Preparation method of probiotic-fortified fermented styrax for treating functional dyspepsia

Abstract

The preparation method of probiotic-enhanced fermented Liushenqu for treating functional dyspepsia includes the following steps: Step 1: Prepare seed solutions of *Lactobacillus plantarum* HL-1, *Lactobacillus acidophilus*, and *Lactobacillus rhamnosus*, and inoculate these solutions into Liushenqu matrix to form fermentation blocks. *Lactobacillus plantarum* HL-1 has the preservation number CGMCC NO.29919. The OD values ​​of the *Lactobacillus plantarum*, *Lactobacillus acidophilus*, and *Lactobacillus rhamnosus* seed solutions are 0.6–0.8, and the volume ratio is 10–1:1–10:1–10. Step 2: After fermentation, the fermentation blocks are dried to obtain the Liushenqu product. The active ingredients of Liushenqu and the effects of lactic acid bacteria work synergistically to promote gastrointestinal digestion, regulate the intestinal environment, and achieve a good therapeutic effect on functional dyspepsia.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese veterinary medicine, and particularly relates to a preparation method of probiotic-strengthened fermented Liu Shenqu for treating functional dyspepsia. BACKGROUND

[0002] Functional dyspepsia (FD) belongs to the most common functional gastrointestinal diseases in clinic. The epidemiological investigation worldwide shows that the universal prevalence rate of functional dyspepsia in adults is 10%-30%, the universal prevalence rate in children is 3.5%-27%, and the prevalence rate in China is 8%-23%. Functional dyspepsia has the characteristics of repeated attack and long duration, which causes great inconvenience to the life and work of patients, and there is no particularly effective treatment drug at present.

[0003] Liu Shenqu, also known as Shenqu, is a kind of medicinal leavening agent prepared by mixing Artemisia vulgaris, Polygonum hydropiper, Herba Xanthii, bitter apricot kernel, and red bean with flour and wheat bran. Liu Shenqu has the effects of food digestion and middle regulation, and is widely used in clinic due to its remarkable effects of invigorating the spleen and stomach.

[0004] In the prior art, the raw materials are directly mixed and then placed at room temperature for natural fermentation when preparing Liu Shenqu. The Liu Shenqu is prepared by using the bacteria carried by the raw materials through a rough fermentation method, and the Liu Shenqu has the defects of slow fermentation speed, easy pollution by some bacteria, poor repeatability, and more dependence on experience.

[0005] The process conditions of Liu Shenqu have no clear records and specific related regulations, so the process conditions of production are quite different in different regions and different companies. The specific process parameters are important ways to ensure the quality of Liu Shenqu, and the fermentation time, fermentation temperature, and liquid-material ratio directly affect the quality of Liu Shenqu.

[0006] With the increasingly in-depth research on probiotics, probiotics have been widely used in food, medicine and other fields. It has become a very effective method to use probiotics to strengthen the efficacy of Chinese herbal medicines and develop new functions.

[0007] At present, the fermentation of traditional Chinese medicine is limited to the research on the fermentation method of single traditional Chinese medicine by probiotics, and there is little research on the addition of probiotics to fermented Chinese herbal medicines. The addition of probiotics to Liu Shenqu has a good interaction with the fermentation process and the full use of the raw material matrix, can improve the extraction rate and accumulation of effective components of Liu Shenqu, and may produce new active substances, so as to achieve the effect of enhancing the efficacy.

[0008] Probiotics are active microorganisms that are beneficial to the host, and have been reported to effectively relieve functional gastrointestinal diseases. Lactic acid bacteria play multiple important functions in the human and animal body, such as maintaining microbial balance, enhancing immunity, and promoting digestion and absorption, and have similar effects to those of Fuling. SUMMARY

[0009] In order to overcome the above shortcomings, the present application provides a preparation method of probiotic fortified fermented Fuling for treating functional dyspepsia.

