Preparation method of Lactobacillus casei IOB-P9 metagenic powder and application of Lactobacillus casei IOB-P9 metagenic powder in adjusting corticosterone level

Abstract

The invention provides a preparation method of lactobacillus casei IOB-P9 after-generation powder and application of the lactobacillus casei IOB-P9 after-generation powder in adjusting the corticosterone level, and belongs to the technical field of microbial fermentation. The preparation method comprises the following steps: performing solid-state gradient temperature fermentation on a first-stage seed solution of the casei casei IOB-P9, and then drying and crushing the fermented raw materials to obtain the casei casei IOB-P9 after-generation powder. The lactobacillus casei IOB-P9 metaactive powder fermented at the solid gradient temperature can regulate the body mass of obese rats, reduce the levels of serum leptin and serum corticosterone, reduce the four levels of blood fat, regulate the damaged state of liver cells and improve related symptoms caused by obesity of the rats.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and specifically relates to a preparation method of postbiotic powder of Lactobacillus casei IOB-P9 and its application in regulating cortisol levels. Background Art

[0002] Obesity is a global public health problem. Research shows that obesity is closely related to various chronic diseases, such as cardiovascular diseases, diabetes, fatty liver, and certain cancers. Obesity not only affects the quality of life of patients but also increases the medical burden. Therefore, the treatment of obesity is of great significance.

[0003] Obesity can lead to dyslipidemia and liver damage, further causing cardiovascular diseases and liver diseases such as atherosclerosis and coronary heart disease. At the same time, obese patients usually exhibit high leptin levels and high cortisol levels. Regulating cortisol levels helps relieve the related symptoms caused by obesity. Cortisol is a stress hormone, and obese patients often have elevated cortisol levels due to chronic stress, which further promotes fat accumulation and metabolic disorders.

[0004] Research shows that probiotics and postbiotics can reduce the secretion of leptin by adipocytes by improving the composition of the gut microbiota, reducing the proportion of pro-inflammatory microbiota, and increasing the abundance of beneficial bacteria. Probiotics and postbiotics can also reduce cortisol levels by regulating the hypothalamic-pituitary-adrenal (HPA) axis. Probiotics can reduce the concentration of cortisol in the feces of obese mice, thus improving the stress response related to obesity. Certain components in postbiotics, such as the outer membrane proteins of lactic acid bacteria, can also regulate cortisol secretion through the vagus nerve pathway. Compared with traditional drugs for treating related diseases in obese patients, probiotics and postbiotics are safer, more stable, and less likely to cause drug resistance. Postbiotics do not require viable bacteria colonization, avoiding the defect of unstable colonization that probiotics may have. In addition, the metabolites of postbiotics can directly act on host cells to regulate metabolism and immune function, thus more effectively improving the metabolic disorders related to obesity.

[0005] In summary, it is of great significance to find a postbiotic that can regulate cortisol levels. Summary of the Invention

[0006] In view of the technical problems existing in the prior art, the present invention aims to provide a preparation method of postbiotic powder of Lactobacillus casei IOB-P9 and its application in regulating cortisol levels.

[0007] Lactobacillus casei ( Lacticaseibacillus casei ) IOB-P9 is classified and named as Lactobacillus casei, and its Latin scientific name is Lacticaseibacillus casei, it was deposited on December 27, 2021 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, abbreviated as CGMCC, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 24195.

[0008] One of the objectives of the present invention is to provide a method for preparing postbiotic powder of Lactobacillus casei IOB-P9, which is characterized in that the preparation method includes: Performing solid-state gradient temperature fermentation on the first-stage seed liquid of Lactobacillus casei IOB-P9 to obtain the fermented raw material; drying and pulverizing the fermented raw material to obtain the postbiotic powder of Lactobacillus casei IOB-P9; The solid-state gradient temperature fermentation includes: Controlling the fermentation temperature at 42-43 °C and maintaining it for about 8-10 h; Lowering the fermentation temperature to 37-40 °C and maintaining it for about 10-12 h; Adjusting the fermentation temperature to 34-38 °C and maintaining it for 10-12 h to obtain the fermented raw material.

[0009] Preferably, the solid-to-liquid ratio of the solid medium for the solid-state gradient temperature fermentation is 1:1-2, and the inoculation amount is 1×10 6 ~5×10 9 cfu / mL.

[0010] Preferably, the component of the solid medium is oats.

