Application of enteric microorganism in medicine or health care product participating in black fungus melanin for combined treatment of iron-deficiency anemia

Abstract

The invention discloses application of enteric microorganisms in medicines or health care products participating in black fungus melanin for combined treatment of iron-deficiency anemia, and belongs to the technical field of biology. In the invention, the intestinal microorganism ackermann bacteria and black fungus melanin are used for intragastric administration to treat iron-deficiency anemia mice, and a synergistic interaction effect is generated, so that the iron-deficiency anemia symptoms of the mice can be obviously improved. Compared with the prior art, the intestinal microorganisms can effectively improve blood routine indexes, iron metabolism indexes and inflammation indexes of IDA mice and repair colon injury in the process of treating iron-deficiency anemia by participating in black fungus melanin. The iron-supplementing prebiotic preparation is expected to be specifically applied to treatment of iron-deficiency anemia and further replace a traditional iron-supplementing agent to become a next-generation novel iron-supplementing prebiotic preparation, and a potential new strategy is provided for treatment of iron-deficiency anemia patients clinically.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of intestinal microorganisms in a drug or health product for the combined treatment of iron deficiency anemia with black fungus melanin. Background Art

[0002] A major primary factor of iron deficiency anemia (IDA) is the reduction of iron intake or excessive loss and the metabolic imbalance of other hematopoietic nutrients, which in turn affects the normal production of red blood cells. IDA is the most common nutritional deficiency in the world, affecting up to 2 billion people. It can affect multiple organs in the human body, such as the liver, spleen, colon, etc., damaging the functions of all stages of life, and seriously affecting people's health. Iron supplementation is currently the main method for treating IDA. However, most oral iron supplements have serious side effects, causing intestinal damage and affecting the absorption of gastrointestinal iron. Therefore, it is crucial to find new safe and effective methods to treat IDA.

[0003] Black fungus (Auricularia auricula-judae) is an edible fungus with extremely wide distribution in China and unique nutritional value and health care functions. Traditional Chinese medicine believes that black fungus has the effect of nourishing qi and blood, treating qi and blood deficiency, and is a high-quality natural blood tonic that can promote hematopoiesis. It contains a variety of bioactive compounds, melanin (Melanin; M) is one of the main bioactive ingredients, and is regarded as one of the most important functional ingredients in black food with extremely high safety. According to literature reports, black fungus melanin can also protect against radiation, anti-tumor, anti-oxidation, and improve the body's immunity. It has broad application prospects in food processing, cosmetics development, disease prevention and treatment.

[0004] Gut microbes and their products are indispensable to the growth and development of the host and can have a multifaceted impact on intestinal health, as gut microbes not only regulate host physiological processes in the gastrointestinal tract, but also regulate extra-intestinal organs such as the liver, brain, and immune system. Studies have shown that IDA can cause intestinal microbial disorders, and low iron conditions are believed to be the cause of major changes in microbial composition.

[0005] Verrucomicrobia is a relatively abundant group of intestinal microorganisms that colonize the gastrointestinal tract. Akkermansia muciniphila (Akk), which belongs to the Verrucomicrobia genus, can specifically degrade mucins and use mucins as the only carbon and nitrogen source to produce short-chain fatty acids to provide energy for the host. It was first isolated from human feces by Derrien using mucin culture medium in 2004. It is widely colonized in the human intestine and is one of the most abundant members of human intestinal microorganisms, accounting for about 3%-5% of the total.

[0006] At present, most studies on Akk are related to obesity. The abundance of Akk in mice fed a high-fat diet is significantly different from that in normal mice. Schneeberger et al. have shown that intestinal bacteria undergo specific changes during obesity, and the abundance of Akk decreases under high-fat diet (HFD). As obesity worsens, Akk levels are strongly positively correlated with almost all parameters involved in fatty acid oxidation and fat browning. In contrast, during this process, it was also found that Akk levels were negatively correlated with inflammatory markers, lipid synthesis, and several plasma markers of insulin resistance, cardiovascular risk, and obesity.

