Preparation method of postbiotics produced by segmented fermentation of Pediococcus acidilactici IOB701 and its application in regulating serum leptin levels

Abstract

The invention relates to a preparation method of a segmented fermentation of lactic acid Pediococcus acidilactici IOB701 postbiotic and an application thereof in regulating serum leptin level. The method comprises the following steps: inoculating lactic acid Pediococcus acidilactici IOB701 into an activation medium at a volume ratio of 1:25-35 for activation culture, and sealing and statically culturing at 36±2°C for 22±2h to obtain seed liquid; inoculating the seed liquid into a liquid fermentation medium at a volume ratio of 1:60-80, and culturing at 36±2°C at a rotation speed of 120-140r / min for 18±2h to obtain fermentation liquid; inoculating the fermentation liquid into a sterilized solid fermentation medium at an inoculation amount of 1:1.5-1.75 of a material-liquid ratio, and statically culturing at 36±2°C under anaerobic conditions for 32±2h to obtain a fermentation product; and high-temperature inactivating bacterial components, metabolites and solid fermentation substrates after fermentation, drying and crushing to obtain solid fermentation lactic acid Pediococcus acidilactici IOB701 postbiotic powder. The method can effectively reduce serum leptin level.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular to a method for preparing postbiotics of segmented fermentation Pediococcus acidilactici IOB701 and an application thereof in regulating serum leptin levels. Background Art

[0002] With the rapid development of society and the improvement of people's living standards, the intake of high-fat and high-sugar foods has led to obesity in some people. Obesity has become a common problem in the current society, and the incidence of obesity has increased year by year. Obesity not only affects people's pursuit of physical beauty, but also poses a huge threat to human health. Studies have found that obesity is closely related to chronic diseases such as hypertension, hyperlipidemia, and hyperglycemia, which seriously endangers human health and affects the quality of life.

[0003] At present, the scientifically recognized healthy weight loss methods are exercise and dietary intervention. The existing dietary intervention methods are to reduce energy intake, such as controlling food intake, adjusting appetite, etc.; reduce energy absorption in the body, such as blocking the absorption of carbon water or fat, etc.; increase energy consumption, such as exercise plans, promoting fat consumption, etc.; change energy metabolism, such as ketogenic diet, etc. However, since the dietary intervention method has a long cycle to alleviate obesity and requires a reasonable diet, mainly controlling the intake of sugars and lipids, many obese patients will choose to give up because they cannot persist for a long time. The existing meal replacement foods on the market are becoming more and more popular among obese people because of their low calories and convenience. However, most of these meal replacement foods lack nutrition and have a short fullness time. In addition, various flavors, pigments and preservatives are added, which greatly reduces the nutrition and safety of meal replacement foods.

[0004] Probiotics are a general term for active microorganisms that colonize the human intestine and reproductive system and can produce definite health effects, thereby improving the host microecological balance and exerting beneficial effects on the host. They are widely used in the fields of bioengineering, food safety, and life health. Many studies have found that probiotics can reduce blood lipids by inhibiting the synthesis and absorption of lipids and promoting cholesterol excretion, thereby achieving a weight loss effect. Compared with conventional weight loss drugs, probiotics have fewer side effects and do not affect the body's immunity and normal metabolic functions. In the prior art, probiotics are used in weight loss products, but most probiotics only play an auxiliary role in regulating intestinal flora, promoting intestinal peristalsis, preventing constipation, etc., and do not directly target weight management or appetite control. In addition, the current research on the weight loss effect of lactic acid bacteria is generally a single strain compound prebiotic, most of which rely on the functional characteristics of lactic acid bacteria and are prepared and processed.

[0005] According to the search, Chinese patent publication number: CN112501046B discloses a fermented lactobacillus strain with weight loss function and its application, and specifically discloses that the fermented lactobacillus strain can effectively reduce serum leptin levels to restore the body's leptin sensitivity, effectively prevent and treat obesity, etc.

[0006] Chinese Patent Publication No.: CN116421632A, discloses the use of Pediococcus acidilactici in the preparation of foods, health products or medicines with fat-reducing effects, and specifically discloses that Pediococcus acidilactici NSS0302 has been experimentally verified to be able to reduce fat accumulation and reduce fat coefficient to inhibit weight gain in rats, thereby achieving the effect of reducing fat and fat, and thus the purpose of weight loss.

[0007] Chinese Patent Publication No.: CN117547029B discloses a Lactobacillus plantarum J26 postbiotic freeze-dried powder with weight loss and lipid-lowering effects, and its preparation method and application. Specifically, Lactobacillus plantarum J26 is fermented in liquid state and then added with a composite carrier, and then pasteurization-ultrasound combined inactivation is adopted. After inactivation, okra extract, monoglyceride, and sucrose ester are added, and freeze-dried to obtain the Lactobacillus plantarum J26 postbiotic freeze-dried powder, which has a significant weight loss and lipid-lowering effect.

