 g / mL, and then adding 8% (v / v) of Lactobacillus fermentans ZJ20 seed solution, Lactobacillus kefir ZJ13 seed solution, Lactobacillus paracasei ZJ22 seed solution, and Lactobacillus rhamnosus ZJ18 seed solution, the mixture was mixed, and the mixture was statically cultured at 42°C for 96 hours to obtain a culture;

[0084] (5) Add strain ZW081 to the culture obtained in step (4) until the concentration of strain ZW081 is 10 8 cuf / mL, mix well, and culture at 37°C for 24 hours to obtain a microbial composition.

[0085] The obtained microbial composition was placed in a boiling water bath for 10 minutes and then colony counted. The survival rate of strain ZW081 was calculated to be 30.7%.

[0086] Comparative Example 4

[0087] A method for preparing a microbial composition comprises the following steps:

[0088] The difference from Example 2 is that before adding Acetobacter ZJ10, strain ZW081 was added, specifically as follows:

[0089] (1) Same as step (1) in Example 2;

[0090] (2) Same as step (2) in Example 2;

[0091] (3) Add strain ZW081 to the culture obtained in step (2) until the concentration of strain ZW081 is 10 8 cuf / mL, mix well, and culture at 37°C for 24 hours to obtain the culture;

[0092] (4) Adding 15% (v / v) Acetobacter ZJ10 seed solution to the culture obtained in step (3), mixing well, and culturing at 37°C for 24 hours to obtain a culture;

[0093] (5) Adding whole milk powder to the culture obtained in step (4) at a weight-to-volume ratio of 1.5:100 g / mL, and then adding 8% (v / v) of Lactobacillus fermentans ZJ20 seed solution, Lactobacillus kefir ZJ13 seed solution, Lactobacillus paracasei ZJ22 seed solution, and Lactobacillus rhamnosus ZJ18 seed solution, the mixture was mixed, and statically cultured at 42°C for 96 hours to obtain a culture;

[0094] The obtained microbial composition was placed in a boiling water bath for 10 minutes and then colony counted. The survival rate of strain ZW081 was calculated to be 82.4%.

[0095] Effect Example 1

[0096] The strain ZW081 culture prepared in Example 1 was used for mouse experiments, as follows:

[0097] 1. Experimental Materials and Methods

[0098] (1) Animal model construction

[0099] Obesity model mice were obtained by feeding 8-week-old Kunming male mice with a high-fat diet.

[0100] (2) Animal experiment design

[0101] All mice were fed a commercial basal diet for one week and then randomly divided into three groups of 15 mice each. During the experiment, the mice's food and water intake were not restricted by any factors. During the modeling period, one group was fed only a basal diet, and the other two groups were fed a high-fat diet for 6 weeks. The high-fat diet ingredients (%) were: glucose 15%, lard 20%. The ordinary feed was crushed and added to the high-fat ingredients, mixed evenly, and dried at 60°C. Intervention treatment began in the third week. Except for Group 1 (control group), which continued to be fed a basal diet, the other two groups received the following interventions:

[0102] Group 2 (obese group) was gavaged with 0.5 mL / day of normal saline;

[0103] In group 3 (treatment group), the strain ZW081 obtained by centrifugation in Example 1 was first diluted with physiological saline to a concentration of 1×10 8 cfu / mL, 1×10 8 cfu / mL of strain ZW081 0.5mL;

[0104] All gavage treatments were performed once a day.

[0105] (3) Analysis of mouse blood and tissue physiological indicators

[0106] At the end of the animal experiment, in accordance with relevant animal ethics regulations, all mice were anesthetized with 1% sodium pentobarbital, and blood was collected by enucleation of the eyeballs. The mice were then sacrificed. Fresh blood samples were allowed to rest at 37°C for 1 hour and then centrifuged at 3000 rpm for 10 minutes to obtain mouse serum.

[0107] Mouse liver and serum samples were collected in sterile PE tubes. Liver tissue sections were preserved in 4% paraformaldehyde, stained with HE, and examined under a microscope.

[0108] Serum liver function indicators ALT (alanine aminotransferase), AST (aspartate aminotransferase) and ALP (alkaline phosphatase) were detected using ELISA kits.

[0109] Liver, epididymal fat, inguinal fat and perirenal fat tissue were dissected and separated, weighed and recorded respectively. Abdominal fat index was the value obtained by dividing the above fat tissue by body weight.

[0110] (4) Data analysis

[0111] All experimental data were expressed as mean ± standard deviation (SD) and plotted using Excel software.

[0112] 2. Experimental results

[0113] (1) Weight and abdominal fat index

[0114] Depend on Figure 1 、 Figure 2 It can be seen that after feeding high-fat feed, the weight and abdominal fat index of mice were significantly increased compared with the control group. However, after receiving treatment with strain ZW081, although it was still higher than the control group, it was significantly better than the obese group. The weight and abdominal fat index of the treatment group decreased significantly, indicating that strain ZW081 can have a significant lipid-lowering and weight-loss effect.