[0007] As the duration of dietary treatment of mice increased, the abundance of Akk gradually decreased, and this effect was exacerbated in HFD. During the aging process, the composition of intestinal microorganisms undergoes major changes, such as a decrease in beneficial bacteria and an increase in pathogenic bacteria. Among them, the significant decrease in Akk has attracted widespread attention. Studies have shown that the distribution of Akk is related to age and decreases with age. Compared with adults, the colonization of this bacterium in the intestine of the elderly is significantly reduced. Akk is also closely related to premature aging in animals, and Akk plays the role of a probiotic in it, which can prolong the lifespan of premature animals. In addition, studies have shown that Akk also plays an important role in the treatment of patients with inflammatory bowel disease, type 2 diabetes, alcoholic fatty liver, host immune response, Parkinson's disease, etc. However, the role of black fungus melanin in improving iron deficiency anemia has not been reported. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide an intestinal microorganism that is "food and medicine of the same origin", can be taken for a long time, has high safety performance, good biocompatibility, and has small toxic and side effects, and its application in the treatment of iron deficiency anemia with black fungus melanin.

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0010] On the one hand, the intestinal microorganisms provided by the present invention are used in drugs or health products participating in the combined treatment of iron deficiency anemia with black fungus melanin.

[0011] Furthermore, each ml of the medicine or health product contains 30 mg of black fungus melanin and 10 8~10 cfu intestinal microorganisms.

[0012] Furthermore, each ml of the medicine or health product contains 10 9 cfu intestinal microorganisms.

[0013] Furthermore, the intestinal microorganism is Akkermansia.

[0014] Furthermore, the preparation method of black fungus melanin comprises:

[0015] (1) Crush the black fungus and extract the powder in concentrated hydrochloric acid;

[0016] (2) adding sodium hydroxide to step (1) to adjust the pH value, and centrifuging to obtain the supernatant;

[0017] (3) adding hydrochloric acid to step (2) to adjust the pH value, allowing to stand, and collecting the precipitate;

[0018] (4) The precipitate in step (3) is placed in concentrated hydrochloric acid, the precipitate is taken out in a water bath, and freeze-dried to obtain the precipitate.

[0019] Furthermore, in the step (1), 25 g of black fungus powder was dissolved in 500 mL of 3 mol / L concentrated hydrochloric acid and extracted in a water bath at 70° C. for 1.5 h.

[0020] Furthermore, in step (2), the pH value is adjusted to 12 with sodium hydroxide, and the mixture is centrifuged at 4000 rpm for 10 min; and in step (3), the pH value is adjusted to 2 with hydrochloric acid.

[0021] Furthermore, in step (4), the precipitate is placed in 150 mL of 7 mol / L concentrated hydrochloric acid, boiled in a boiling water bath for 2 h, centrifuged at 10,000 rpm for 20 min, and freeze-dried to obtain the product.

[0022] The present invention has the following beneficial effects:

[0023] The black fungus melanin in the present invention can effectively improve the blood routine indexes, iron metabolism indexes, inflammatory indexes, and repair colon damage of IDA mice. After the bacterial flora is eliminated, the melanin improves the functional deficiency of IDA; the intestinal microorganisms transplanted into anemic mice cannot improve the symptoms of IDA; after the intestinal microorganisms improved by melanin are transplanted, it is found that the black fungus melanin has restored the medicinal effect. The results showed that the pharmacodynamic pathway of black fungus melanin in improving IDA requires intestinal microorganisms; high-throughput sequencing determined that Akkermansia was the key intestinal microorganism affecting the improvement of IDA by black fungus melanin; black fungus melanin significantly increased the abundance of Akkermansia muciniphila in the intestine of IDA mice, and in vitro experiments showed that black fungus melanin had a positive promoting effect on it within a certain concentration range; gene expression verified that black fungus melanin utilized exogenous melanin iron by affecting the Feo iron transport system in Akkermansiamuciniphila at the optimal concentration; at the same time, oral administration of Akkermansiamuciniphila and melanin could significantly restore the blood indicators, iron metabolism indicators, and colon inflammation indicators of anemic mice; in short, Akkermansia and melanin in the present invention had a synergistic effect on each other, and oral administration could significantly improve the symptoms of iron deficiency anemia in mice, and it is expected to be used in the treatment of iron deficiency anemia in a targeted manner and further replace traditional iron supplements to become the next generation of new supplementary prebiotic preparations, providing a potential new strategy for the treatment of patients with iron deficiency anemia in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The blood routine indexes of mice in each model group in Example 1 of the present invention were measured;

