Application of probiotic-prebiotic compound in preparation of product for regulating spleen deficiency type intestinal flora

Abstract

The invention relates to an application of a probiotic-prebiotic compound in preparation of a product for regulating spleen deficiency type intestinal flora. Tests prove that the probiotic-prebiotic compound disclosed by the invention can be used for improving intestinal flora disorder caused by spleen deficiency. Therefore, the probiotic-prebiotic compound disclosed by the invention has the effect of regulating the spleen deficiency type intestinal flora and can be used for preparing products for regulating the spleen deficiency type intestinal flora.

Description

Technical Field

[0001] The present invention belongs to the technical field of probiotics, and relates to the application of a probiotic-prebiotic complex in the preparation of a product for regulating the intestinal flora of the spleen-deficiency type. Background Art

[0002] According to the research data, in accordance with the diagnostic criteria of the Expert Consensus on Traditional Chinese Medicine Diagnosis and Treatment of Spleen Deficiency Syndrome (2017), the proportion of adult consumers with spleen deficiency symptoms in China is as high as 75.8%, and the proportion of spleen deficiency people who meet the diagnostic criteria of spleen deficiency syndrome reaches 41.3%; more than half of the spleen deficiency people have irregular diets, more than eight out of ten spleen deficiency people like sweet, fried, and stimulating foods, more than six out of ten spleen deficiency people are in a state of bad emotions for a long time, with normal stress and anxiety, and more than six out of ten spleen deficiency people lack a healthy lifestyle, and it is normal not to exercise and stay up late. Spleen deficiency is a disease name in traditional Chinese medicine, manifested as fatigue, loss of appetite, weight loss, dry and falling hair, etc. Cheng Yue et al. from Liaoning University of Traditional Chinese Medicine published a literature titled "Discussion on the Essence of Traditional Chinese Medicine of Intestinal Microecology under the Guidance of the Theory of Spleen and Stomach" in "Lishizhen Medicine and Materia Medica", in which it is mentioned that "when the spleen transports effectively, the flora is nourished, and the body is peaceful", and "disorder of the spleen in transportation is often accompanied by disorders of the intestinal flora". The literature "Microecological Regulatory Effect of Jianpi Zhixie Granule on Spleen Deficiency Syndrome and Antibiotic-induced Intestinal Flora Dysbiosis Model Mice", the authors of which are Li Qiuming et al. from Changchun University of Traditional Chinese Medicine, also corroborates this theory, and this literature was published in "Journal of Basic Chinese Medicine".

[0003] Intestinal flora dysbiosis refers to an imbalance state caused by changes in the composition of the intestinal flora, changes in bacterial activity, or changes in the flora distribution. Intestinal flora dysbiosis is a common clinical disease that can occur in people of all ages, especially in the elderly and infants. There are many factors causing this imbalance state, such as digestive system diseases, metabolic diseases (such as hyperglycemia / hyperlipidemia), anti-infective drugs (especially broad-spectrum anti-infective drugs), and unhealthy dietary structures, etc., which can all induce intestinal flora dysbiosis. Clinically, most cases of regulating flora disorders will choose microecological preparations, with lactobacilli and bifidobacteria as typical probiotic raw materials. However, current research shows that there are significant differences in the genomes of different species of probiotics, and there are also differences even between different strains of the same species of probiotic. Since different strains of the same species of probiotic contain or express different functional genes and can exert different probiotic effects, probiotics need to be characterized and described at the microbial strain level (a literature published by the Probiotics Branch of the Chinese Institute of Food Science and Technology in the Journal of Chinese Institute of Food Science and Technology with the title "Scientific Consensus on Probiotics (2020 Edition)"). In addition, specific strains are often used to intervene in intestinal flora dysbiosis caused by different reasons. For example, Lactobacillus reuteri for improving Helicobacter pylori infection, Bifidobacterium Bb-12 for regulating constipation, Streptococcus thermophilus for improving lactose intolerance, Bacillus subtilis for improving eczema, etc. (a literature published by the Pediatric Group of the Microecology Branch of the Chinese Preventive Medicine Association in the Chinese Journal of Practical Pediatrics with the title "Expert Consensus on the Application of Microecological Preparations in Pediatrics (October 2010)" (hereinafter referred to as the "Consensus")). At the same time, the "Consensus" points out that probiotics are not a "panacea" that can cure all diseases. The flora disorders caused by different diseases are different, and it is necessary to select probiotic preparations with clear functions for targeted regulation. Therefore, for the flora disorders caused by special diseases, it is necessary to screen specific highly active strains.