[0010] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0011] The preparation method of probiotic fortified fermented Fuling for treating functional dyspepsia comprises the following specific steps:

[0012] Step one, Lactobacillus plantarum HL-1, Lactobacillus acidophilus and Lactobacillus rhamnosus are prepared into seed liquid, and are inoculated into Fuling substrate soft material to form koji blocks for fermentation treatment, wherein the classification name of Lactobacillus plantarum HL-1 is Lactobacillus plantarum, which has been preserved in the China General Microbiological Culture Collection Center on February 29, 2024, at the address of Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, with the preservation number of CGMCC NO.29919, and the OD value of the Lactobacillus plantarum seed liquid, the Lactobacillus acidophilus seed liquid and the Lactobacillus rhamnosus seed liquid is 0.6-0.8, and the volume ratio is 10-1:1-10:1-10;

[0013] Step two, after the koji blocks after fermentation treatment are dried, Fuling products are obtained.

[0014] Further optimization, the screening method of Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus rhamnosus in step one is: according to any one or more of the properties of beta-glucosidase activity, amylase activity, protease activity, saccharifying enzyme activity and antioxidant performance, the strains with excellent performance are screened out.

[0015] Further optimization, the preparation method of the Fuling soft material in step one is: in a sterile environment, the Fuling is ground into powder and mixed with premix, and then the mixed filtrate of Artemisia vulgaris, Polygonum hydropiper and Xanthium sibiricum is added and stirred uniformly.

[0016] Further optimization, the premix comprises adzuki bean powder, bitter almond powder, wheat bran and flour.

[0017] Further optimization, the preparation method of the mixed filtrate of Artemisia, Polygonum hydropiper and Xanthium is as follows: Artemisia, Polygonum hydropiper and Xanthium are cut into small pieces, soaked with water, and then decocted for 1 hour, filtered, concentrated, cooled, and high-temperature sterilized.

[0018] Further optimization, the added amount of the six-shenqu is 1-10% of the weight of the six-shenqu matrix soft material.

[0019] Further optimization, the total inoculation amount of the Lactobacillus plantarum seed liquid, Lactobacillus acidophilus seed liquid and Lactobacillus rhamnosus seed liquid is 1-10% of the weight of the six-shenqu matrix soft material.

[0020] Further optimization, the drying temperature in step two is 35-60 DEG C, and the drying time is 8-20 hours.

[0021] Further optimization, the weight ratio of the six-shenqu to the six-shenqu matrix soft material is 1-60%.

[0022] Further optimization, the volume ratio of the Lactobacillus plantarum seed liquid, Lactobacillus acidophilus seed liquid and Lactobacillus rhamnosus seed liquid to the weight of the six-shenqu matrix soft material is 1-55%.

[0023] The beneficial effects of the present application are:

[0024] The fermentation method of the six-shenqu provided in the present application clearly inoculates seed koji, so that the substrate is inoculated after sterilization, thereby avoiding the influence of miscellaneous bacteria contained in the substrate; high-quality six-shenqu is selected as the koji for fermentation, compared with traditional natural fermentation, not only the fermentation process is accelerated, the amylase activity is improved, but also the stability of the amylase activity between batches is improved; the Lactobacillus plantarum seed liquid, Lactobacillus acidophilus seed liquid and Lactobacillus rhamnosus seed liquid are added to assist the fermentation of the six-shenqu, so as to achieve the purpose of strengthening, improve the content of effective components in the six-shenqu, and stabilize the quality of the six-shenqu; the Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus rhamnosus have the effects of immune regulation, inhibition of pathogenic bacteria, reduction of serum cholesterol content, prevention of cardiovascular diseases, maintenance of intestinal flora balance, promotion of nutrient absorption, inhibition of tumor cell formation, etc.; the functional components of the six-shenqu and the effects of the lactic acid bacteria can well synergize to promote gastrointestinal digestion, regulate the intestinal environment, and have a good therapeutic effect on functional indigestion. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The effect of different fermentation days on the enzyme activity of the probiotic bacteria-strengthened fermented six-shenqu in the single-factor test of the fermentation process;

[0026] Figure 2 The effect of different liquid-material ratios on the enzyme activity of the probiotic bacteria-strengthened fermented six-shenqu in the single-factor test of the fermentation process;

[0027] Figure 3 Effect of different inoculation amount on enzyme activity of probiotic bacteria fortified fermentation of Liu Shenqu in fermentation process single factor experiment;