[0008] It can be seen that the above patented technologies all use liquid fermentation preparation in the preparation of lactic acid bacteria or strain compositions and postbiotics. In addition, most of the above patented technologies use lactic acid bacteria composites or lactic acid bacteria postbiotics combined with other prebiotics that are beneficial to weight loss to obtain compositions that control obesity. There is no application of segmented fermentation of lactic acid Pediococcus postbiotic powder in regulating serum leptin levels. Summary of the invention

[0009] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for preparing a postbiotic of Pediococcus acidilactici IOB701, which can effectively reduce serum leptin levels, reduce body fat percentage and Lee's index, inhibit weight gain, and effectively control the occurrence and development of obesity, and its application in regulating serum leptin levels.

[0010] The present invention solves the technical problem by adopting the following technical solutions:

[0011] A method for preparing postbiotics from segmented fermentation of Pediococcus acidilactici IOB701 comprises the following steps:

[0012] S1. Strain activation: Pediococcus acidilactici ( Pediococcus acidilactici )IOB701 was inoculated into the activation culture medium at a volume ratio of 1:25-35 for activation culture at a temperature of 36±2℃ and sealed and statically cultured for 22±2h to obtain the seed solution;

[0013] S2, first stage fermentation: inoculate the seed liquid into the liquid fermentation medium at a volume ratio of 1:60-80, at a temperature of 36±2°C, a rotation speed of 120r / min-140r / min, and culture for 18±2h to obtain the fermentation liquid;

[0014] S3, second stage fermentation: inoculate the obtained fermentation liquid into the sterilized solid fermentation medium at an inoculation amount of 1:1.5-1.75 of the solid-liquid ratio, culture at 36±2°C, anaerobic conditions, and culture for 32±2h to obtain the fermentation product;

[0015] S4. Preparation of postbiotics: The fermented bacterial components, metabolites and solid-state fermentation substrates are inactivated at high temperature, dried and crushed to obtain solid-state fermented lactic acid Pediococcus acidilactici ( Pediococcus acidilactici ) IOB701 postbiotic powder;

[0016] The activation medium in step S1 is a modified MRS medium, the formula of which is: 8g-10g soy protein isolate, 3g-4g yeast extract, 3g-5g sucrose, 1g-2g dipotassium hydrogen phosphate, 1g-2g triammonium citrate, MgSO 4 7H 2 O 4g-5g, MnSO 4 0.05g-0.1g, agar 8g-10g, Tween 80 0.8g-1g; pH = 6.5±0.2, dilute to 1L, sterilize at 110-130℃ for 10-20min;

[0017] The liquid fermentation medium in step S2 is a modified MRS liquid fermentation medium, and the formula is: 40g-50g of soy protein isolate, 15g-20g of yeast extract, 10g-15g of sucrose, 5g-10g of dipotassium hydrogen phosphate, 5g-10g of triammonium citrate, MgSO 4 7H 2 O 4g-5g, MnSO4 0.25g-0.5g, Tween 80 0.4g-0.5g; pH = 6.5±0.2, dilute to 1L, sterilize at 110-130℃ for 10-20min;

[0018] The solid-state fermentation medium in step S3 is: taro-soybean protein isolate complex, which is sterilized at 110-130° C. for 20-30 min and then cooled to room temperature for standby use.

[0019] Moreover, the high temperature inactivation is 75℃±2℃, and the drying temperature is 55℃±2℃.

[0020] Furthermore, the Pediococcus acidilactici ( Pediococcus acidilactici )IOB701, strain name: IOB701, classification name: Pediococcus acidilactici ( Pediococcus acidilactici )， The deposit number is: CGMCCNo.16077, the deposit date is: July 10, 2018, the deposit unit is: General Microbiology Center of China Culture Collection Administration, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0021] An application of the segmented fermentation Pediococcus acidilactici IOB701 postbiotics in regulating serum leptin levels.

[0022] An application of the segmented fermentation Pediococcus acidilactici IOB701 postbiotics in the preparation of relevant drugs for improving liver tissue damage.

[0023] A segmented fermentation of Pediococcus acidilactici IOB701 postbiotics comprises Pediococcus acidilactici ( Pediococcus acidilactici ) Use of the proecological preparation of IOB701 in the preparation of a method for preventing or treating Type II diabetes.

[0024] The advantages and positive effects of the present invention are:

[0025] 1. The lactic acid Pediococcus IOB701 postbiotics provided by the present invention are obtained by segmented fermentation. Compared with the solid-state fermentation lactic acid Pediococcus IOB701 postbiotics, the first fermentation stage obtains bioactive substances rich in small molecule peptides, polysaccharides, etc., and the second fermentation stage not only effectively retains the original flavonoids, enzymes, vitamins, minerals and other nutrients in the solid-state fermentation substrate, but also produces active metabolites such as polysaccharides, polypeptides, and unsaturated fatty acids through fermentation. While being able to efficiently promote the proliferation of lactic acid Pediococcus IOB701, it can also protect the strain activity and increase the intracellular active ingredient content of lactic acid Pediococcus IOB701, thereby enriching the types of lactic acid Pediococcus IOB701 postbiotics, increasing the content of active metabolites, and not being restricted by gastric acid and bile salts, and can play a prebiotic function in the intestine.