[0004] Currently, for the development of products to improve intestinal flora dysbiosis, many researchers mainly use the intestinal flora dysbiosis model induced by antibiotics, and rely on the flora disorders induced by different pathologies as the indicators for evaluating drug efficacy, such as the intestinal flora dysbiosis model induced by loperamide, the intestinal flora dysbiosis model induced by alcohol, the intestinal flora dysbiosis model of mice induced by inflammatory bowel disease, etc. However, there is no report on these products for intestinal flora dysbiosis of the spleen-deficiency type, and there are almost no probiotic products for regulating the flora disorder of the spleen-deficiency type so far. The conventional traditional Chinese medicine therapy mainly takes traditional Chinese medicine for strengthening the spleen and stomach. The treatment plan can only be determined after being diagnosed by a doctor before taking traditional Chinese medicine, which is relatively complicated. As a class of drugs recognized internationally as relatively safe, if probiotic products can improve the intestinal flora disorder caused by spleen deficiency, it will provide great reference for clinical diagnosis and treatment.

[0005] In view of this, developing a product that can effectively regulate the intestinal flora of the spleen-deficiency type has become an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] To solve the problems existing in the prior art, the present invention provides an application of a probiotic-prebiotic complex in the preparation of a product for regulating spleen-deficiency type intestinal flora. The technical solution adopted by the present invention is as follows:

[0007] An application of a probiotic-prebiotic complex in the preparation of a product for regulating spleen-deficiency type intestinal flora.

[0008] In a preferred embodiment of the present invention, the regulation of spleen-deficiency type intestinal flora includes improving the composition structure of spleen-deficiency type intestinal flora, and further includes shortening the sample distance of intestinal flora from the healthy group and reducing the species difference from the healthy group.

[0009] In a preferred embodiment of the present invention, the regulation of spleen-deficiency type intestinal flora includes, at the family classification level,

[0010] increasing the abundance of the following families:

[0011] Bacteroidales_BS11_gut_group; and,

[0012] Tannerellaceae; and,

[0013] p-2534-18B5_gut_group;

[0014] and / or,

[0015] decreasing the abundance of the following families:

[0016] Campylobacteraceae; and,

[0017] norank_o_RF39.

[0018] In a preferred embodiment of the present invention, the regulation of spleen-deficiency type intestinal flora includes, at the species classification level,

[0019] increasing the abundance of the following species:

[0020] Porphyromonadaceae_bacterium_DJF_B175; and

[0021] uncultured_rumen_bacterium_f_Eubacterium_coprostanoligenes_group; and

[0022] uncultured_bacterium_f_p-2534-18B5_gut_group; and

[0023] uncultured_bacterium_g_Prevotellaceae_NK3B31_group; and

[0024] uncultured_Clostridiales_bacterium_g_Monoglobus;

[0025] and / or,

[0026] reduce the abundance of the following bacterial species:

[0027] uncultured_bacterium_o_Clostridia_UCG-014; and

[0028] Ruminococcus_sp._N15.MGS-57_g_Ruminococcus.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] The probiotic-prebiotic complex described in the present invention has been proven by experiments. On the one hand, it can regulate the intestinal flora disorder caused by spleen deficiency (adjust the flora structure to a level similar to that of the normal, increase the abundance of beneficial bacteria and reduce the abundance of harmful bacteria at the family level, and increase the abundance of beneficial bacteria and reduce the abundance of harmful bacteria at the species level). On the other hand, it can improve the spleen deficiency symptoms of spleen-deficient mice (increase the daily food intake of spleen-deficient mice, increase the body weight of spleen-deficient mice, increase the spontaneous activity ability of spleen-deficient mice, and improve the disordered / dull / yellowish hair of spleen-deficient mice). Therefore, the probiotic-prebiotic complex of the present invention has the effect of regulating the spleen-deficiency type intestinal flora and can be used to prepare products for regulating the spleen-deficiency type intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the dilution curve of the intestinal flora in the fecal samples of each group of the present invention;