[0028] Figure 4 Effect of different fermentation temperature on enzyme activity of probiotic bacteria fortified fermentation of Liu Shenqu in fermentation process single factor experiment;

[0029] Figure 5 Effect of different volume of Shenqu on enzyme activity of probiotic bacteria fortified fermentation of Liu Shenqu in fermentation process single factor experiment;

[0030] Figure 6 Effect of fermentation days and liquid material ratio interaction on enzyme activity of probiotic bacteria fortified fermentation of Liu Shenqu;

[0031] Figure 7 Effect of fermentation days and inoculation amount interaction on enzyme activity of probiotic bacteria fortified fermentation of Liu Shenqu;

[0032] Figure 8 Effect of fermentation days and fermentation temperature interaction on enzyme activity of probiotic bacteria fortified fermentation of Liu Shenqu;

[0033] Figure 9 Effect of liquid material ratio and inoculation amount interaction on enzyme activity of probiotic bacteria fortified fermentation of Liu Shenqu;

[0034] Figure 10 Effect of liquid material ratio and fermentation temperature interaction on enzyme activity of probiotic bacteria fortified fermentation of Liu Shenqu;

[0035] Figure 11 Effect of inoculation amount and fermentation temperature interaction on enzyme activity of probiotic bacteria fortified fermentation of Liu Shenqu;

[0036] Figure 12 Content of ferulic acid and rutin in probiotic bacteria fortified fermentation of Liu Shenqu, unfermented Liu Shenqu and commercially available Liu Shenqu;

[0037] Figure 13 Content of quercitrin, quercetin and luteolin in probiotic bacteria fortified fermentation of Liu Shenqu, unfermented Liu Shenqu and commercially available Liu Shenqu;

[0038] Figure 14 Figure of week change of mouse weight;

[0039] Figure 15 Figure of week change of mouse food intake;

[0040] Figure 16 Measurement of mouse gastric emptying rate;

[0041] Figure 17 Measurement of mouse small intestine propulsion rate;

[0042] Figure 18 Results of mouse pepsin activity assay. DETAILED DESCRIPTION

[0043] In order to enable persons skilled in the art to more clearly understand the above-mentioned objects, features and advantages of the present application, the present application will be described in detail below in conjunction with specific examples, which are implemented on the premise of the technical solutions of the present application, and give detailed implementation manners and specific operation processes. However, the present application can also be implemented in other manners different from those described herein, and therefore the protection scope of the present application is not limited to the following examples.

[0044] The preparation method of the probiotic fermented Fuling for treating functional dyspepsia comprises the following steps:

[0045] Step one, grind and sieve the adzuki beans and bitter almond, then uniformly mix the wheat bran and flour to prepare a premix, and sterilize at high temperature;

[0046] Cut the Artemisia annua, Polygonum hydropiper and Xanthium into small pieces, soak them in water, and then decoct for 1 hour. Filter the decoction, concentrate the filtrate, cool it, and sterilize it at high temperature to obtain a concentrated medicinal juice;

[0047] Under a sterile environment, uniformly mix the commercially available high-quality Fuling as a starter culture and ground into Fuling powder with the premix according to a weight ratio of 1-60%, and then mix and stir the concentrated medicinal juice to prepare a Fuling matrix soft material;

[0048] Prepare seed liquids of Lactobacillus plantarum HL-1 with the preservation number of CGMCC NO.29919, Lactobacillus acidophilus and Lactobacillus rhamnosus, and inoculate them into the Fuling matrix soft material to prepare koji blocks for fermentation treatment. The volume ratio of the seed liquids of the three strains to the mass of the Fuling matrix soft material is 1-55%, the relative humidity of the fermentation is 70-95%, the relative humidity of the fermentation is 85-95%, the fermentation time is 1-7 days, and the fermentation temperature is 26-42℃. The OD values of the seed liquids of the Lactobacillus plantarum, the Lactobacillus acidophilus and the Lactobacillus rhamnosus are 0.6-0.8, and the ratio is 10-1:1-10:1-10. The β-glucosidase activity, amylase activity, protease activity, saccharifying enzyme activity and antioxidant performance of the multiple strains added according to the above ratio are all higher than those of a single strain;

[0049] Step two, dry the fermented koji blocks at a temperature of 35-60℃ for 8-20 hours to obtain Fuling products.