[0026] 2. The present invention adopts segmented fermentation technology to prepare postbiotics in the preparation and treatment method. No material is added in the entire fermentation and post-treatment process. Not only the probiotics' teichoic acid, peptidoglycan and other bacterial components, polypeptides, polyphenols, short-chain fatty acids, extracellular polysaccharides and other metabolites are utilized, but also the fermentation substrate's dietary fiber, small molecule active peptides, polysaccharides and other nutrients are utilized. Animal experiments have verified that the complex is a postbiotic obtained by segmented fermentation of Pediococcus acidilactici IOB701, and the segmented fermentation of Pediococcus acidilactici IOB701 postbiotics provided can reduce serum leptin levels, body fat rate, TG, TC, Lee's index, reduce fat accumulation, reduce body weight, and effectively prevent and control obesity.

[0027] 3. The segmented fermentation of lactic acid Pediococcus acidilactici IOB701 postbiotics provided by the present invention can regulate serum alkaline phosphatase (ALP) levels, reduce serum triglyceride (TG), total cholesterol (TC) content, low-density lipoprotein cholesterol (LDL) content, increase high-density lipoprotein cholesterol (HDL) content, and improve liver tissue damage; at the same time, it can also reduce fasting blood sugar content and improve intestinal health. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Figure 2 is a graph showing the body weights of mice in different groups according to the present invention.

[0029] Figure 2 Figure 2 is a diagram of body fat percentage of mice in different groups according to the present invention.

[0030] Figure 3 The Lee's index diagram of different groups of mice in the present invention.

[0031] Figure 4 Graph showing serum leptin levels in different groups of mice of the present invention.

[0032] Figure 5 Graph showing blood lipid indexes of mice in different groups according to the present invention.

[0033] Figure 6 Graph showing serum alkaline phosphatase levels in different groups of mice of the present invention.

[0034] Figure 7 Graph showing fasting blood glucose levels of mice in different groups according to the present invention.

[0035] Figure 8 HE staining images of liver tissues of mice in different groups of the present invention.

[0036] Fig. 9 The Oil Red O staining images of liver tissues of mice in different groups of the present invention are shown. DETAILED DESCRIPTION

[0037] The present invention is further described in detail below through specific examples. The following examples are only illustrative and not restrictive, and the protection scope of the present invention cannot be limited thereto.

[0038] A method for preparing postbiotics from segmented fermentation of Pediococcus acidilactici IOB701 comprises the following steps:

[0039] S1. Strain activation: inoculate Pediococcus acidilactici IOB701 into activation culture medium at a volume ratio of 1:25-35 for activation culture at a culture temperature of 36±2°C and culture in a sealed static manner for 22±2h to obtain seed solution;

[0040] S2, first stage fermentation: inoculate the seed liquid into the liquid fermentation medium at a volume ratio of 1:60-80, at a temperature of 36±2°C, a rotation speed of 120r / min-140r / min, and culture for 18±2h to obtain the fermentation liquid;

[0041] S3, second stage fermentation: inoculate the obtained fermentation liquid into the sterilized solid fermentation medium at an inoculation amount of 1:1.5-1.75 of the solid-liquid ratio, culture at 36±2°C, anaerobic conditions, and culture for 32±2h to obtain the fermentation product;

[0042] S4. Preparation of postbiotics: The fermented bacterial components, metabolites and solid-state fermentation substrates are subjected to high-temperature inactivation, drying and pulverization to obtain solid-state fermentation Pediococcus acidilactici IOB701 postbiotic powder.

[0043] The lactic acid bacteria used in the present invention Pediococcus acidilactici ) IOB701 was independently selected from naturally fermented sourdough. This strain has been preserved and tested for physical and chemical indicators. It was tested for bacterial identification at China Food Fermentation Industry Research Institute Co., Ltd. on January 8, 2020. The colonies are milky white, round, moist, opaque, and have neat edges.