[0032] Figure 2 is the PCoA schematic diagram of the intestinal flora of each group of the present invention at the OTU level;

[0033] Figure 3 is the microbial community composition diagram (family level) of the test animals in each group of the present invention;

[0034] Figure 4 is the microbial community composition diagram (species level) of the test animals in each group of the present invention;

[0035] Figure 5 is the schematic diagram of the significant difference analysis of the intestinal flora species of the test animals in each group of the present invention (family level);

[0036] Figure 6Schematic diagram of the significant difference analysis of the intestinal flora species of the test animals in each group of the present invention (species level);

[0037] Figure 7 Schematic diagram of the statistical analysis of the hair condition scores of the test animals in each group of the present invention;

[0038] Figure 8 Schematic diagram of the statistical analysis of the average daily food intake of the test animals in each group of the present invention in the 3rd week;

[0039] Figure 9 Schematic diagram of the statistical analysis of the weight changes of the test animals in each group of the present invention before and after the experiment;

[0040] Figure 10 Schematic diagram of the statistical analysis of the evaluation of the spontaneous activity of the test animals in each group of the present invention;

[0041] Figure 11 Photographs of the behavioral states of the test animals in each group of the present invention;

[0042] In the figure, NC represents the blank group, MC represents the model group, and FHW represents the complex group. Detailed implementation mode

[0043] The present invention will be specifically described below in conjunction with specific drawings and embodiments. It is necessary to point out here that the following embodiments are only used for further illustration of the present invention and cannot be understood as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the content of the present invention still fall within the protection scope of the present invention.

[0044] The raw materials used in the embodiments are all conventional commercially available raw materials. Among them, the manufacturer of the rhubarb medicinal material is Bozhou Baichuan Pharmaceutical Co., Ltd.

[0045] The probiotic-prebiotic complex of the present invention is the probiotic-prebiotic complex in the Chinese patent application "Probiotic-Prebiotic Complex and Its Preparation Method and Application" (publication number: CN118749658A), specifically the probiotic-prebiotic complex prepared in Example 5.

[0046] In the present invention, the dilution curve can directly reflect the rationality of the sequencing data volume. When the curve tends to be flat, it indicates that the sequencing data volume is gradually reasonable, and more data volume will only generate a small number of new species (OTUs). The dilution curve is constructed with the number of sequences drawn and the number of OTUs they can represent.

[0047] The instruments used in the embodiments are all conventional commercially available instruments.

[0048] Example 1

[0049] 1. Preparation of the test samples

[0050] 1.1 Rheum palmatum Decoction: Take 250 g of raw Rheum palmatum, add 2000 ml of water, soak for 0.5 h, decoct over low heat for 15 min, cool naturally, filter through four layers of gauze, combine the decoction, and concentrate it to 250 ml using a rotary evaporator, equivalent to 1 g / ml of raw medicine.

[0051] 1.2 Test sample of the complex: Weigh 2.7 g of the probiotic-prebiotic complex, dissolve it in 10 mL of physiological saline to obtain a test sample of the probiotic-prebiotic complex with a concentration of 270 g / mL.

[0052] 2. Animal grouping, model establishment and drug administration

[0053] 2.1 Test animals and animal grouping

[0054] The test animals were selected as mice of the KM strain (SPF level). All test animals were of clean grade, with the certificate number SCXK(Jing)2021-0011, a total of 30. The test animals were all fed in cage racks, 5 in each cage, and separated by gender. The room temperature was maintained at about 24 °C, the relative humidity was 40-60%, and the light cycle was 12 h. During the whole test process, the animals were given ordinary feed and free access to water. The mice were adaptively fed for 1 week, weighed, and randomly divided into 3 groups according to their body weight. Then, the model was established and drugs were administered. The method of model establishment referred to "The Effect of Guizhi Baizhu Decoction on the Expression of Immune Factors and Aquaporins in Spleen-deficient Mice" (published by Gansu University of Chinese Medicine in Lishizhen Medicine and Materia Medica).