[0025] Figure 2 HE staining images of colon tissues of mice in each model group in Example 1 of the present invention;

[0026] Figure 3 The PCA analysis diagram and Venn diagram of intestinal microorganisms of mice in each model group in Example 2 of the present invention;

[0027] Figure 4 The composition ratios of the main phyla and genus levels of the bacterial flora in the fecal samples of each model group in Example 2 of the present invention;

[0028] Figure 5 The growth curve (48h) and the viable count (20h) of Akkermansia muciniphila at different melanin concentrations in Example 3 of the present invention are shown in FIG. 10 8 );

[0029] Figure 6The effect of black fungus melanin (100 μg / mL) on iron transport-related genes in Akkermansia muciniphila in Example 3 of the present invention;

[0030] Figure 7 is the relative content of Akkermansia muciniphila in the feces of each group of mice in Example 3 of the present invention;

[0031] Figure 8 The effect of Akkermansia muciniphila on tissue iron content in the model group mice in Example 3 of the present invention;

[0032] Fig. 9 The effect of Akkermansia muciniphila on the expression of iron transport genes in the model group mice in Example 3 of the present invention;

[0033] Fig.10 The blood routine indexes of mice in each model group in Example 4 of the present invention were measured;

[0034] Fig.11 This is the HE staining image of the colon tissue of mice in each model group in Example 4 of the present invention. DETAILED DESCRIPTION

[0035] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments. However, the present invention is by no means limited to these examples. The following is only a preferred embodiment of the present invention, which is only used to explain the present invention and cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that any modification, equivalent substitution and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0036] Unless otherwise specified, the components used in the present invention are all commercially available products, and the experimental test conditions are not particularly specified, and are all conventional technical operations in the art.

[0037] Among them, Akkermansia muciniphila strain number: CICC 24917;

[0038] Black fungus was provided by Dongning Black Fungus Industrial Base, strain collection number: HDXZ-1.

[0039] The black fungus melanin used in the present invention is obtained by a method of hydrochloric acid hydrolysis-filtration-alkali dissolution-acid precipitation.

[0040] The specific preparation method is as follows: after the black fungus is crushed, it is passed through an 80-mesh sieve, 25 g of black fungus powder is taken in 500 mL of 3 mol / L concentrated hydrochloric acid, and extracted in a water bath at 70°C for 1.5 h, and then sodium hydroxide is added to adjust the pH value to 12, and the mixture is centrifuged at 4000 rpm for 10 min after stirring. 3 mol / L hydrochloric acid is added to the supernatant to adjust the pH value to 2, and after standing for a period of time, the mixture is centrifuged at 4000 rpm for 20 min, and the precipitate is obtained to obtain the crude melanin. The crude product is placed in 150 mL-7 mol / L concentrated hydrochloric acid, in a boiling water bath for 2 h, and centrifuged at 10000 rpm for 20 min. The obtained precipitate is washed twice with chloroform, ethyl acetate and ethanol, washed 7 times with deionized water, and the solid is freeze-dried to obtain purified black fungus melanin.

[0041] In view of the safety problems such as side effects during long-term use of conventional iron supplements in the prior art, the present invention provides an intestinal microorganism that is both medicinal and edible, has good safety performance, and has few toxic and side effects and can significantly improve iron deficiency anemia, and is used in the treatment of iron deficiency anemia with black fungus melanin.

[0042] Example 1 Experiment on improving IDA mouse model by intestinal microorganisms in collaboration with black fungus melanin

[0043] (1) IDA mouse modeling and mouse grouping

[0044] The experimental animals used in this experiment were all Kunming male mice aged 24 days. The mice were adapted for 1 week and randomly divided into a normal control group (Control, fed with normal feed and distilled water) and an iron deficiency anemia group (IDA, fed with low-iron feed and deionized water). Plastic cages and non-iron cage covers must be used throughout the experiment to prevent exogenous iron from interfering with the test results. After feeding for 2 weeks, blood was collected from the tail, and blood indicators such as mouse Hb were measured using an automatic blood cell analyzer. The model was successfully established when the Hb mass concentration of the experimental mouse was no higher than 100 g / L.