[0044] The Pediococcus acidilactici ( Pediococcus acidilactici )IOB701, strain name: IOB701, classification name: Pediococcus acidilactici ( Pediococcus acidilactici ) ， The deposit number is: CGMCC No.16077, the deposit date is: July 10, 2018, the deposit unit is: General Microbiology Center of China Culture Collection Administration, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Example 1

[0045] A method for preparing postbiotics by segmented fermentation of Pediococcus acidilactici IOB701, this embodiment is a method for preparing postbiotics of taro by solid-state fermentation of Pediococcus acidilactici IOB701, comprising the following steps:

[0046] S1. Strain activation: Take out Pediococcus acidilactici IOB701 from the strain freezing tube, inoculate it into the activation culture medium at a volume ratio of 1:30, and perform strain activation culture at a temperature of 36°C, in a sealed container, and let it stand for 24 hours. The activated strain is the seed liquid;

[0047] The activation medium is a modified MRS medium, and its formula is: 9g soy protein isolate, 4g yeast extract, 5g sucrose, 2g dipotassium hydrogen phosphate, 2g triammonium citrate, 2g MgSO 4 7H 2 O 5g, MnSO4 0.07g, agar 9g, Tween 80 0.9g, pH=6.5, dilute to 1L, 121℃, sterilize for 15min;

[0048] S2, fermentation: the activated seed liquid is inoculated into taro at a solid-liquid ratio of 1:1.75, the temperature is 36°C, and the closed culture is kept static for 32 hours to obtain a fermentation product;

[0049] S3. Preparation of postbiotics: The fermented bacteria, metabolites and solid-state fermentation substrates are subjected to high-temperature inactivation, drying and pulverization to obtain solid-state fermentation Pediococcus acidilactici IOB701 postbiotic powder; wherein the high-temperature inactivation temperature is 75°C and the drying temperature is 55°C. Example 2

[0050] A method for preparing postbiotics of segmented fermentation of Pediococcus acidilactici IOB701, in this embodiment, is a method for preparing a taro-soybean protein isolate complex, comprising the following steps:

[0051] S1, dissolving taro powder in phosphate buffer of pH=9, the solution concentration is 2%, and stirring at constant temperature to make it completely dissolved;

[0052] S2. The taro and soy protein isolate are mixed at a ratio of 2.5:1, and mixed evenly by magnetic stirring. The sample is placed in a water bath at 50°C, and the reaction time is 60 min. The sample after the reaction is centrifuged at 4000 r / min for 15 min to remove insoluble substances. The clear liquid is precooled at -30°C for 2.5 h, and freeze-dried to obtain a taro-soy protein isolate complex for later use. Example 3

[0053] A method for preparing postbiotics by segmented fermentation of Pediococcus acidilactici IOB701, this embodiment is a method for preparing postbiotics by solid-state fermentation of taro-soybean protein isolate complex by Pediococcus acidilactici IOB701, comprising the following steps:

[0054] S1. Strain activation: Take out Pediococcus acidilactici IOB701 from the strain freezing tube, inoculate it into the activation culture medium at a volume ratio of 1:30, and perform strain activation culture at a temperature of 36°C, in a sealed container, and let it stand for 24 hours. The activated strain is the seed liquid;

[0055] The activation medium is a modified MRS medium, and its formula is: 9g soy protein isolate, 4g yeast extract, 5g sucrose, 2g dipotassium hydrogen phosphate, 2g triammonium citrate, 2g MgSO 4 7H 2 O 5g, MnSO 40.07g, agar 9g, Tween 80 0.9g, pH=6.5, dilute to 1L, 121℃, sterilize for 15min;

[0056] S2, fermentation: the activated seed liquid is inoculated into taro-soy protein isolate at a solid-liquid ratio of 1:1.75, and the temperature is 36°C, and the closed culture is kept static for 32 hours to obtain a fermentation product;

[0057] S3. Preparation of postbiotics: The fermented bacterial components, metabolites and solid-state fermentation substrates are subjected to high-temperature inactivation, drying and crushing to obtain solid-state fermentation taro-soybean protein isolate complex Pediococcus acidilactici IOB701 postbiotic powder; wherein the high-temperature inactivation temperature is 75°C and the drying temperature is 55°C. Example 4

[0058] A method for preparing postbiotics from segmented fermentation of Pediococcus acidilactici IOB701 comprises the following steps:

[0059] S1. Strain activation: inoculate Pediococcus acidilactici IOB701 into activation culture medium at a volume ratio of 1:30 for activation culture at a temperature of 36°C and culture in a sealed static manner for 22 hours to obtain seed solution;

[0060] The activation medium is a modified MRS medium, and its formula is: 9g soy protein isolate, 4g yeast extract, 5g sucrose, 2g dipotassium hydrogen phosphate, 2g triammonium citrate, 5g MgSO4·7H2O, 0.07g MnSO4, 9g agar, 0.9g Tween 80, pH=6.5, fixed to 1L, 121°C, sterilized for 15min;

[0061] S2, first stage fermentation: seed liquid was inoculated into liquid fermentation medium at a volume ratio of 1:70, the temperature was 36°C, the rotation speed was 130r / min, and the fermentation liquid was obtained after culturing for 18h;