[0055] Table 1 Test animals and animal grouping

[0056] Group Test animals Gender Body weight Number of animals Blank group KM mice Half male and half female (20±2)g 10 Model group KM mice Half male and half female (20±2)g 10 Complex group KM mice Half male and half female (20±2)g 10

[0057] 2.2 Model establishment and drug administration of test animals

[0058] Table 2 Model establishment and drug administration of test animals

[0059]

[0060]

[0061] 3 Detection of test animals

[0062] 3.1 Recording of external signs of test animals in each group

[0063] During the experiment, the body weight of the animals was recorded daily. During the third week, the hair condition, behavior state, food intake, and body weight of the test animals in each group were recorded. Among them, in the evaluation of the behavior state of the test animals in each group (i.e., the evaluation of the spontaneous activity of the animals), the test animals in each group were taken turns to be placed in the spontaneous activity recorder daily and the number of activities within a unit time was counted.

[0064] The animal hair was scored with reference to the "Expert Consensus on the Diagnosis and Treatment of Traditional Chinese Medicine for Spleen Deficiency Syndrome (2023)" and "Methodology of Traditional Chinese Medicine Experimental Animal Models". The appearance signs of spleen-deficient mice were scored according to the following criteria: ① Hair condition: smooth, shiny, no yellowing, no piloerection, scored 0 points; messy, dull, slightly yellowing, no piloerection, scored 1 point; messy, dirty, withered, yellowing, no piloerection, scored 2 points; messy, dirty, withered, yellowing, piloerection, scored 3 points.

[0065] 3.2 Intestinal flora of each group of test animals (16S rRNA sequencing analysis)

[0066] After continuous administration for 2 weeks, the intestinal flora of mice in each group was analyzed. After collecting the fecal samples of each animal, they were sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd. for flora detection. The species composition and richness of the flora in each group were analyzed using the online data analysis platform provided by Majorbio.

[0067] 4 Result analysis

[0068] 4.1 Intestinal flora of test animals in each group

[0069] Figure 1 The dilution curves of the intestinal flora of each group of test animals in the present invention are shown. From Figure 1 it can be seen that the Shannon-curves have entered the plateau phase, indicating that the sequencing data volume is sufficient to cover all taxa and can reflect the vast majority of microbial information, meeting the analysis requirements.

[0070] 4.1.1 Sample distance of intestinal flora among test animals in each group .

[0071] Figure 2 The PCoA analysis based on the OTU level is shown, which is used to display the sample distance of the intestinal flora of each group of test animals and measure the similarity of the microbial composition between groups; among them, the X-axis and Y-axis represent two selected principal coordinate axes, and the percentage represents the explanatory value of the principal coordinate axes for the sample composition difference; the scales of the X-axis and Y-axis are relative distances and have no practical significance; points of different colors or shapes represent samples of different groups, and the closer the two sample points are, the more similar the species composition of the two samples is. From Figure 2 it can be seen that the model group and the blank group are completely separated (P < 0.01), indicating that the composition of the intestinal flora of spleen-deficient mice has changed significantly. The compound group and the model group are completely separated (P < 0.01) and partially overlap with the blank group (P > 0.05), indicating that the intervention of the compound regulates the flora composition of spleen-deficient mice and makes its intestinal flora composition tend to be normal.

[0072] 4.1.2 Microbial community composition and difference analysis of intestinal flora of test animals in each group .

[0073] Figure 3The microbial community composition map (relative abundance) of each group of test animals at the family level is shown. From Figure 3 it can be seen that at the family level, the top 12 families with the highest proportions are: Muribaculaceae, Prevotellaceae, Eubacteriaceae, Ruminococcaceae, norank_o_ClostridiaUCG-014, Erysipelotrichaceae, Lachnospiraceae, p-251-05, Oscillospiraceae, Rikenellaceae, Eubacterium coprostanoligenes group, F082.

[0074] Compared with the blank group, the relative abundances of Muribaculaceae, Eubacteriaceae, Ruminococcaceae, and Erysipelotrichaceae in the model group increased; compared with the model group, the relative abundances of Muribaculaceae, Eubacteriaceae, Ruminococcaceae, and Erysipelotrichaceae in the complex group decreased. Compared with the blank group, the relative abundances of Prevotellaceae, p-251-05, and Rikenellaceae in the model group decreased; compared with the model group, the relative abundances of Prevotellaceae, p-251-05, and Rikenellaceae in the complex group increased.