[0045] The mice were randomly divided into 6 groups, with 15 mice in each group: control group (Control), iron deficiency anemia group (IDA), iron deficiency anemia plus melanin group (IDA+M), iron deficiency anemia plus antibiotic cocktail plus melanin group (IDA+ABX+M), fecal microbiota transplantation from the iron deficiency anemia group to the iron deficiency anemia plus melanin group after the microbiota was eliminated [IDA-IDA(ABX)+M], and fecal microbiota transplantation from the iron deficiency anemia plus melanin group to the iron deficiency anemia plus melanin group after the microbiota was eliminated [IDA+M-IDA(ABX)+M].

[0046] The Control and IDA groups were gavaged with saline, the IDA+M group was gavaged with melanin for 3 weeks (gavage dose: 200 mg / kg·d), the IDA+ABX+M group was gavaged with black fungus melanin for 3 weeks after the bacterial flora was eliminated for 1 week (gavage dose of antibiotic cocktail: neomycin sulfate, metronidazole, ampicillin are all 200 mg / kg·d, vancomycin is 100 mg / kg·d; melanin dose: 200 mg / kg·d), and the IDA+ABX+M group was continuously gavaged with antibiotic cocktail for 1 week to eliminate intestinal microorganisms, and then the mixed antibiotics (ampicillin, neomycin sulfate and metronidazole are all 1 g / L, vancomycin 0.5 g / L) were freely administered in drinking water (the Control group was fed with normal feed and distilled water throughout the experimental period; the other groups were fed with low-iron feed and deionized water). To avoid exogenous iron contamination, the experiment used plastic cages and non-iron cage covers.

[0047] The fecal microbiota transplantation groups were divided into the following groups: the fecal microbiota transplantation in the iron deficiency anemia group was given to the iron deficiency anemia plus melanin group after the microbiota was eliminated [IDA-IDA(ABX)+M]; the fecal microbiota transplantation in the iron deficiency anemia plus melanin group was given to the iron deficiency anemia plus melanin group after the microbiota was eliminated [IDA+M-IDA(ABX)+M]. One week after the bacterial flora of the IDA-IDA(ABX)+M group was eliminated, the mice were gavaged with fecal bacteria of the IDA group and black fungus melanin for 3 weeks (the gavage dose of antibiotic cocktail: the same as above; the gavage dose of fecal bacteria: 200 μL; the gavage dose of melanin: 200 mg / kg·d). One week after the bacterial flora of the IDA+M-IDA(ABX)+M group was eliminated, the mice were gavaged with fecal bacteria of the IDA+M group and black fungus melanin for 3 weeks (the gavage dose of antibiotic cocktail: the same as above; the gavage dose of fecal bacteria: 200 μL, the gavage dose of melanin: 200 mg / kg·d). Both groups of mice were always fed with low-iron feed and deionized water.

[0048] (2) Elimination of mouse intestinal microorganisms

[0049] After the IDA model was successfully established, antibiotic cocktail treatment was performed. The drugs were vancomycin 100 mg / kg, neomycin sulfate, metronidazole and ampicillin 200 mg / kg each. In the early stage, it was gavaged once a day for 7 consecutive days to eliminate intestinal microorganisms. Feces were collected after 7 days, and the qRTPCR results were used as indicators to detect the elimination effect. In the later stage, ampicillin, neomycin sulfate and metronidazole were freely administered in drinking water at 1 g / L each, and vancomycin 0.5 g / L to prevent rapid recovery of the flora;

[0050] (3) Fecal microbiota transplantation

[0051] Take appropriate amount of feces from donor mice (IDA and IDA+M):

[0052] (1) Add stool sample (1 g) to a centrifuge tube, then add PBS buffer (2 mL) to dissolve the stool, and centrifuge at 700 r / min for 5 min.