[0062] The liquid fermentation medium is a modified MRS liquid fermentation medium, and its formula is: 45g soy protein isolate, 20g yeast extract, 15g sucrose, 8g dipotassium hydrogen phosphate, 9g triammonium citrate, MgSO 4 7H 2 O 5g, MnSO 4 0.35g, Tween 80 0.5g, pH=6.5, dilute to 1L, 121℃, sterilize for 15min;

[0063] S3, second stage fermentation: sterilize the taro-soy protein isolate complex at an inoculation amount of 1:1.75 of solid-liquid ratio, cool to room temperature, inoculate the obtained fermentation liquid into the sterilized solid fermentation medium at an inoculation amount of 5% by mass, culture at 36°C, under anaerobic conditions, and culture for 36 hours to obtain a fermentation product;

[0064] S4. Preparation of postbiotics: The fermented bacterial components, metabolites and solid-state fermentation substrates are subjected to high-temperature inactivation, drying and pulverization to obtain solid-state fermentation Pediococcus acidilactici IOB701 postbiotic powder; wherein the high-temperature inactivation temperature is 75°C and the drying temperature is 55°C.

[0065] Example 5

[0066] A method for preparing Pediococcus acidilactici IOB701 bacterial powder comprises the following steps:

[0067] S1. Strain activation: Take out Pediococcus acidilactici IOB701 from the strain freezing tube, inoculate it into the activation culture medium at a volume ratio of 1:30, and perform strain activation culture at a temperature of 36°C, in a sealed container, and let it stand for 24 hours. The activated strain is the seed liquid;

[0068] The activation culture medium is MRS culture medium, and its formula is: peptone 10.0g, beef extract powder 8.0g, yeast extract powder 4.0g, glucose 20.0g, dipotassium hydrogen phosphate 2.0g, diammonium hydrogen citrate 2.0g, sodium acetate 5.0g, magnesium sulfate 0.2g, manganese sulfate 0.04g, agar 10.0g, Tween 80 1.0g, fixed volume 1L, pH=6.5, water addition amount is 1L, 121°C, sterilization for 15min.

[0069] S2, fermentation: the activated seed liquid is inoculated into the liquid fermentation medium at a volume ratio of 1:70, the temperature is 36°C, the rotation speed is 130r / min, and the fermentation liquid is obtained by culturing for 18h;

[0070] The liquid fermentation medium is an MRS medium, and its formula is: 50.0g of peptone, 40.0g of beef extract powder, 20.0g of yeast extract powder, 100.0g of glucose, 10.0g of dipotassium hydrogen phosphate, 10.0g of diammonium hydrogen citrate, 25.0g of sodium acetate, 1.0g of magnesium sulfate, 0.2g of manganese sulfate, 50.0g of agar, 5.0g of Tween 80, constant volume 5L, pH=6.5, 121°C, sterilization for 15min.

[0071] S3. Preparation of bacterial powder: The fermentation liquid obtained after the fermentation is centrifuged at 4°C and 4000 r / min for 15 min to obtain the supernatant and bacterial mud respectively. The obtained bacterial mud is precooled and then vacuum freeze-dried to obtain the bacterial powder of Pediococcus acidilactici IOB701.

[0072] Experimental Results

[0073] The postbiotics of Pediococcus acidilactici IOB701 obtained in Examples 1, 3, 4 and 5 were tested for nutritional components, and the test reference standards were as follows: the test method for polysaccharides was SN / T 24260-2015 Determination of crude polysaccharides in exported plant-based foods by phenol-sulfuric acid method; the test method for dietary fiber was GB 5009.88-2023 Food Safety National Standard Determination of dietary fiber in foods; the test method for soy isoflavones was GB / T 23788-2009 Determination of soy isoflavones in health foods by high performance liquid chromatography; the test method for peptide content was GB / T 22492-2008 Appendix B Determination of peptide content. The test results are shown in the following table:

[0074] Table 1 Nutrient composition test results of Pediococcus acidilactici IOB701 postbiotic powder

[0075]

[0076] As shown in Table 1, compared with the solid-state fermented taro lactic acid Pediococcus IOB701 postbiotics and solid-state fermented taro-soybean protein isolate complex lactic acid Pediococcus IOB701 postbiotics and lactic acid Pediococcus IOB701 powder nutrients, the content of bioactive substances such as polysaccharides, soy isoflavones, and small molecule peptides increased significantly during the segmented fermentation process, and it is rich in dietary fiber, which can promote intestinal peristalsis and digestive gland secretion, increase the number of intestinal bifidobacteria, improve intestinal flora, and promote metabolism of substances in the body. In addition, many studies have reported that soy isoflavones can inhibit lipid accumulation, improve visceral fat metabolism, regulate metabolism and balance, and may potentially prevent and treat obesity.

[0077] Regulatory effects of postbiotics from Pediococcus acidilactici IOB701 on obese mice

[0078] 1. Experimental Materials

[0079] Experimental animals: clean grade male mice.