[0075] It can be seen from this that the microbial community composition (at the family level) of spleen-deficient mice has changed significantly. After 2 weeks of intervention with the probiotic-prebiotic complex, it regulated the microbial community composition of spleen-deficient mice, making their microbial community composition tend to be normal.

[0076] Figure 4 The microbial community composition map (relative abundance) of each group of test animals at the species level is shown. From Figure 4It can be seen that at the family level, the top 14 species with the highest proportions are: species under Muribaculaceae (norank_f_Muribaculaceae), species under Eubacteriaceae (norank_f_Eubacteriaceae), species under ClostridiaUCG-014 (norank_o_Clostridia UCG-014), Prevotella spp., norank_f_p-251-05, Eubacterium_siraeum_group, norank_f_Eubacterium_coprostanoligenes_group, Rikenellaceae_RC9_gut_group, Ruminococcus spp., Prevotellaceae_UCG-001, norank_f_F082, species under Lachnospiraceae (unclassified_f_Lachnospiraceae), Leibacterium, species under Prevotellaceae (unclassified_f_Prevotellaceae).

[0077] Compared with the blank group, the relative abundances of species under Muribaculaceae (norank_f_Muribaculaceae), Eubacteriaceae (norank_f_Eubacteriaceae), Eubacterium_siraeum_group, and Leibacterium were increased in the model group; compared with the model group, the relative abundances of species under Muribaculaceae (norank_f_Muribaculaceae), Eubacteriaceae (norank_f_Eubacteriaceae), Eubacterium_siraeum_group, and Leibacterium were decreased in the complex group. Compared with the blank group, the relative abundances of Prevotella, norank_f_p-251-05, Rikenellaceae_RC9_gut_group, Prevotellaceae_UCG-001, and unclassified species under Prevotellaceae were decreased in the model group; compared with the model group, the relative abundances of Prevotella, norank_f_p-251-05, Rikenellaceae_RC9_gut_group, Prevotellaceae_UCG-001, and unclassified species under Prevotellaceae were increased in the complex group.

[0078] It can be seen that the microbial community composition (species level) of spleen-deficient mice has changed significantly. After 2 weeks of intervention with the probiotic-prebiotic complex, it regulated the microbial community composition of spleen-deficient mice and made their microbial community composition tend to be normal.

[0079] 4.1.3 Analysis of species difference significance of intestinal flora of test animals in each group 。

[0080] 4.1.3.1 Analysis of the significance of species differences in the intestinal flora of the test animals in each group (family level).

[0081] Figure 5 The figure shows the schematic diagram of the analysis of the significance of species differences in the intestinal flora at the family level of the test animals in each group. As Figure 5 can be seen, compared with the blank group, the abundances of all three beneficial bacteria were significantly decreased (P < 0.05), and the abundances of both harmful bacteria were significantly increased (P < 0.05). Among them, the three beneficial bacteria are respectively:

[0082] Bacteroidales_BS11_gut_group, Tannerellaceae, p-2534-18B5_gut_group; the two harmful bacteria are: Campylobacteraceae, norank_o_RF39. Bacteroidales_BS11_gut_group participates in the fermentation of monosaccharides in the intestine and produces short-chain fatty acids (SCFAs); Tannerellaceae is beneficial to inhibiting intestinal inflammatory responses;

[0083] p-2534-18B5_gut_group helps to maintain the diversity and stability of the intestinal flora. Compared with the model group, the abundances of the three beneficial bacteria in the complex group were all significantly increased (P < 0.05), and the abundances of the two harmful bacteria were all significantly decreased (P < 0.05).

[0084] It can be seen therefrom that at the family classification level, in the intestinal flora of spleen-deficiency mice, the abundances of multiple beneficial bacteria were all significantly decreased, and the abundances of multiple harmful bacteria were all significantly increased. After intervention with the probiotic-prebiotic complex for 2 weeks, it was able to significantly reverse the dysregulation of "the abundances of multiple beneficial bacteria were all significantly decreased, and the abundances of multiple harmful bacteria were all significantly increased" in the intestinal flora of spleen-deficiency mice, such that the abundances of multiple beneficial bacteria were all significantly increased and the abundances of multiple harmful bacteria were all significantly decreased, basically returning to the normal level.