[0050] The screening method of Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus rhamnosus is to screen strains with excellent performance according to any one or more of the β-glucosidase activity, amylase activity, protease activity, saccharifying enzyme activity and antioxidant performance. Specifically,

[0051] Step one, 0.3g of escin, 0.05g of ferric citrate, 0.2g of NaCl, 0.05g of MgSO4·7H2O, 0.1g of KH2PO4 and 2g of agar were mixed to prepare 100mL of escin culture medium;

[0052] Step two, the escin culture medium was heated and mixed, then placed in a high-pressure steam sterilization pot for sterilization, and a 96-well standard enzyme plate was opened and sprayed with disinfectant alcohol, and placed in an ultraviolet sterilization open clean bench for sterilization for about 40min;

[0053] Step three, after the escin culture medium was sterilized, it was taken out and cooled to room temperature, and 200μL of escin culture medium was taken out with a pipette gun in the clean bench and added to the wells of the 96-well standard enzyme plate, and then the ultraviolet sterilization was started;

[0054] Step four, take the bacterial liquid of the 28 strains to be screened, and use a pipette gun to take 100μL of the strain to the well plate in the clean bench, and make three groups for each strain, and then the 96-well standard enzyme plate is packaged with a self-sealing bag and placed in a 28℃ incubator for 24h, and the color change is observed, and the strains with obvious color deepening are further tested, and three strains are preliminarily screened, which are identified as Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus rhamnosus. The β-glucosidase activity of the three strains screened is tested, and the test results are shown in Table 1.

[0055] Table 1 β-glucosidase activity of strains

[0056]

[0057] The instruments, reagents, sample preparation methods, detection methods and the like used in the present application are described as follows:

[0058] 1. Instruments and reagents

[0059] 1.1 Instruments

[0060] 1260Infinity high-performance liquid chromatograph, Agilent Technology Co., Ltd.; UV-5500 ultraviolet-visible spectrophotometer, Shanghai Unico Instrument Co., Ltd.; DNP9162 constant temperature incubator, Shanghai Jinghong Experimental Equipment Co., Ltd.; HH-2 constant temperature water bath, Licheng Instrument Technology Co., Ltd.; YXO-LS-50S II high-pressure steam sterilization pot, Shanghai Boxun Industry Co., Ltd. Medical Equipment Factory; Pico-21 centrifuge, Thermo Fsher.

[0061] 1.2 Reagents

[0062] Flour was purchased from Hebei Jinshahai Flour Industry Group Co., Ltd., wheat bran was purchased from Xuzhou Farmhouse Shop, Artemisia annua, Polygonum hydropiper, Xanthium sibiricum, and Phaseolus roseus were purchased from Anhui Xin'an Jishidasheng Chain Co., Ltd., Xingfengrui Sixiangqu was purchased from Sichuan Huaxia Pharmaceutical Chain Co., Ltd., 3,5-dinitrosalicylic acid was purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd., rutin, quercetin, quercitrin, and luteolin were purchased from Hefei Bomei Biological Technology Co., Ltd., and ferulic acid was purchased from Shanghai Yuanye Biological Technology Co., Ltd. Pepsin kit was purchased from Iseki Biological Technology Co., Ltd.

[0063] 2. Preparation method of sample and detection method

[0064] 2.1 Preparation of culture medium

[0065] MRS broth medium was prepared according to the instructions to culture Lactobacillus plantarum seed liquid, Lactobacillus acidophilus seed liquid, and Lactobacillus rhamnosus seed liquid, respectively.

[0066] 2.2 Preparation of seed liquid

[0067] The Lactobacillus plantarum seed liquid, Lactobacillus acidophilus seed liquid, and Lactobacillus rhamnosus seed liquid were inoculated into MRS broth medium, respectively, and cultured, activated, and subcultured at 37°C. The OD value of the bacterial liquid was dynamically monitored during the process, and stopped when it reached about 0.7, ready for use.