[0080] Experimental materials: postbiotics from taro solid-state fermentation by Pediococcus acidilactici IOB701, postbiotics from taro-soy protein isolate complex solid-state fermentation by Pediococcus acidilactici IOB701, and postbiotics from segmented fermentation by Pediococcus acidilactici IOB701.

[0081] 2. Rearing environment: 12h light / 12h dark cycle, temperature 22±2℃, humidity 40%-60%, free access to water and diet

[0082] 3. Mouse Modeling and Grouping

[0083] 3.1 Mouse modeling

[0084] Clean grade male mice, 3-4 weeks old, were selected. Animal breeding conditions were 22℃, 60% relative humidity, and 12h lighting. The mice were raised in separate cages, free drinking water, and cages were cleaned regularly. The experiment began after 5 days of adaptation. The obese mouse model was established by feeding with a high-fat diet. Ten mice were randomly selected as the blank group, and 40 mice were randomly selected and fed with a high-fat diet for 6 weeks. Serum total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, blood glucose, fat / body mass ratio, and Lee's index were measured to evaluate the high-fat obesity model.

[0085] 3.2 Grouping of mice

[0086] After the model was successfully established, 40 mice were randomly divided into model group, experimental group 1, experimental group 2, experimental group 3, and experimental group 4. The blank group and the model group were gavaged with normal saline, 0.5 ml / mouse, once a day, for 4 weeks; the experimental group 1 was gavaged with solid-state fermented taro lactic acid Pediococcus IOB701 postbiotics, 0.5 ml / mouse, gavage concentration 0.055 g / ml, once a day, for 4 weeks; the experimental group 2 was gavaged with solid-state fermented taro-soybean protein isolate complex lactic acid Pediococcus IOB701 postbiotics, 0.5 ml / mouse, gavage concentration 0.055 g / ml, once a day, for 4 weeks; the experimental group 3 was gavaged with segmented fermentation lactic acid Pediococcus IOB701 postbiotic powder, 0.5 ml / mouse, gavage concentration 0.055 g / ml, once a day, for 4 weeks; the experimental group 4 was gavaged with lactic acid Pediococcus IOB701 bacterial powder, 0.5 ml / mouse, gavage concentration 0.055 g / ml, bacterial activity 1.2×10 12 / g, once a day.

[0087] 4. Detection indicators

[0088] 4.1 Morphological observation of mice: Observe and record the weight, coat color, feces, mental state and movement status of the rats, and record the conditions on days 0, 7, 14 and 28 respectively.

[0089] 4.2 Measurement of mouse body length: Before the mice were killed, their body length (length from nose to anus) was measured under anesthesia, and then the Lee's value was calculated. The calculation formula is: Lee's index = [body weight (g) × 103 / body length (cm)] 1 / 3 .

[0090] 4.3 Collection of mouse tissues: At the end of the experiment, the mice in the four groups were fasted for 12 hours overnight and then weighed. Blood samples were collected by eyeball bleeding. The blood samples were centrifuged at 3000×g for 10 min at 4℃, and the serum was collected and stored at -80℃ for later use. The mice were killed by cervical dislocation, and the liver, kidneys and all fat around the genitals were quickly removed and weighed to calculate the fat / body ratio (g fat / 100g bw). Liver tissue was taken for Oil Red O staining and HE staining.

[0091] 4.4 Determination of serum and blood lipid indexes in mice: The kit was used to measure the indexes such as TC, TG, high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), blood glucose, and serum leptin (LEP) in the serum.

[0092] 4.5 Determination of liver and kidney function indices in mice: The test kit was used to determine the alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and other indices in the liver and kidney.

[0093] 5 Experimental Results

[0094] 5.1 Mouse morphology

[0095] By observing the activities and mental state of the mice, it was found that the mice in the blank group and the experimental group had normal activities and good mental state, indicating that the mice were in a better mental state after consuming Pediococcus acidilactici IOB701 powder, liquid fermentation Pediococcus acidilactici IOB701 postbiotics, and segmented fermentation Pediococcus acidilactici IOB701 postbiotics.

[0096] 5.2 Mouse body weight

[0097] The mortality rate of mice during the experiment was zero, and the number of mice in each group increased during the experiment. Figure 1 As shown in the figure, compared with the model group, the weight gain of obese mice after intervention with Pediococcus acidilactici IOB701 powder and postbiotics was not obvious, indicating that both Pediococcus acidilactici IOB701 powder and postbiotics can effectively control the weight of mice, and the effect of segmented fermentation of Pediococcus acidilactici IOB701 postbiotics is more obvious, gradually approaching the weight of mice in the blank group, see for details. Figure 1 .