[0011] Preferably, the method for preparing the first-stage seed liquid of Lactobacillus casei IOB-P9 includes: Inoculating the IOB-P9 strain in the cryopreservation tube into the slant medium and placing it in an incubator at 35-39 °C for 16-24 h to obtain the slant strain; Taking the slant strain and inoculating it into the culture solution containing 2% oat powder, and culturing it at 35-39 °C for 16-20 h to obtain the first-stage seed liquid.

[0012] Preferably, the components of the slant medium are: 20-25 g of yeast powder, 25-30 g of glucose, 1-3 ml of Tween 80, 2-5 g of dipotassium hydrogen phosphate, 5-7 g of sodium acetate, 2-5 g of ammonium citrate, 0.2-0.5 g of magnesium sulfate, 0.05-0.07 g of manganese sulfate, 15-17 g of agar powder, and 1000 ml of distilled water.

[0013] Preferably, the drying temperature is 55-60 °C, and the moisture content after drying is ≤10%.

[0014] Another objective of the present invention is to provide the application of the postbiotic powder of Lactobacillus casei IOB-P9 in the preparation of drugs for improving obesity.

[0015] The third object of the present invention is to provide an application of the postbiotic powder of Lactobacillus casei IOB-P9 in the preparation of a drug for improving hepatocyte damage caused by obesity.

[0016] The fourth object of the present invention is to provide an application of the postbiotic powder of Lactobacillus casei IOB-P9 in the preparation of a drug for increasing the corticosterone level.

[0017] Beneficial effects of the present invention: The present invention provides a preparation method of the postbiotic powder of Lactobacillus casei IOB-P9. Using oats as a solid fermentation medium, the primary seed liquid of Lactobacillus casei IOB-P9 is subjected to solid-state gradient temperature fermentation, and then the fermented raw material is dried and pulverized to obtain the postbiotic powder of Lactobacillus casei IOB-P9. In the present invention, the postbiotic powder of Lactobacillus casei IOB-P9 can regulate the body weight of obese rats, reduce the levels of serum leptin and serum corticosterone, reduce the levels of four blood lipid items, regulate the damaged state of liver cells, and improve the related symptoms caused by obesity in rats. Description of the drawings

[0018] Figure 1 Shows the effect of the postbiotic of Lactobacillus casei IOB-P9 on the body weight of rats; Figure 2 Shows the effect of the postbiotic of Lactobacillus casei IOB-P9 on the LEE' index of rats; Figure 3 Shows the effect of the postbiotic of Lactobacillus casei IOB-P9 on the fat / body ratio of rats; Figure 4 Shows the effect of the postbiotic of Lactobacillus casei IOB-P9 on the serum leptin of rats; Figure 5 Shows the effect of the postbiotic of Lactobacillus casei IOB-P9 on the serum corticosterone of rats; Figure 6 Shows the effect of the postbiotic of Lactobacillus casei IOB-P9 on the TC content of rats; Figure 7 Shows the effect of the postbiotic of Lactobacillus casei IOB-P9 on the TG content of rats; Figure 8 Shows the effect of the postbiotic of Lactobacillus casei IOB-P9 on the LDL-C content of rats; Figure 9 Shows the effect of the postbiotic of Lactobacillus casei IOB-P9 on the HDL-C content of rats; Figure 10 Shows the effect of the postbiotic of Lactobacillus casei IOB-P9 on the Glu content of rats; Figure 11 Shows the effect of the postbiotic of Lactobacillus casei IOB-P9 on the ALP content of rats; Figure 12 Effect of postbiotics of Lactobacillus casei IOB-P9 on the ALT content in rats; Figure 13 Effect of postbiotics of Lactobacillus casei IOB-P9 on the AST content in rats; Figure 14 Results of HE staining and Oil Red staining.

[0019] The Lactobacillus casei provided by the present invention ( Lacticaseibacillus casei ) IOB-P9 is classified and named as Lactobacillus casei, and its Latin scientific name is Lacticaseibacillus casei , which was deposited in the China General Microbiological Culture Collection Center on December 27, 2021, abbreviated as CGMCC, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 24195. Specific Embodiments

[0020] The present invention provides a method for preparing a postbiotic powder of Lactobacillus casei IOB-P9 and its application in regulating the corticosterone level.