[0053] (2) Pour the upper solution into a new centrifuge tube;

[0054] (3) Add 1 mL of PBS to the remaining residue and resuspend at 700 r / min for 5 min;

[0055] (4) mixing the secondary stool suspension and packaging;

[0056] (5) During intragastric administration, 200 μL of the solution was drawn at a time and 100 μL was applied to the back of the mouse three times.

[0057] (4) Routine blood test of mice

[0058] Blood was collected from the eyeballs of the experimental model mice, and 20 μL of whole blood was added to the bullet containing the diluent. After mixing, various indicators were detected using a blood cell automatic analyzer, and the changes in the content of Hb, MCH, MCHC and other indicators of the two model mice were recorded;

[0059] (5) Mouse colon histopathology

[0060] After the test period, the test mice were quickly dissected, the colon segments were cut, rinsed with cold saline, fixed in formaldehyde, prepared into paraffin blocks, cut and stained with hematoxylin-eosin (HE), and then the changes in colon tissue damage were observed under a microscope. Inflammation was determined by measuring the degree of inflammatory cell infiltration, epithelial damage and crypt loss. The specific steps are as follows:

[0061] The colon was fixed by immersion in neutral formalin solution for 5 h; dehydrated with 85% ethanol for 90 min, 95% ethanol for 40 min, and anhydrous ethanol for 15 min to remove water from the tissue block; the block was placed in xylene opacifier for 10 min to make it transparent. This step was repeated twice.

[0062] The transparent tissue block was placed in paraffin, immersed and embedded; the embedded block was placed in a cutter and cut into 4 μm thickness; the slices were baked at 70°C for 45 min.

[0063] Dewaxing with xylene for 10 min, repeated three times, and then rehydrated and washed with ethanol of different concentrations; Harris hematoxylin staining for 5 min, washing with hematoxylin stain in running water for 1 min; differentiation with 0.5% hydrochloric acid ethanol for 10 s, washing with running water for 15 min; reblueing with 50℃ hot water; restaining with eosin stain for 1 min, washing twice with distilled water; elution with 85% ethanol, 95% ethanol, and anhydrous ethanol for 1 min each; dehydration by washing with xylene for 2 min to make it transparent; sealing with neutral gum; observation and photography under a microscope.

[0064] according to Figure 1 As shown in the figure, after 3 weeks of gavage with black fungus melanin, the Hb, MCH, and MCHC of mice in the IDA+M group were significantly increased compared with those in the IDA group, reaching normal levels. After the bacterial flora of the IDA+ABX+M group was eliminated, gavage with black fungus melanin could not effectively improve the anemia symptoms of IDA mice. Compared with the IDA-IDA(ABX)+M group, which was transplanted with anemia mouse feces, the Hb, MCH, and MCHC of mice in the IDA+M-IDA(ABX)+M group, which was transplanted with black fungus melanin, were significantly increased and returned to normal levels.

[0065] according to Figure 2 As shown in the figure, after the bacterial flora was eliminated, compared with the IDA+M group, the mucosal layer of the colon tissue of the mice in the IDA+ABX+M group was atrophic, the epithelial cells of the mucosal layer were defective, the crypts were irregularly arranged, and there were different degrees of necrosis and defects. Compared with the IDA-IDA(ABX)+M group, which was transplanted with anemic mouse feces, the epithelial cells of the colon tissue of the mice in the IDA+M-IDA(ABX)+M group, which was transplanted with black fungus melanin, were arranged more neatly, the crypts were arranged more regularly, and the lamina propria remained intact; the colon tissue of the mice in the IDA+M-IDA(ABX)+M group was similar to that of the Control group and the IDA+M group in terms of overall structure; this shows that intestinal microorganisms play a vital role in the process of melanin improving IDA.

[0066] Example 2 Determination of key components in intestinal microorganisms

[0067] (1) Mouse modeling and grouping

[0068] Forty-five male Kunming mice with an initial weight of 14.0 ± 1.7 g and 24 days of age were randomly divided into three groups, with 15 mice in each group, namely the normal control group (Control), IDA group, and IDA+M group (melanin treatment group). The experiment began after one week of adaptive feeding. The IDA group and IDA+M group were fed a low-iron diet and had free access to deionized water. After two weeks of modeling, the mice were gavaged when they gained weight to 30 g. During the experimental treatment period, the IDA+M group was gavaged with melanin (concentration of 200 mg / kg·d), fed a low-iron diet, and drank deionized water, while the IDA group was gavaged with deionized water and fed a low-iron diet. The Control group was fed a normal diet and free access to distilled water throughout the experimental period, and the experiment lasted for 3 weeks. To avoid exogenous iron contamination, plastic cages and non-iron cage covers were used in the experiment. Since the beginning of the experiment, mice in each group were weighed every week and recorded.