[0085] 4.1.3.2 Analysis of the significance of species differences in the intestinal flora of each group of test animals (species level).

[0086] Figure 6 The schematic diagram of the analysis of the significance of species differences in the intestinal flora of each group of test animals of the present invention at the species level (species level) is shown. It can be Figure 6 seen that compared with the blank group, the abundances of the five beneficial bacteria were all significantly decreased (P < 0.05), and the abundances of the two harmful bacteria were all significantly increased (P < 0.05). Among them, the five beneficial bacteria are: Porphyromonadaceae_bacterium_DJF_B175,

[0087] uncultured_rumen_bacterium_f_Eubacterium_coprostanoligenes_group, uncultured_bacterium_f_p-2534-18B5_gut_group,

[0088] uncultured_bacterium_g_Prevotellaceae_NK3B31_group, uncultured_Clostridiales_bacterium_g_Monoglobus; the two harmful bacteria are respectively: uncultured_bacterium_o_Clostridia_UCG-014,

[0089] Ruminococcus_sp._N15.MGS-57_g_Ruminococcus. Among them, unculturedbacteriumg_Prevotellaceae_NK3B31_group is beneficial to promoting the proliferation of immune cells in the intestine;

[0090] uncultured_rumen_bacterium_f_Eubacterium_coprostanoligenes_group and Ruminococcus_sp._N15.MGS-57_g_Ruminococcus are both beneficial bacteria that produce short-chain fatty acids. Compared with the model group, the abundances of the 5 beneficial bacteria in the complex group were all significantly increased (P < 0.05), and the abundances of the 2 harmful bacteria were all significantly decreased (P < 0.05).

[0091] It can be seen from this that at the species classification level, in the intestinal flora of spleen-deficiency mice, the abundances of multiple beneficial bacteria were all significantly decreased, and the abundances of multiple harmful bacteria were all significantly increased. After 2 weeks of intervention with the probiotic-prebiotic complex, it was able to significantly reverse the imbalance of "the abundances of multiple beneficial bacteria were all significantly decreased, and the abundances of multiple harmful bacteria were all significantly increased" in the intestinal flora of spleen-deficiency mice, resulting in a significant increase in the abundances of multiple beneficial bacteria and a significant decrease in the abundances of multiple harmful bacteria, basically returning to normal levels.

[0092] 4.2 Normalized physical signs of each group of test animals

[0093] As mentioned above, "during the 3rd week, the appearance signs, behavior status, food intake, and body weight of each group of mice were recorded daily". According to the above scoring criteria, the hair conditions of the mice in each group were scored, and after scoring, statistical analysis was performed using GraphPad Prism 8.0 software. The final data was expressed as mean ± standard deviation (one-way ANOVA was used for comparison among multiple groups); similarly, the behavior status, food intake, and body weight of each group of mice were also analyzed statistically using GraphPad Prism 8.0 software (one-way ANOVA was used for comparison among multiple groups). The results are as Figures 7 - 10as shown; where, # indicates a significant difference between this group and the blank group (P < 0.05), ## indicates a highly significant difference between this group and the blank group (P < 0.01); * indicates a significant difference between this group and the model group (P < 0.05).

[0094] Figure 7 shows the schematic diagram of the statistical analysis of the hair condition scores of the test animals in each group within the 3rd week. From Figure 7 it can be seen that for the test animals in the blank group, most of their hair is smooth, shiny, and without yellowing. Compared with the blank group, some of the test animals in the model group have messy, dull, and slightly yellowed hair, some have dirty, dry, and yellowed hair, and individual test animals also show piloerection. For the hair condition scores, there is a highly significant difference between the model group and the blank group (P < 0.01). Compared with the model group, the test animals in the complex group also showed messy, dull, and slightly yellowed hair at the end of the 1st week, but after the experiment ended, the aforementioned hair conditions were significantly improved. Except for individual test animals still having messy and yellowed hair, the hair conditions of the remaining test animals were basically comparable to those of the blank group. For the hair condition scores, there is a significant difference between the complex group and the model group (P < 0.05). Thus, it can be known that the hair of spleen-deficiency mice is messy, dull, yellowed, dirty, and dry. After intervention with the probiotic-prebiotic complex for 2 weeks, it can significantly reverse the "messy, dull, yellowed, dirty, and dry hair" condition of spleen-deficiency mice, making the hair of the mice basically return to being smooth, shiny, and without yellowing.