[0021] According to the first aspect of the present invention, there is provided a method for preparing a postbiotic powder of Lactobacillus casei IOB-P9, characterized in that the preparation method includes: Performing solid-state gradient temperature fermentation on the primary seed liquid of Lactobacillus casei IOB-P9 to obtain the fermented raw material; drying and pulverizing the fermented raw material to obtain the postbiotic powder of Lactobacillus casei IOB-P9; The solid-state gradient temperature fermentation includes: Controlling the fermentation temperature at 42-43°C and maintaining for about 8-10 h; Lowering the fermentation temperature to 37-40°C and maintaining for about 10-12 h; Adjusting the fermentation temperature to 34-38°C and maintaining for 10-12 h to obtain the fermented raw material.

[0022] In the present invention, in the initial stage, the fermentation temperature is controlled at 42-43°C and maintained for about 8-10 h to activate the activity of the bacterial strain. The high temperature of 42-43°C can quickly break the dormant state of the bacterial strain, promote the activation of the cell enzyme system, enable it to quickly adapt to the fermentation environment, shorten the lag phase, and lay a foundation for subsequent growth. Controlling the fermentation temperature at 42-43°C and maintaining for about 8-10 h in the initial stage can accelerate the reproduction of the bacterial strain, improve the initial fermentation efficiency, and ensure the yield of subsequent metabolites. Activate the glycolytic enzymes related to energy metabolism to accumulate sufficient metabolic substrates for the logarithmic growth phase. The activated bacterial strain activity at this stage can promote the secretion of early metabolites such as short-chain fatty acids and small molecule peptides, and these components may initially regulate the balance of the intestinal flora and lay a foundation for subsequent corticosterone regulation.

[0023] During the logarithmic growth phase, the fermentation temperature is reduced to 37 - 40 °C and maintained for about 10 - 12 h to maintain metabolic balance. 37 - 40 °C is close to the optimal growth temperature of the strain, which can not only ensure rapid cell proliferation but also avoid enzyme inactivation caused by high temperature, maintaining the stability of the metabolic pathway. Reducing the fermentation temperature to 37 - 40 °C and maintaining it for about 10 - 12 h can maximize the accumulation of bacterial biomass, providing rich cell components for postbiotics; it can also promote the synthesis of secondary metabolites such as organic acids and vitamins, and these components can reduce the corticosterone level by regulating the hypothalamic - pituitary - adrenal (HPA) axis. The metabolites produced at this stage can directly act on the host through the vagus nerve pathway, inhibiting corticosterone secretion, while improving the intestinal barrier function, reducing the entry of inflammatory factors into the blood, and indirectly reducing the stress hormone level.

[0024] During the stationary phase, the fermentation temperature is adjusted to 34 - 38 °C and maintained for 10 - 12 h to optimize product accumulation. Lowering the temperature to 34 - 38 °C can slow down the metabolic rate of the bacteria, reduce nutrient consumption, promote the accumulation of secondary metabolites such as functional peptides and polysaccharides, and avoid product degradation caused by cell autolysis. Adjusting the fermentation temperature to 34 - 38 °C and maintaining it for 10 - 12 h can induce the strain to produce stress - resistant substances, and these components can enhance the stability and biological activity of postbiotics. It can also promote cell apoptosis or autolysis, releasing effective components such as nucleotides and amino acids in the cells, and improving the functional diversity of postbiotics. The functional components accumulated during the stationary phase can regulate the intestinal flora abundance, reduce the proportion of pro - inflammatory bacteria, and thus reduce leptin secretion, synergistically regulating the corticosterone level.

[0025] In the present invention, segmented temperature control enables the strain to express differential metabolites at different growth stages, promoting the accumulation of functional components, such as activation products in the initial stage, enzymes and organic acids in the logarithmic phase, and polysaccharides and peptides in the stationary phase, forming a system with multi - component synergistic effects. Postbiotics fermented with gradient temperature can significantly reduce the corticosterone level in rat serum, and its effect is better than that of ordinary fermentation. Through temperature gradient control, the problems of bacterial growth stagnation or metabolite degradation under a single temperature are avoided, the utilization rate of the fermentation raw material (oat) is improved, and the production cost is reduced. The low - temperature stationary phase can reduce the damage of heat - sensitive components, ensure the retention of active components during the drying and storage of postbiotic powder, and enhance the stability of the product. Postbiotics fermented with gradient temperature can more effectively regulate blood lipids and repair liver damage. This fermentation process enables postbiotics to simultaneously possess multiple functions of regulating intestinal flora, inhibiting corticosterone secretion, and improving metabolic disorders, achieving comprehensive intervention for obesity and its complications.