[0068] 2.3 Preparation of solid matrix

[0069] Based on the prescription of Sixiangqu in the 19th volume of “Ministry of Health of the People's Republic of China Pharmaceutical Standards · Chinese Patent Medicine Prescriptions”, the fermentation soft material was prepared, made into blocks, and compacted to make koji.

[0070] 2.4 Fermentation of probiotic bacteria to strengthen fermentation of Sixiangqu

[0071] The prepared koji blocks were placed in a constant temperature and humidity box for culture. After fermentation, they were taken out, dried at low temperature, and stored for use.

[0072] 2.5 Determination of enzyme activity

[0073] The amylase activity was determined by 3,5-dinitrosalicylic acid (DNS) spectrophotometry.

[0074] In specific embodiments, the inoculation ratio refers to the volume ratio of koji. The inoculation amount refers to the mass g / mass g fraction of koji and matrix soft material, for example, an inoculation amount of 1% means 1g of koji is inoculated into 100g of soft material.

[0075] In specific embodiments, the inoculation ratio refers to the volume ratio of Lactobacillus plantarum seed liquid, Lactobacillus acidophilus seed liquid and Lactobacillus rhamnosus seed liquid. The inoculation amount refers to the volume mL / quality g ratio of seed liquid and substrate soft material, for example, an inoculation amount of 1% means that 1 mL of seed liquid is inoculated into 100 g of soft material.

[0076] Optimization of fermentation process

[0077] 1. Single factor test of fermentation process

[0078] The koji, Lactobacillus plantarum seed liquid, Lactobacillus acidophilus seed liquid and Lactobacillus rhamnosus seed liquid were inoculated into the solid substrate to explore the influence of different fermentation factors on the amylase activity of probiotic bacteria fortified fermentation of Shenghuangqiu, see Figures 1 to 5 .

[0079] According to the results of Figure 1 , it can be seen that with the increase of fermentation time, the amylase activity shows a trend of first increasing and then decreasing, and reaches the maximum enzyme activity on the 4th day; since Shenghuangqiu is mainly used to digest rice and flour, the amylase activity is considered as the index, and the optimal fermentation time of probiotic bacteria fortified fermentation of Shenghuangqiu is preliminarily determined as 4 days.

[0080] According to the results of Figure 2 , it can be seen that with the increase of liquid material ratio, the amylase activity shows a trend of first increasing and then decreasing, and then slightly increasing, and when the liquid material ratio is 1:1, the amylase reaches the maximum enzyme activity, and the optimal liquid material ratio of probiotic bacteria fortified fermentation of Shenghuangqiu is preliminarily determined as 1:1.

[0081] According to the results of Figure 3 , it can be seen that with the increase of inoculation amount, the amylase activity shows a trend of first increasing and then gradually stabilizing, and when the inoculation amount is 2%, it gradually reaches the maximum enzyme activity, and the optimal inoculation amount of probiotic bacteria fortified fermentation of Shenghuangqiu is preliminarily determined as 2%.

[0082] According to the results of Figure 4 , it can be seen that with the increase of fermentation temperature, the amylase activity shows a trend of first increasing and then decreasing, and when the temperature reaches 32℃, the amylase activity reaches the maximum, and the optimal fermentation temperature of probiotic bacteria fortified fermentation of Shenghuangqiu is preliminarily determined as 32℃.

[0083] According to the results of Figure 5 , it can be seen that with the increase of koji block volume, the amylase activity shows a trend of first increasing and then decreasing, and when the koji block volume reaches 8cm 3 , the amylase activity reaches the maximum, and the optimal koji block volume of probiotic bacteria fortified fermentation of Shenghuangqiu is preliminarily determined as 8cm 3 .

[0084] 2. Box-Behnken response surface test optimization screening process.

[0085] Plackett-Burman design was used to select fermentation time (A), liquid-solid ratio (B), inoculum size (C), fermentation temperature (D) and koji volume (E) as the factors. The results are shown in Table 1.

[0086] Table 1 Plackett-Burman design and results

[0087]

[0088] The results of Plackett-Burman design are shown in Table 2.

[0089] Table 2 Statistical analysis of Plackett-Burman design

[0090]

[0091] Note: * means significant influence (p<0.05); ** means extremely significant influence (p<0.01). The following tables are the same.