[0115] Elevated levels of ALT, AST, and ALP generally occur during liver inflammation, fatty liver, and a series of chronic liver diseases. Figure 3 It can be seen that after feeding high-fat feed, the liver function indicators ALT, AST and ALP of mice were significantly increased compared with the control group. However, after receiving strain ZW081 treatment, although they were still higher than the control group, they were significantly better than the obese group. The ALT, AST and ALP of the treatment group all decreased significantly, indicating that strain ZW081 has a significant effect on improving liver function.

[0116] (2) Observation of liver tissue morphology

[0117] Depend on Figure 4 It can be seen that obvious fat particles appeared in the livers of the obese mice that consumed a high-fat diet, which is a typical symptom of fatty liver. After the treatment group was treated with strain ZW081, the symptoms of fatty liver were significantly alleviated, which is also consistent with the changing trend of liver function test indicators ALT, AST and ALP.

[0118] The above experimental results show that feeding strain ZW081 can significantly reduce obesity, fatty liver and liver function damage caused by consuming high-fat feed, which lays the foundation for the development of weight loss probiotic products based on strain ZW081.

[0119] Effect Example 2

[0120] The strain ZW081 culture prepared in Example 1 and the microbial compositions prepared in Example 2 and Comparative Examples 1-4 were subjected to rat experiments, as follows:

[0121] 1. Experimental Materials and Methods

[0122] (1) Animal model construction

[0123] Five-week-old SD rats were fed a high-fat diet to establish an obesity model.

[0124] (2) Animal experiment design

[0125] All rats were fed a commercial basal diet for one week and then randomly divided into groups of 15 rats per group. During the experiment, the rats' food and water intake were not restricted by any factors. During the modeling period, the blank group was fed only the basal diet, while the other nine groups were fed a high-fat diet. The high-fat diet ingredients (%) were: glucose 15%, lard 20%. The high-fat ingredients were added to the crushed ordinary feed, mixed evenly, and dried at 60°C. The intervention treatment began in the third week and lasted for 10 weeks. Except for the blank group, which continued to be fed the basal diet, the other nine groups received the following interventions:

[0126] The obese group was gavaged with 1 mL / kg normal saline once a day.

[0127] In Example 1, the strain ZW081 obtained by centrifugation in Example 1 was first diluted with physiological saline to a concentration of 1×10 8 cfu / ml, 1×10 8 cfu / ml of strain ZW081 1mL / kg, all gavage treatments were once a day.

[0128] The Example 2 group was gavaged with 1 mL / kg of the microbial composition prepared in Example 2 each time, and all gavage treatments were performed once a day.

[0129] The comparative example 1 group was gavaged with 1 mL / kg of the microbial composition prepared in comparative example 1 each time, and all gavage treatments were performed once a day.

[0130] The comparative example 2 group was gavaged with 1 mL / kg of the microbial composition prepared in comparative example 2 each time, and all gavage treatments were performed once a day.

[0131] The comparative example 3 group was gavaged with 1 mL / kg of the microbial composition prepared in comparative example 3 each time, and all gavage treatments were performed once a day.

[0132] The comparative example 4 group was gavaged with 1 mL / kg of the microbial composition prepared in comparative example 4 each time, and all gavage treatments were performed once a day.

[0133] In Experimental Example 1, the microbial composition prepared in Example 2 was gavaged at 1 mL / kg each time. Unlike the Example 2 group, the microbial composition was gavaged every three days.

[0134] In experimental group 2, 1×10 8The strain ZW081 with a cfu / ml of 1 mL / kg was administered orally, and the difference from the group in Example 1 was that the mice were gavaged once every three days.

[0135] Body weight was measured at the beginning and end of the experiment.

[0136] The experimental results are shown in Table 1:

[0137] Table 1 Body weight of rats in different groups

[0138]

[0139] The single intake of strain ZW081 in Experimental Example 2 and Example 1 was the same, except that the intake of strain ZW081 was every 3 days or every day, respectively. This may be because strain ZW081 could not colonize well in the intestine, so the lipid-lowering and weight-loss effects in Experimental Example 2 were significantly reduced.

[0140] The difference between Experimental Example 1 and Example 2 is that the microbial composition was taken every three days or daily, respectively, with the same single intake. This may be because strain ZW081, when combined with other microbial species, is able to colonize the intestine, extending the duration of action. The microbial composition provided by the present invention has significant lipid-lowering and weight-loss effects and a long-lasting effect.

[0141] It can be seen from Experimental Example 1 and Experimental Example 2 that the number of spores of strain ZW081 ingested by the two groups is similar, and both groups ingested probiotics every 3 days, but the effect of Experimental Example 1 group was significantly better than that of Experimental Example 2 group, which further illustrates that the microbial composition provided by the present invention has a longer duration of action and a significant lipid-lowering and weight-loss effect.

[0142] Through the experimental results of Example 2 and Comparative Example 1, the inventors found that although the combination of other microbial species as probiotics can reduce weight gain to a certain extent, the strain ZW081 should still play the main lipid-lowering and weight-loss effects.