[0026] In a preferred embodiment of the present invention, the solid - to - liquid ratio of the solid medium for solid - state gradient temperature fermentation is 1:1 - 2, and the inoculum size is 1×10 6 ~5×10 9cfu / mL.

[0027] In a preferred embodiment of the present invention, the solid medium consists of oats.

[0028] In a preferred embodiment of the present invention, the method for preparing the primary seed solution of Lactobacillus casei IOB-P9 includes: Inoculate the IOB-P9 strain in the cryotube into the slant medium and place it in an incubator at 35-39 °C for 16-24 h to obtain the slant strain. Take the slant strain and inoculate it into the culture solution containing 2% oat powder, and culture it at 35-39 °C for 16-20 h to obtain the primary seed solution.

[0029] In a preferred embodiment of the present invention, the composition of the slant medium is: 20-25 g of yeast powder, 25-30 g of glucose, 1-3 ml of Tween 80, 2-5 g of dipotassium hydrogen phosphate, 5-7 g of sodium acetate, 2-5 g of ammonium citrate, 0.2-0.5 g of magnesium sulfate, 0.05-0.07 g of manganese sulfate, 15-17 g of agar powder, and 1000 ml of distilled water.

[0030] In a preferred embodiment of the present invention, the drying temperature is 55-60 °C, and the moisture content after drying is ≤10%.

[0031] According to the second aspect of the present invention, there is provided an application of the postbiotic powder of Lactobacillus casei IOB-P9 in the preparation of a drug for improving obesity.

[0032] According to the third aspect of the present invention, there is provided an application of the postbiotic powder of Lactobacillus casei IOB-P9 in the preparation of a drug for improving hepatocyte damage caused by obesity.

[0033] According to the fourth aspect of the present invention, there is provided an application of the postbiotic powder of Lactobacillus casei IOB-P9 in the preparation of a drug for increasing the corticosterone level.

[0034] To further elaborate on the technical means and effects adopted by the present invention, the technical solutions in the present invention will be clearly and completely described below in conjunction with the embodiments in the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0035] Example 1: Preparation of the postbiotic powder of Lactobacillus casei IOB-P9 by ordinary fermentation (1) Activation and cultivation of the strain: Inoculate the IOB-P9 strain in the cryotube into the slant medium, and place it in an incubator at 35-39 °C for 16-24 h. After the slant seeds are cultured to be uniform in size, consistent in morphology, and robust, take fresh slant seeds and inoculate them into a bottle containing 25 mL of 2% oatmeal powder, and culture at 35-39 °C for 16-20 h to obtain the primary seed liquid; inoculate the primary seed liquid into the solid medium (with oatmeal as the substrate) at a solid-to-liquid ratio of 1:1-2 and an inoculation amount of 1×10 6 ~5×10 9 cfu / mL, and inoculate it into the solid medium, and incubate statically at 34-38 °C for 24-48 h to obtain the fermented raw material.

[0036] (2) Drying: Dry the fermented raw material. The drying temperature is 55-60 °C, and the moisture content is controlled at ≤10% to obtain the postbiotic powder of Lactobacillus casei IOB-P9.

[0037] Among them, the composition of the slant medium is: 25 g of yeast powder, 25 g of glucose, 1 mL of Tween 80, 2 g of dipotassium hydrogen phosphate, 5 g of sodium acetate, 2 g of ammonium citrate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 15 g of agar powder, and 1000 mL of distilled water.

[0038] Example 2: Preparation of postbiotic powder by gradient temperature fermentation of Lactobacillus casei IOB-P9 (1) Activation and cultivation of the strain: Inoculate the IOB-P9 strain in the cryotube into the slant medium, and place it in an incubator at 35-39 °C for 16-24 h. After the slant seeds are cultured to be uniform in size, consistent in morphology, and robust, take a loop of fresh slant seeds and inoculate them into a bottle containing 25 mL of 2% oatmeal powder, and culture at 35-39 °C for 16-20 h to obtain the primary seed liquid; inoculate the primary seed liquid into the solid medium (with oatmeal as the substrate) at a solid-to-liquid ratio of 1:1-2 and an inoculation amount of 1×10 6 ~5×10 9 cfu / mL, and inoculate it into the solid medium for gradient temperature fermentation. Initial stage: Control the fermentation temperature at 42-43 °C and maintain it for about 8-10 h to activate the strain activity.