[0069] (2) Sample collection

[0070] 24 h after the last gavage, mouse feces were collected, 0.5 g from each group, placed in 2 ml cryovials, and stored at -80 °C for subsequent microbial high-throughput sequencing analysis.

[0071] (3) High-throughput sequencing of mouse intestinal microbiota

[0072] About 0.2 g of mouse feces samples were taken, and total DNA was extracted using Stool Genomic DNA Extraction Kit (TIANGEN, Shanghai, China) in strict accordance with the instructions. The concentration and purity of the extracted DNA were detected using NanoDrop ultra-micro spectrophotometer (N50, China). Qualified DNA samples were stored in cryovials, frozen in liquid nitrogen, and then placed in dry ice and sent to the sequencing company. The samples were commissioned to Shanghai Meiji Biomedical Technology Co., Ltd. to use Illumina Miseq PE250 / PE300 for 16s rDNA sequencing, and the bacterial flora of each group of samples were analyzed and identified at the phylum and genus levels.

[0073] according to Figure 3 As shown in the figure, PCA analysis showed that the Control group and the IDA+M group clustered together and were significantly separated from the IDA group, indicating that the intestinal microbial structures of the Control group and the IDA+M group were highly similar, and both were significantly different from the flora structure of the IDA group, indicating that melanin has an improving effect on the intestinal microbial structure of anemic mice. Venn diagrams can be used to count the number of common and unique OTUs in samples, and intuitively show the similarity and overlap of the number composition of OTUs in different environmental samples. Figure 3B shows that there are 130 common OTUs in the three groups of samples, and 174 unique OTUs in the IDA group, which is the least among the three groups of samples. After gavage with melanin, the number of unique OTUs increased to 233. This shows that melanin can increase the number of unique OTUs in the intestinal microorganisms of IDA mice and increase the diversity of the flora.

[0074] according to Figure 4 As shown, supplementation with black fungus melanin can alleviate the changes in the main dominant bacterial phyla caused by IDA, increase the abundance of beneficial bacteria, reduce the abundance of harmful bacteria, and restore them to normal levels. This shows that intestinal microorganisms play an important role in participating in the process of black fungus melanin improving anemia. At the genus level, the abundance of Akkermansia muciniphila in IDA mice was significantly reduced, and supplementation with black fungus significantly increased the abundance of Akkermansia muciniphila, indicating that Akkermansiamuciniphila plays an important role in the process of black fungus melanin improving IDA.

[0075] Example 3 Akkermansia assists black fungus melanin in promoting iron absorption and transport

[0076] (1) Akkermansia in vitro culture experiment

[0077] Akkermansia was anaerobically cultured at 37°C for 3-5 days in the above-mentioned normal culture medium, iron-depleted culture medium, and iron-depleted culture medium plus different concentrations of Auricularia auricularia melanin (1 μg / mL, 10 μg / mL, 100 μg / mL, 1000 μg / mL). The growth curve of Akk in the above-mentioned culture medium was detected by an ELISA instrument and the number of viable bacteria was calculated.

[0078] according to Figure 5 As shown in the figure, when the amount of melanin added is too low, it will not promote the growth of bacteria. When the concentration of melanin added is too high, it will inhibit the growth of bacteria. When the amount of melanin added is 100 μg / mL, it is the optimal amount of melanin added for in vitro intervention of Akkermansia muciniphila, and the growth of bacteria is the best at this time.

[0079] This indicates that melanin has a positive promoting effect on Akk in vitro within a certain concentration range.