[0143] The number of spores of strain ZW081 ingested daily in Group 1 and Group 2 was similar, and the weight gain of the two groups of rats was also relatively close. Compared with the obesity model group, both groups showed better lipid-lowering and weight-loss effects, further demonstrating that strain ZW081 should be the main lipid-lowering and weight-loss effect in the microbial composition.

[0144] The microbial species of Comparative Example 2, Comparative Example 3, and Comparative Example 4 are the same as those of Example 2. The difference is that the strain ZW081 is added to the first step, the last step, or different times in the middle of the fermentation in Comparative Example 2, Comparative Example 3, and Comparative Example 4, respectively. Compared with the group in Example 2, the weight loss effect is significantly reduced. This may be because the strain ZW081 in the comparative example 2 group germinates spores in the pasteurized fresh milk. Although it will still be encapsulated into other microbial species through the subsequent fermentation process, its survival rate is 61.8% in the form of vegetative cells, which is lower than 95.6% in Example 2. In Comparative Example 3, the strain ZW081 is added in the last step. Since it cannot be encapsulated into other microbial species, some spores germinate at the same time, and its survival rate is only 30.7%. In Comparative Example 4, the strain ZW081 is added at a different stage, from being added after Acetobacter ZJ10 to being added before, and the spore rate still decreases slightly to 82.4%. The inventors speculate that this may have affected the survival rate of strain ZW081 in gastrointestinal fluid, etc., ultimately reducing the lipid-lowering and weight-loss effects.

[0145] In summary, the strain ZW081 provided by the present invention has significant lipid-lowering and weight-loss effects, significantly reducing obesity, fatty liver disease, and liver damage caused by consuming a high-fat diet. This paves the way for the development of weight-loss probiotic products based on strain ZW081. The microbial composition provided by the present invention also has significant lipid-lowering and weight-loss effects, with a long-lasting effect.

Claims

1. A strain of Hendrixella coagulans ( Heyndrickxia coagulans ) ZW081, deposited in the China Center for Type Culture Collection on October 21, 2024, with the deposit address being Wuhan University, Wuhan, China, with the deposit number being CCTCC NO: M 20242271.

2. A microbial composition, characterized in that The following strains are included: The coagulant bacteria according to claim 1 ( Heyndrickxia coagulans ) ZW081, Lactobacillus kefir ZJ13, Acetobacter aceticus ZJ10, Lactobacillus paracasei ZJ22, Lactobacillus rhamnosus ZJ18, Leuconostoc mesenteroides ZJ09, Lactobacillus fermentum ZJ20, and Kluyveromyces ZJ19; Heindrixella coagulans ( Heyndrickxia coagulans ) ZW081 is referred to as strain ZW081; The preparation method of the microbial composition comprises the following steps: ① Preparation of seed solution: Lactobacillus kefir ZJ13, Acetobacter ZJ10, Lactobacillus paracasei ZJ22, Lactobacillus rhamnosus ZJ18, Leuconostoc mesenteroides ZJ09, Lactobacillus fermentans ZJ20 and Kluyveromyces ZJ19 were inoculated into pasteurized fresh milk, incubated at 28°C for 72 hours, and diluted with pasteurized fresh milk to make a bacterial count of 10 6 cfu / mL of seed solution; ② Add the Kluyveromyces ZJ19 seed solution and the Leuconostoc mesenteroides ZJ09 seed solution prepared in step ① to pasteurized fresh milk at a volume ratio of 8%, mix well, and incubate at 30° C. for 24 hours to obtain a culture; ③ Add the Acetobacter ZJ10 seed solution prepared in step ① to the culture obtained in step ② at a volume ratio of 15%, mix well, and incubate at 37° C. for 24 hours to obtain a culture; ④ Add strain ZW081 to the culture obtained in step ③ until the concentration of strain ZW081 is 10 8 cuf / mL, mix well, and culture at 37°C for 24 hours to obtain the culture; ⑤ To the culture obtained in step ④, whole milk powder was added at a weight-to-volume ratio of 1.5:100 g / mL, and then the fermented Lactobacillus mucilaginosus ZJ20 seed solution, kefir Lactobacillus ZJ13 seed solution, Lactobacillus paracasei ZJ22 seed solution, and Lactobacillus rhamnosus ZJ18 seed solution prepared in step ① were added at a volume ratio of 8%, mixed, and statically cultured at 42°C for 96 hours to obtain a microbial composition; The strain ZW081 in step ④ is cultured in MRS medium, statically cultured at 35-40° C. until the spore rate reaches 99.5%, solid-liquid separation is performed, and the solid is collected to obtain the strain ZW081.

3. The coagulant Bacillus hendrickii of claim 1 ( Heyndrickxia coagulans ) ZW081 or the microbial composition of claim 2, for use in any of the following: a. Application in the preparation of weight loss products; b. Application in the preparation of lipid-lowering products; c. Application in the preparation of drugs for preventing or treating fatty liver.

4. The use according to claim 3, characterized in that The lipid-lowering product and weight-loss product are health foods or pharmaceutical preparations.

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