[0039] Logarithmic growth phase: Lower the fermentation temperature to 37-40 °C to maintain metabolic balance and maintain it for about 10-12 h.

[0040] Stationary phase: After the fermentation enters the stationary phase, the temperature can be adjusted to 34-38 °C and continue fermentation for 10-12 h until the fermentation is completed to finally obtain the fermented raw material.

[0041] (2) Drying: Dry the fermented raw materials. The drying temperature is 55 - 60 °C, and the moisture content is controlled at ≤ 10%, then the probiotic powder of Lactobacillus casei IOB - P9 is obtained.

[0042] Among them, the composition of the slant medium is: 20 g yeast powder, 15 g glucose, 8 g sucrose, 1 ml Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g ammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 15 g agar powder, 1000 ml distilled water.

[0043] Example 3: Establishment and treatment of a rat obesity model After 5 days of adaptation, the rats are divided into 4 groups, with 10 rats in each group, including a blank control group, a model group, experimental group 1 and experimental group 2. Experimental group 1 is the probiotic powder of IOB - P9 by ordinary fermentation, and experimental group 2 is the probiotic powder of IOB - P9 by gradient temperature fermentation. The blank control group is given maintenance feed, and the rest of the rats are given high - calorie feed to prepare obese rats. After the modeling is completed, the experimental rats are divided into a model group, a blank group, experimental group 1, and experimental group 2. During the experiment, the model group and the blank control group are given an equal amount of normal saline, experimental group 1 is given the probiotic powder of IOB - P9 by ordinary fermentation, and experimental group 2 is given the probiotic powder of IOB - P9 by gradient temperature fermentation. Once a day, continuous gavage is carried out for 10 weeks. The rats are anesthetized once a week to record the weight changes of the rats. After 10 weeks, the rats are sacrificed uniformly for index detection.

[0044] Drug dosage: Probiotic powder of IOB - P9 by ordinary fermentation: 2.5×10 9 CFU / mL Probiotic powder of IOB - P9 by gradient temperature fermentation: 2.5×10 9 CFU / mL Example ④ Determination of rat body weight, LEE' value and fat - to - body ratio During the experiment, observe the health status of the rats, measure the body weight of the rats every week, and observe the changes in body weight during the experiment; before sacrificing the rats, measure the body length (the length from the nose to the anus) under anesthesia, and then calculate the LEE' value.

[0045]

[0046] After sacrificing the rats and bleeding them, dissect and take all the fat around the kidneys and genitals, weigh it, and calculate the fat - to - body ratio (g fat / 100 g bw).

[0047] To observe the effect of the probiotic powder of IOB - P9 on rats, observe the state of the rats and measure the body weight of the rats every week. The mental state of the rats is good during the experiment, such as Figure 1As shown, the body weights of rats in each group showed a slow growth trend. The body weight of the model group was higher than that of the blank control group and the experimental group, and there was no significant difference in body weight among the rats in each group. This indicates that the IOB-P9 postbiotics powder has no adverse effect on the growth of rats.

[0048] The LEE’ value and the fat / body ratio can intuitively reflect the obesity status of rats during the growth process. The experimental results are as Figure 2 and Figure 3 shown. Compared with the blank control group, the LEE’ index and the fat / body ratio of the model group were significantly increased. The LEE’ index and the fat / body ratio of the rats in experimental group 1 and experimental group 2 showed a decreasing trend compared with those of the rats in the model group, indicating that the IOB-P9 postbiotics powder can significantly improve the obesity status of rats, and the effect of the IOB-P9 postbiotics powder fermented at gradient temperatures is more significant.

[0049] Determination of the contents of serum leptin and serum corticosterone in rats in Example 5 After the last gavage of rats, the rats were fasted but given water. After the rats were sacrificed, whole blood of the rats was collected by the method of eye enucleation. The blood was centrifuged at 3500 r·min -1 in a centrifuge for 15 min, and the upper-layer serum was taken. The contents of serum leptin (Leptin, LEP) and serum corticosterone (Corticosterone, CORT) in the rats were determined according to the instructions of the kit.