[0098] 5.3 Changes in mouse adipose tissue

[0099] Body fat percentage and Lee's index are the main criteria for reflecting obesity. Figure 2-3As shown in the figure, the body fat rate and Lee's index of obese mice were significantly lower than those of the model group after oral administration of lactic acid Pediococcus IOB701 powder and postbiotics. This indicates that lactic acid Pediococcus IOB701 powder and postbiotics can effectively reduce the body fat rate and Lee's index of obese mice and alleviate the obesity of obese mice. In addition, the body fat rate and Lee's index of obese mice were significantly reduced after intervention with segmented fermentation of lactic acid Pediococcus IOB701 postbiotics. For details, see Figure 2 , Figure 3 .

[0100] 5.4 Serum leptin levels in mice

[0101] Leptin is a hormone secreted by adipose tissue. Its content in serum is proportional to the size of adipose tissue. When body fat decreases or is in a low-energy state, the leptin content in serum will decrease significantly.

[0102] Serum leptin levels in different groups of mice Figure 4 As shown in the figure, compared with the model group, the serum leptin level of obese mice decreased significantly after the intervention of fermented lactic acid Pediococcus IOB701 powder and postbiotics, indicating that both lactic acid Pediococcus IOB701 powder and postbiotics can regulate the leptin level in serum to control obesity, but the leptin level of obese mice decreased most significantly after the intervention of segmented solid-state fermentation of lactic acid Pediococcus IOB701 postbiotics. Figure 4 .

[0103] 5.5 Blood lipid indexes in mice

[0104] Blood lipid indexes of mice in different groups Figure 5 As shown in the figure, compared with the model group, after oral administration of Pediococcus acidilactici IOB701 powder and postbiotics, the levels of triglyceride (TG), total cholesterol (TC), and low-density lipoprotein (LDL) in obese mice were significantly reduced, and the level of high-density lipoprotein (HDL) was significantly increased, indicating that both Pediococcus acidilactici IOB701 powder and postbiotics can effectively reduce the cholesterol content in obese mice, help alleviate obesity caused by hyperlipidemia symptoms, and help control the occurrence and development of obesity. In addition, the effect of segmented fermentation of Pediococcus acidilactici IOB701 postbiotics on controlling obesity caused by hyperlipidemia is more obvious. For details, see Figure 5 .

[0105] 5.6 Mouse Alkaline Phosphatase Levels

[0106] Alkaline phosphatase (ALP) is an enzyme that catalyzes the hydrolysis of organic phosphates in an alkaline environment. Clinically, the determination of ALP is mainly used for the diagnosis and differential diagnosis of hepatobiliary diseases. Eating a large amount of greasy food (high-fat diet or high-sugar diet) can lead to a large release of ALP in the intestine, causing an increase in the ALP level in the blood. However, a significant increase in the ALP level in the blood can cause liver tissue damage and intestinal diseases.

[0107] Serum ALP levels of mice in different groups Figure 6 As shown in the figure, compared with the model group, the serum ALP level of obese mice decreased significantly after intervention with lactic acid Pediococcus acidilactici IOB701 powder and postbiotics, indicating that lactic acid Pediococcus acidilactici IOB701 powder and postbiotics can regulate the ALP level in obese serum, effectively improve liver tissue damage and intestinal health, and the ALP level of obese mice decreased most significantly after intervention with segmented solid-state fermentation lactic acid Pediococcus acidilactici IOB701 postbiotic powder. Figure 6 .

[0108] 5.7 Fasting blood glucose levels in mice

[0109] Obesity is an important risk factor for type 2 diabetes. The free fatty acids and fasting insulin levels in the blood of obese people are increased. Figure 7 As shown in the figure, compared with the model group, the fasting blood glucose level of obese mice decreased significantly after oral administration of lactic acid Pediococcus acidilactici IOB701 powder and postbiotics, indicating that lactic acid Pediococcus acidilactici IOB701 powder and postbiotics can reduce the fasting blood glucose level of obese mice and effectively improve the further development of obesity. After oral administration of segmented solid-state fermentation lactic acid Pediococcus acidilactici IOB701 postbiotic powder, the fasting blood glucose level of obese mice decreased most significantly. For details, see Figure 7 .

[0110] 5.8 HE staining and Oil Red O staining of mouse liver tissue

[0111] HE staining and Oil Red O staining were used to observe the pathological changes of the liver of mice in each group. Figure 8 and Fig. 9 As shown. Observed under a 400x optical microscope, the morphology and structure of the liver tissue of the blank group mice were normal in HE staining and Oil Red O staining. There were a large number of lipid vacuoles in the liver cells of the model group mice, accompanied by more lipid accumulation. After the intervention of fermented lactic acid Pediococcus IOB701 powder and postbiotics, the lipid vacuoles in the liver tissue of obese mice were significantly reduced, and lipid accumulation was significantly alleviated, indicating that lactic acid Pediococcus IOB701 powder and postbiotics can effectively reduce lipid accumulation in the liver tissue of obese mice, which is beneficial to reduce the body fat rate of mice, thereby achieving the purpose of controlling the development of obesity. In addition, the segmented fermentation of lactic acid Pediococcus IOB701 postbiotics has a more significant effect on reducing fat accumulation in the liver tissue of obese mice. For details, see Figure 8 and Fig. 9 .