[0080] (2) Determination of the effect of black fungus melanin on iron transport in Akkermansia muciniphila

[0081] Akk grown to the logarithmic phase in normal culture medium was transferred to normal culture medium, iron-depleted culture medium, and iron-depleted culture medium plus black fungus melanin. After 4 h, the qRT-PCR method was used to detect the changes in the mRNA levels of iron transport-related indicators (FeoA, FeoB, 4Fe-4S, Ferredoxin, and Ferroxidase) of Akk in different culture media.

[0082] according to Figure 6 As shown in the figure, after adding black fungus melanin, the expression levels of FeoA (p<0.001), FeoB (p<0.05), 4Fe-4S (p<0.01), Ferredoxin (p<0.05), and Ferroxidase (p<0.05) in the iron-free group were significantly increased compared with the iron-free group. This shows that Akk uses exogenous melanin iron through the Feo transport system.

[0083] (3) Determination of the relative content of Akkermansia muciniphila in mouse feces

[0084] Fresh feces were collected for DNA extraction and detection, and Akk-specific primers (upstream primer AM1: 5'-CAGCACGTGAAGGTGGGGAC-3', downstream primer AM2: 5'-CCTTGCGGTTGGCTTCAGAT-3') were used for qRT-PCR detection.

[0085] according to Figure 7 As shown, the relative content of Akk in the feces of the IDA group and the IDA+ABX+M group was significantly lower than that of the control group, and the relative content of Akk in the feces of the group using the fecal bacteria of the IDA+M group as the donor was significantly higher than that of the group using the fecal bacteria of the IDA group as the donor. The results showed that intragastric administration of black fungus melanin helped to increase the abundance of Akk in IDA mice.

[0086] Example 4 Experiment on improving IDA mouse model with Akkermansia synergistic with black fungus melanin

[0087] (1) Mouse modeling and grouping

[0088] Iron deficiency anemia plus antibiotic cocktail plus melanin group (IDA+ABX+M), iron deficiency anemia plus antibiotic cocktail plus melanin plus key intestinal microbiome (IDA+ABX+M+Akk). The IDA+ABX+M group was gavaged with black fungus melanin for 3 weeks after the bacterial flora was eliminated for 1 week (antibiotic cocktail gavage dose: the same as above; melanin dose: 200 mg / kg·d), and the IDA+ABX+M+Akk group was gavaged with Akk and black fungus melanin for 3 weeks after the bacterial flora was eliminated for 1 week (antibiotic cocktail gavage dose: the same as above; Akk bacteria concentration: 1×10 9 cfu / mL, 200 μl per gavage; melanin concentration was 30 mg / ml, 200 μl per gavage, gavage dose: 200 mg / kg·d). Both groups were always fed with low-iron feed and deionized water.

[0089] (2) Determination of tissue iron index in mouse model

[0090] Tissue iron: For detailed methods for determining the iron content in mouse liver, spleen, and duodenum, please refer to the instructions in the tissue iron concentration detection kit provided by Beijing Solebow Biotechnology Co., Ltd. and perform the determination strictly in accordance with the instructions.

[0091] according to Figure 8 As shown, compared with the IDA+ABX+M group, the iron content in the liver, spleen and duodenum of mice in the IDA+ABX+M+Akk group was significantly increased, by 69.87% (p<0.01), 71.52% (p<0.01) and 83.68% (p<0.05), respectively. The results showed that simultaneous oral administration of Akk and melanin could significantly restore the iron content in the tissues of IDA mice, indicating that Akk helps to assist melanin in improving the symptoms of IDA.

[0092] (3) Expression determination of iron transport genes in mouse models

[0093] The mRNA contents of iron transport-related indicators (DMT1, FPN1, Hp) were determined by fluorescence quantitative PCR (qRT-PCR).

[0094] according to Fig. 9 As shown in the figure, compared with the IDA+ABX+M group, the mRNA levels of DMT1 (p<0.05), FPN1 (p<0.05), and Hp (p<0.05) in the IDA+ABX+M+Akk group were significantly increased. The results showed that simultaneous oral administration of Akk and melanin could significantly restore the iron metabolism of IDA mice, indicating that Akk plays an important role in the improvement of IDA by melanin.