[0050] Serum corticosterone is a glucocorticoid secreted by the adrenal cortex. Corticosterone directly causes obesity by promoting appetite and fat accumulation. Chronic elevation of corticosterone levels is closely related to the occurrence and development of obesity. The levels of serum leptin and serum corticosterone in rats of different groups are respectively as Figure 4 and Figure 5 shown. Compared with the blank group, the contents of serum leptin and corticosterone in the rats of the model group were significantly increased. After intervention with the IOB-P9 postbiotics powder, both experimental group 1 and experimental group 2 decreased the levels of serum leptin and serum corticosterone in obese rats. Compared with the model group, the level of serum corticosterone in experimental group 2 was significantly decreased. This indicates that the IOB-P9 postbiotics powder fermented at gradient temperatures can significantly regulate the levels of leptin and serum corticosterone in serum to achieve the effect of controlling obesity.

[0051] Determination of the four serum lipid items and fasting blood glucose content in rats in Example 6 After the last gavage in rats, food was withheld but water was available. After sacrificing the rats, whole blood was collected from the rats by the method of eye enucleation, and the contents of total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), and fasting blood glucose value (Glucose, Glu) were measured according to the kit instructions.

[0052] As Figures 6 - 8 shown, compared with the blank control group, the levels of total cholesterol (TC), triglyceride (TG), and low density lipoprotein cholesterol value (LDL-C) in the serum of the model group were significantly increased, indicating that the blood lipid level of the model group was significantly increased. Compared with the model group, both experimental group 1 and experimental group 2 decreased the above serum indexes. Among them, the above four indexes in experimental group 2 were significantly decreased. As Figure 9 shown, the level of high density lipoprotein cholesterol value (HDL-C) in the model group was significantly lower than that in the blank control group, while the serum HDL-C levels in experimental group 1 and experimental group 2 were significantly increased, and the increase effect in experimental group 2 was more obvious, indicating that the postbiotic powder fermented by IOB-P9 gradient temperature has the effect of regulating lipid metabolism disorder in obese rats. As Figure 10 shown, the postbiotic powder fermented by IOB-P9 gradient temperature can reduce the fasting blood glucose value of rats and effectively improve the further development of obesity.

[0053] Determination of liver function indexes of rats in Example 7 After the last gavage in rats, food was withheld but water was available. After sacrificing the rats, whole blood was collected from the rats by the method of eye enucleation, and the contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), which are liver and kidney function indexes, were measured according to the kit instructions.

[0054] ALT (alanine aminotransferase), AST (aspartate aminotransferase), and ALP (alkaline phosphatase) are mainly present in liver cells and are sensitive indicators of liver cell damage. Metabolic disorders caused by obesity, such as insulin resistance, inflammation, and oxidative stress, can lead to liver cell damage, and then cause an increase in the levels of ALT, AST, and ALP.

[0055] The levels of serum ALP, ALT, and AST in different groups are as Figures 11 - 13As shown in the figure. After the intervention treatment with the probiotic powder fermented by IOB-P9 through ordinary fermentation and the probiotic powder fermented by IOB-P9 at gradient temperatures, the levels of ALP, ALT, and AST in experimental group 1 and experimental group 2 were significantly decreased, and the decreasing effect in experimental group 2 was more obvious, indicating that the probiotic powder fermented by IOB-P9 at gradient temperatures can significantly improve liver tissue damage caused by obesity.

[0056] Determination of the morphological structure of rat liver tissue in Example 8 Fix the liver tissue specimens in 4% paraformaldehyde solution for 24 h, dehydrate the tissue, impregnate with wax and embed, cut the tissue into 5-μm sections, affix the sections on slides and bake, stain with HE, stain with Oil Red O staining solution for 10 minutes, and finally observe under a microscope and take pictures of 3 fields of view each.