[0092] From Table 2, fermentation time (A), fermentation temperature (D), liquid-solid ratio (B) and inoculum size (C) have significant influence on the amylase activity of Fufangqi (p<0.05), and koji volume has no significant influence on the amylase activity.

[0093] Box-Behnken design was used to optimize the fermentation conditions, and the results are shown in Table 3.

[0094] Table 3 Box-Behnken design and results

[0095]

[0096] The results of Table 3 were processed by Design Expert 11 software to obtain a reliable model, analyze the interaction between factors, and obtain the three-dimensional surface graph of response surface, as shown in Figures 6 to 11 .

[0097] The optimal fermentation conditions predicted by Box-Behnken response surface model are as follows: fermentation time 4.26 d, liquid-solid ratio 1.06:1, inoculum size 3.14%, fermentation temperature 32.57℃, and the amylase activity predicted by the model is 464.723 U / g. Considering the actual situation, the optimized fermentation time is 4 d, the liquid-solid ratio is 1.1:1, the inoculum size is 3%, and the fermentation temperature is 32℃.

[0098] The fermentation of probiotic fermented Shenzhou-6 was carried out under the determined optimal process, and three batches were prepared. The average activity of amylase was 459.22±6.39 U / g, which only had an error of 1.184% compared with the theoretical predicted value. The data repeatability was good, which confirmed that the model constructed by DesignExpert 11 software with amylase activity as the response value was effective and reliable.

[0099] According to the determined optimal process, Shenzhou-6 was prepared, and the content of components was determined compared with unfermented Shenzhou-6 and commercially available high-quality Shenzhou-6.

[0100] As shown in Figure 12 , the contents of ferulic acid and rutin in probiotic fermented Shenzhou-6, unfermented Shenzhou-6 and commercially available Shenzhou-6 were determined. It can be seen that each component has significant difference (p<0.05). The contents of ferulic acid and rutin in probiotic Shenzhou-6 are significantly higher than those in commercially available Shenzhou-6 and unfermented Shenzhou-6. Compared with commercially available Shenzhou-6, ferulic acid increases by 35.98%, and rutin increases by 191.91%, which indicates that the added probiotics have the effect of promoting the accumulation of ferulic acid and rutin.

[0101] As shown in Figure 13 , the contents of flavonoids such as quercitrin, quercetin and luteolin in probiotic fermented Shenzhou-6, unfermented Shenzhou-6 and commercially available Shenzhou-6 were determined. It can be seen that each component has significant difference (p<0.05). The contents of quercitrin, quercetin and luteolin in probiotic Shenzhou-6 are significantly higher than those in commercially available Shenzhou-6 and unfermented Shenzhou-6. Compared with commercially available Shenzhou-6, quercitrin increases by 241.93%, quercetin increases by 65.25%, and luteolin increases by 50.08%. Among the determined flavonoids, the content of unfermented Shenzhou-6 is the lowest, and quercetin and luteolin are not detected, which indicates that fermentation is beneficial to the production and accumulation of flavonoids in Shenzhou-6, especially after the addition of probiotics, under the synergistic effect of probiotics and other strains, flavonoids are accumulated more.

[0102] Animal experiment

[0103] The following experiment is the experiment of functional dyspepsia probiotic fermented Shenzhou-6 prepared by using the embodiment of the present application for treating functional dyspepsia. The influence of Shenzhou-6 of the present application on the body weight, food intake, gastrointestinal motility and pepsin of functional dyspepsia mice is investigated.

[0104] Test mice: 60 female KM mice aged 6 weeks, each weighing 10-15 g, provided by Henan Skb Bioscience Co., Ltd., production license number SCXK(YU)2020-0005; environmental conditions: temperature 22-25℃, relative humidity 55%-70%, lighting time 12h / d.

[0105] Modeling of functional dyspepsia mice: after one week of adaptive feeding, all mice were given 0.2 mL of 0.1% iodoacetamide solution by gavage in combination with fasting every other day for modeling, and the modeling time lasted for two weeks.