[0095] Figure 8 shows the average daily food intake of the test animals in each group. From Figure 8 it can be seen that compared with the blank group, the average daily food intake of the test animals in the model group is extremely significantly reduced (P < 0.01). Compared with the model group, the test animals in the complex group also showed a decrease in average daily food intake at the end of the 1st week, but after the experiment ended, the average daily food intake of the test animals in the complex group was extremely significantly increased (P < 0.05). Thus, it can be known that the average daily food intake of spleen-deficiency mice is significantly reduced. After intervention with the probiotic-prebiotic complex for 2 weeks, it can significantly increase the average daily food intake of spleen-deficiency mice.

[0096] Figure 9 shows the changes in body weight of the test animals in each group before and after the experiment. From Figure 9 it can be seen that the weight gain of the test animals in the model group is significantly lower than that of the blank group (P < 0.05). The weight gain of the test animals in the complex group is significantly higher than that of the model group (P < 0.05) during the experiment and is similar to that of the blank group. Thus, it can be known that the weight gain rate of spleen-deficiency mice is significantly slowed down. After intervention with the probiotic-prebiotic complex for 2 weeks, it can significantly increase the weight gain of spleen-deficiency mice, making it basically return to the level of the blank group.

[0097] Figure 10 The evaluation of the spontaneous activity of each group of test animals is shown, specifically the number of activities of mice within 5 min. It can be Figure 10 seen that compared with the blank group, the number of activities of the test animals in the model group within 5 min decreased extremely significantly (P < 0.01). Compared with the model group, the number of activities in the complex group increased significantly (P < 0.05), and basically recovered to the level of the blank group. Thus, it can be known that the number of activities per unit time of spleen-deficient mice decreased significantly. After 2 weeks of intervention with the probiotic-prebiotic complex, it can significantly increase the number of activities per unit time of spleen-deficient mice and basically recover it to the level of the blank group.

[0098] In addition, Figure 11 the photos of the behavioral states of each group of test animals are shown, which were taken on the first day after the end of the experiment. It can be Figure 11 seen that the mice in the blank group were active normally; the mice in the model group were listless, lazy to move, and liked to huddle together, which corroborated that the mice in the model group showed significant spleen-deficiency symptoms; after 2 weeks of intervention with the probiotic-prebiotic complex, it could significantly improve the behaviors of mice such as "listlessness, laziness to move, and liking to huddle together".

Claims

1. Use of a probiotic-prebiotic complex in the preparation of a product for regulating spleen-deficiency type intestinal flora.

2. The application according to claim 1, wherein: The regulation of spleen-deficiency type intestinal flora includes improving the composition and structure of spleen-deficiency type intestinal flora, and further includes shortening the sample distance of intestinal flora from that of the healthy group and reducing the species difference from the healthy group.

3. The application according to claim 1, wherein: The regulation of spleen-deficiency type intestinal flora includes, at the family classification level, increasing the abundances of the following families: Bacteroidales_BS11_gut_group; and, Tannerellaceae; and, p-2534-18B5_gut_group; and / or, decreasing the abundances of the following families: Campylobacteraceae; and, norank_o_RF39.

4. The application according to claim 1, characterized in that: The regulation of spleen-deficiency type intestinal flora includes, at the species classification level, increasing the abundances of the following species: Porphyromonadaceae_bacterium_DJF_B175; and uncultured_rumen_bacterium_f_Eubacterium_coprostanoligenes_group; and uncultured_bacterium_f_p-2534-18B5_gut_group; and uncultured_bacterium_g_Prevotellaceae_NK3B31_group; and uncultured_Clostridiales_bacterium_g_Monoglobus; and / or, decreasing the abundances of the following species: uncultured_bacterium_o_Clostridia_UCG-014; and Ruminococcus_sp._N15.MGS-57_g_Ruminococcus.

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