[0057] As Figure 14 shown, in the HE staining results, the hepatocyte structure in the blank group was normal, the cytoplasm was uniform, and no obvious fatty degeneration was seen; in the model group, fatty degeneration of hepatocytes occurred, the hepatocytes were arranged densely and disorderly, the tissue morphology was irregular, the cell edges were blurred, and the cells were severely damaged. After the intervention treatment with the probiotic powder fermented by IOB-P9 through ordinary fermentation and the probiotic powder fermented by IOB-P9 at gradient temperatures, the hepatocyte structure was improved and the fatty degeneration was alleviated. The number of lipid droplets in hepatocytes and the disordered state of hepatocyte arrangement in the group of probiotic powder fermented by IOB-P9 at gradient temperatures were significantly decreased and gradually restored to the normal level of the blank group. The Oil Red O staining results showed that no obvious lipid deposition was seen in the liver cells of the blank group and the staining was lighter; obvious lipid deposition was seen in the model group, showing large red fat droplets. After the intervention treatment with the probiotic powder fermented by IOB-P9 through ordinary fermentation and the probiotic powder fermented by IOB-P9 at gradient temperatures, the area and volume of the red fat droplets decreased, the lipid deposition decreased. At the same time, the area of the red fat droplets in experimental group 2 decreased significantly and was arranged relatively sparsely, showing a color similar to that of the blank group. It indicates that the probiotic powder fermented by IOB-P9 at gradient temperatures can effectively improve hepatocyte damage caused by obesity.

[0058] In summary, the probiotic powder fermented by IOB-P9 at gradient temperatures of the present invention has a relieving effect on obesity caused by a high-fat diet, has an effect of improving blood lipid abnormalities in obese rats, and can also repair the damaged state of the liver of obese rats, providing a new idea for the development of probiotic powder of Lactobacillus casei IOB-P9.

[0059] In any aspect of the present invention, the above-described embodiments of the present invention can only be considered as an illustration of the present invention and cannot limit the present invention. The claims point out the scope of the present invention, while the above description does not point out the scope of the present invention. Therefore, any change within the meaning and scope equivalent to the claims of the present invention should be considered as included within the scope of the claims of the present invention.

Claims

1. A method for preparing postbiotic powder of Lactobacillus casei IOB-P9, characterized in that, The preparation method includes: Performing solid-state gradient temperature fermentation on the first-stage seed liquid of Lactobacillus casei IOB-P9 to obtain the fermented raw material; drying and pulverizing the fermented raw material to obtain the postbiotic powder of Lactobacillus casei IOB-P9. The solid-state gradient temperature fermentation includes: Controlling the fermentation temperature at 42 - 43 °C and maintaining for about 8 - 10 h; Lowering the fermentation temperature to 37 - 40 °C and maintaining for about 10 - 12 h; Adjusting the fermentation temperature to 34 - 38 °C and maintaining for 10 - 12 h to obtain the fermented raw material.

2. The preparation method according to claim 1, characterized in that, The solid medium for the solid-state gradient temperature fermentation has a solid-to-water ratio of 1:1 to 2, and the inoculation amount is 1×10 6 ~5×10 9 cfu / mL.

3. The preparation method according to claim 2, characterized in that, The composition of the solid medium is oats.

4. The preparation method according to claim 1, characterized in that, The preparation method of the first-stage seed liquid of Lactobacillus casei IOB-P9 includes: Inoculating the IOB-P9 strain in the cryotube into the slant medium and culturing at 35 - 39 °C for 16 - 24 h to obtain the slant strain; Taking the slant strain and inoculating it into the culture solution containing 2% oat powder, and culturing at 35 - 39 °C for 16 - 20 h to obtain the first-stage seed liquid.

5. The preparation method according to claim 4, characterized in that, The composition of the slant medium is: 20 - 25 g of yeast powder, 25 - 30 g of glucose, 1 - 3 ml of Tween 80, 2 - 5 g of dipotassium hydrogen phosphate, 5 - 7 g of sodium acetate, 2 - 5 g of ammonium citrate, 0.2 - 0.5 g of magnesium sulfate, 0.05 - 0.07 g of manganese sulfate, 15 - 17 g of agar powder, and 1000 ml of distilled water.

6. The preparation method according to claim 1, characterized in that, The drying temperature is 55 - 60 °C, and the moisture content after drying is ≤ 10%.

7. Use of the postbiotic powder of Lactobacillus casei IOB-P9 prepared by the preparation method according to any one of claims 1 - 6 in the preparation of a drug for improving obesity.

8. Use of the postbiotic powder of Lactobacillus casei IOB-P9 prepared by the preparation method according to any one of claims 1 - 6 in the preparation of a drug for improving hepatocyte injury caused by obesity.

9. Use of the postbiotic powder of Lactobacillus casei IOB-P9 prepared by the preparation method according to any one of claims 1 - 6 in the preparation of a drug for increasing the corticosterone level.

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