[0106] Drug provision and grouping: the mice were randomly divided into 6 groups: normal control group (gavaged with 0.2 ml of distilled water), FD model control group (gavaged with 0.2 ml of distilled water), FD + positive control group (gavaged with 3.78 mg·kg -1 ·d -1 (multipar), FD + treatment functional dyspepsia probiotic fermented FSH low-dose group (gavaged with treatment functional dyspepsia probiotic fermented FSH decoction, 1 g·kg -1 ·d -1 ), FD + treatment functional dyspepsia probiotic fermented FSH medium-dose group (gavaged with treatment functional dyspepsia probiotic fermented FSH decoction, 2.5 g·kg -1 ·d -1 ), FD + treatment functional dyspepsia probiotic fermented FSH high-dose group (gavaged with treatment functional dyspepsia probiotic fermented FSH decoction, 4 g·kg -1 ·d -1 ), 10 mice in each group.

[0107] Test index: record the body weight and food intake of each group, and determine the influence of gastrointestinal motility, pepsin activity, etc.

[0108] Body weight of each group: the mice were weighed every day at 10 am. Food intake: record the amount of feed and the amount of remaining feed of each group every day. The amount of feed was subtracted from the amount of remaining feed. After the last administration of each group of mice, the mice were fasted for 24 h without water, and the next morning each group of mice was gavaged with 5% (m / m) charcoal paste. Gastric emptying rate determination: accurately weigh the whole stomach, wash the stomach contents with 0.9% saline, dry with filter paper and weigh the empty stomach, as formula (1) as follows:

[0109]

[0110] D = (M - m) / M × 100%

[0111] Small intestine propulsion rate determination: take the whole small intestine from the pylorus to the ileocecal junction, naturally straighten and lay on white filter paper, measure the charcoal propulsion distance and total small intestine length with a ruler. As formula (2) as follows:

[0112]

[0113] P = (S / L) x 100

[0114] The pepsin activity in the stomach tissue of each group of mice was determined using a pepsin kit.

[0115] As shown in Figure 14 , the body weight of the mice increased slightly during the adaptive feeding period, and began to decrease during the modeling period, especially in the second week of modeling, when the body weight decreased significantly. Treatment began in the third week, and the mice's body weight began to recover slowly. The treatment of functional dyspepsia probiotic fermented Fufangju at a medium dose and domperidone was better, and the mice basically returned to normal weight levels. The low and high dose groups of functional dyspepsia probiotic fermented Fufangju were slightly less effective, and the mice not given treatment also had some recovery due to the body's self-repair.

[0116] As shown in Figure 15 , the food intake of the mice began to decrease during the modeling period, especially in the second week of modeling, when the food intake decreased significantly. Treatment began in the third week, and the mice's food intake began to increase slowly. The treatment of functional dyspepsia probiotic fermented Fufangju at a medium dose and domperidone was better.

[0117] Gastric emptying rate is one of the important indicators for evaluating digestive function, as shown in Figure 16 , compared with the normal group, the gastric emptying rate of the model group decreased by 22.13%, indicating that the modeling was successful. Compared with the model group, each treatment group had a certain degree of recovery. The gastric emptying rate of the functional dyspepsia probiotic fermented Fufangju medium dose treatment group increased by 18.89%, and the gastric emptying rate of the functional dyspepsia probiotic fermented Fufangju high dose treatment group increased by 17.39%. The results showed that the medium and high doses of functional dyspepsia probiotic fermented Fufangju could have a certain therapeutic effect on the gastric emptying rate problem caused by functional dyspepsia.

[0118] Small intestinal transit rate is one of the important indicators for evaluating digestive function, as shown in Figure 17 , compared with the normal group, the small intestinal transit rate of the model group decreased by 36.38%, indicating that the modeling was successful. Compared with the model group, each treatment group had a certain degree of recovery. The small intestinal transit rate of the functional dyspepsia probiotic fermented Fufangju medium dose treatment group increased by 33.55%, and the small intestinal transit rate of the functional dyspepsia probiotic fermented Fufangju high dose treatment group increased by 26.48%. The results showed that the medium and high doses of functional dyspepsia probiotic fermented Fufangju could have a certain therapeutic effect on the small intestinal transit rate problem caused by functional dyspepsia.