[0095] (4) Blood indexes and histological determination in mouse models

[0096] The model mice in each group were killed by dislocating the neck after blood was taken from the eyeballs, and blood and tissue samples were collected from each group of mice for routine blood analysis and colon tissue HE staining. Other experimental operating conditions, dosage of each component and index determination method were consistent with Example 1.

[0097] like Fig.10 As shown, when the black fungus melanin and Akkermansia were supplemented simultaneously and compared with the single gavage of black fungus melanin, the Hb, RBC, HCT, and MCV of mice in the IDA+ABX+M+Akk group were significantly increased compared with the IDA+ABX+M group, which were significantly increased by 95.37% (p<0.001), 79.50% (p<0.001), 94.84% (p<0.001), and 7.39% (p<0.05), respectively. The results showed that the simultaneous gavage of Akk and melanin can significantly restore the blood routine of IDA mice, indicating that Akk helps to assist melanin in improving the symptoms of IDA.

[0098] like Fig.11 As shown in the figure, the colon tissues of mice in the IDA+ABX+M+Trp group and the IDA+ABX+M group were stained with HE and observed, and it was found that the colon tissue structure of mice in the IDA+ABX+M group was damaged, the crypt structure and goblet cells were distorted, the mucosal layer of the tissue was atrophied, the arrangement of mucosal epithelial cells was missing, and the upper mucosa and barrier were deformed. In contrast, the colon epithelial cells of mice in the IDA+ABX+M+Trp group were arranged tightly and orderly, with a large number of well-structured crypts and villi, long and clear crypts and villi, and no atrophy of the mucosal layer or defect in the arrangement of epithelial cells, indicating that tryptophan participates in the improvement of black fungus melanin on the colon lesions of IDA mice.

[0099] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The application of intestinal microorganisms in the combined treatment of iron deficiency anemia with black fungus melanin drugs or health products.

2. The use of intestinal microorganisms according to claim 1 in drugs or health products for the combined treatment of iron deficiency anemia with black fungus melanin, characterized in that: The medicine or health product contains 30 mg black fungus melanin and 10 8~ 10 cfu intestinal microorganisms.

3. The use of intestinal microorganisms according to claim 2 in a drug or health product for the combined treatment of iron deficiency anemia with black fungus melanin, characterized in that: Each ml of the medicine or health product contains 10 9 cfu intestinal microorganisms.

4. The use of intestinal microorganisms according to claim 3 in drugs or health products for the combined treatment of iron deficiency anemia with black fungus melanin, characterized in that: The intestinal microorganism is Akkermansia.

5. The use of intestinal microorganisms according to claim 1 in a drug or health product for the combined treatment of iron deficiency anemia with black fungus melanin, characterized in that: The preparation method of black fungus melanin comprises: (1) Crush the black fungus and extract the powder in concentrated hydrochloric acid; (2) adding sodium hydroxide to step (1) to adjust the pH value, and centrifuging to obtain the supernatant; (3) adding hydrochloric acid to step (2) to adjust the pH value, allowing to stand, and collecting the precipitate; (4) The precipitate in step (3) is placed in concentrated hydrochloric acid, the precipitate is taken out in a water bath, and freeze-dried to obtain the precipitate.

6. The use of the intestinal microorganisms according to claim 5 in the drug or health product for the combined treatment of iron deficiency anemia with black fungus melanin, characterized in that: In the step (1), 25 g of black fungus powder was dissolved in 500 mL of 3 mol / L concentrated hydrochloric acid and extracted in a water bath at 70° C. for 1.5 h.

7. The use of the intestinal microorganisms according to claim 5 in a drug or health product for the combined treatment of iron deficiency anemia with black fungus melanin, characterized in that: In the step (2), the pH value is adjusted to 12 with sodium hydroxide, and the mixture is centrifuged at 4000 rpm for 10 min. In the step (3), the pH value is adjusted to 2 with hydrochloric acid.

8. The use of the intestinal microorganisms according to claim 5 in a drug or health product for the combined treatment of iron deficiency anemia with black fungus melanin, characterized in that: In the step (4), the precipitate is placed in 150 mL of 7 mol / L concentrated hydrochloric acid, boiled in a boiling water bath for 2 h, centrifuged at 10,000 rpm for 20 min, and freeze-dried to obtain the precipitate.

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