[0112] In summary, the present invention studies that lactic acid Pediococcus acidi IOB701 powder and postbiotics can not only effectively reduce serum leptin levels, but also reduce body fat rate and Lee's index, inhibit weight gain, and effectively control the occurrence and development of obesity. In addition, it can also regulate serum alkaline phosphatase (ALP) levels, reduce serum triglyceride (TG), total cholesterol (TC) content, low-density lipoprotein cholesterol (LDL) content, increase high-density lipoprotein cholesterol (HDL) content, reduce liver tissue lipid accumulation, effectively improve liver tissue damage and intestinal health; it can also reduce fasting blood sugar content, prevent the occurrence and development of II diabetes, thereby achieving the purpose of controlling obesity. Compared with lactic acid Pediococcus acidi IOB701 powder, solid-state fermented taro lactic acid Pediococcus acidi IOB701 postbiotics, and solid-state fermented taro-soybean protein isolate complex lactic acid Pediococcus acidi IOB701 postbiotics, segmented fermentation lactic acid Pediococcus acidi IOB701 postbiotics have more significant effects on regulating serum leptin levels and controlling obesity. The segmented fermentation of Pediococcus acidilactici IOB701 postbiotic probiotic preparation obtained by the method described in the present invention, as well as the functional verification of the bacterial powder and the postbiotic probiotic preparation, can provide a certain pharmacological basis for assisting in preventing and treating the occurrence and development of obesity, thereby providing a basis for developing new methods for treating obesity.

[0113] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for preparing postbiotics from segmented fermentation of Pediococcus acidilactici IOB701, characterized in that: The steps include: S1. Strain activation: Pediococcus acidilactici ( Pediococcus acidilactici )IOB701 was inoculated into the activation culture medium at a volume ratio of 1:25-35 for activation culture at a temperature of 36±2℃ and sealed and statically cultured for 22±2h to obtain the seed solution; S2, first stage fermentation: inoculate the seed liquid into the liquid fermentation medium at a volume ratio of 1:60-80, at a temperature of 36±2°C, a rotation speed of 120r / min-140r / min, and culture for 18±2h to obtain the fermentation liquid; S3, second stage fermentation: inoculate the obtained fermentation liquid into the sterilized solid fermentation medium at an inoculation amount of 1:1.5-1.75 of the solid-liquid ratio, culture at 36±2°C, anaerobic conditions, and culture for 32±2h to obtain the fermentation product; S4. Preparation of postbiotics: The fermented bacterial components, metabolites and solid-state fermentation substrates are inactivated at high temperature, dried and crushed to obtain solid-state fermentation Pediococcus acidilactici ( Pediococcus acidilactici ) IOB701 postbiotic powder; The activation medium in step S1 is a modified MRS medium, and the formula is: soy protein isolate 8g-10g, yeast extract 3g-4g, sucrose 3g-5g, dipotassium hydrogen phosphate 1g-2g, triammonium citrate 1g-2g, MgSO4·7H2O 4g-5g, MnSO4 0.05g-0.1g, agar 8g-10g, Tween 80 0.8g-1g; pH = 6.5 ± 0.2, dilute to 1L, sterilize at 110-130℃ for 10-20min; The liquid fermentation medium in step S2 is a modified MRS liquid fermentation medium, and the formula is: 40g-50g soy protein isolate, 15g-20g yeast extract, 10g-15g sucrose, 5g-10g dipotassium hydrogen phosphate, 5g-10g triammonium citrate, 4g-5g MgSO4·7H2O, 0.25g-0.5g MnSO4, 0.4g-0.5g Tween 80; pH=6.5±0.2, fixed to 1L, sterilized at 110-130°C for 10-20min; The solid-state fermentation medium in step S3 is: taro-soybean protein isolate complex, sterilized at 110-130° C. for 20-30 min, then cooled to room temperature for standby use; The Pediococcus acidilactici ( Pediococcus acidilactici )IOB701, strain name: IOB701, classification name: Pediococcus acidilactici ( Pediococcus acidilactici ) ， The deposit number is: CGMCC No.16077, the deposit date is: July 10, 2018, the deposit unit is: General Microbiology Center of China Culture Collection Administration, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The method for preparing postbiotics of segmented fermentation of Pediococcus acidilactici IOB701 according to claim 1, characterized in that: The high temperature inactivation is 75℃±2℃, and the drying temperature is 55℃±2℃.

3. A postbiotic produced by segmented fermentation of Pediococcus acidilactici IOB701, characterized in that: The method according to claim 1 is used to prepare the 4. Use of the postbiotics of the segmented fermentation of Pediococcus acidilactici IOB701 according to claim 3 in the preparation of a microecological preparation for lowering fasting blood sugar levels.

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