[0119] The pepsin activity in the gastric tissue homogenate of each group of mice was determined, and the results are shown in Table 1. Figure 18 Compared with the normal group, the pepsin activity of the model group was significantly lower than that of the normal group, indicating that functional dyspepsia was accompanied by a decrease in pepsin activity. Compared with the model group, each treatment group had a certain degree of recovery. The pepsin activity of the high-dose group of the probiotic fermented Liu Shenqu for treating functional dyspepsia was not significantly different from that of the blank control group, indicating that the high-dose probiotic fermented Liu Shenqu for treating functional dyspepsia could alleviate the problem of pepsin activity caused by functional dyspepsia.

[0120] The present application is a safe and effective preparation method for probiotic fermented Liu Shenqu for treating functional dyspepsia. The three probiotics synergistically enhance the fermentation of Liu Shenqu, effectively promote gastrointestinal motility, improve digestive enzyme activity, and have a therapeutic effect on functional dyspepsia.

[0121] The above shows and describes the main features, use method, basic principle and advantages of the present application. It should be understood by those skilled in the art that the present application is not limited by the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made according to the actual situation, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for preparing probiotic fermented Fuling for treating functional dyspepsia, characterized in that, The specific steps are as follows: Step one, Lactobacillus plantarum HL-1, Lactobacillus acidophilus and Lactobacillus rhamnosus are all prepared into seed liquid, and are inoculated into soft material of six God's mixture matrix to make into koji block for fermentation treatment, the total inoculation amount of the Lactobacillus plantarum seed liquid, the Lactobacillus acidophilus seed liquid and the Lactobacillus rhamnosus seed liquid is 3wt% of the soft material of six God's mixture matrix, the volume ratio of the three kinds of seed liquid to the mass of the soft material of six God's mixture matrix is 1.1:1, the fermentation time is 4 days, and the fermentation temperature is 32℃; The preservation number of the Lactobacillus plantarum HL-1 is CGMCC NO.29919, the OD value of the Lactobacillus plantarum seed liquid, the Lactobacillus acidophilus seed liquid and the Lactobacillus rhamnosus seed liquid is 0.6-0.8, and the volume ratio is 10-1:1-10:1-10; step two, the koji block after the fermentation treatment is dried to obtain the six God's mixture product.

2. The preparation method of the functional indigestion probiotic fortified fermented Fuling of claim 1, characterized in that, The screening method of the Lactobacillus plantarum, the Lactobacillus acidophilus and the Lactobacillus rhamnosus in the step one is that any one or more of the following performances is screened to obtain a high-quality strain: β-glucosidase activity, amylase activity, protease activity, saccharifying enzyme activity and antioxidant performance.

3. The preparation method of the probiotic fortified fermented Fuling for treating functional dyspepsia according to claim 1, characterized in that, The preparation method of the soft material of six God's mixture matrix in the step one is that the six God's mixture is ground into powder, is uniformly mixed with premix, and then the mixed filtrate of artemisia, polygonum hydropiper and xanthium is added and stirred uniformly.

4. The preparation method of the probiotic fortified fermented Fuling for treating functional dyspepsia according to claim 3, characterized in that, The premix includes adzuki bean powder, bitter almond powder, wheat bran and flour.

5. The preparation method of the probiotic fortified fermented Fuling for treating functional dyspepsia according to claim 3, characterized in that, The preparation method of the mixed filtrate of artemisia, polygonum hydropiper and xanthium is that the artemisia, the polygonum hydropiper and the xanthium are cut into small pieces, are soaked in water, and then are decocted for 1 hour, are filtered, and the filtrate is concentrated, cooled and high-temperature sterilized.

6. The preparation method of the probiotic fortified fermented Fuling for treating functional dyspepsia according to claim 3, characterized in that, The six God's mixture addition amount is 1-10% of the weight of the soft material of six God's mixture matrix.

7. The preparation method of the probiotic fortified fermented Fuling for treating functional dyspepsia according to claim 1, characterized in that, The drying temperature in the step two is 35-60℃, and the drying time is 8-20 hours.

8. The preparation method of the probiotic fortified fermented Fuling for treating functional dyspepsia according to claim 1, characterized in that, The weight ratio of the six God's mixture to the soft material of six God's mixture matrix is 1-60%.

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