阅读说明：本技术 一种提高肠道益生菌在口服递送过程中存活率的方法 (Method for improving survival rate of intestinal probiotics in oral delivery process ) 是由 曹芳芳 毛峥伟 陈小元 于 2021-10-16 设计创作，主要内容包括：本发明提供一种提高肠道益生菌在口服递送过程中存活率的方法,包括：使用含有过渡金属的疏水纳米材料,并利用过渡金属与肠道益生菌表面的糖和蛋白形成配位作用,进而通过自组装包裹肠道益生菌,阻隔消化液中分子量大且亲水的消化酶与肠道益生菌的接触。本发明还提供一种可抵抗消化酶的肠道益生菌,由肠道益生菌表面通过配位作用吸附含有过渡金属的疏水纳米材料构成,疏水纳米材料包裹益生菌形成疏水保护层。本发明还提供制备可抵抗消化酶的肠道益生菌的方法。本发明可抵抗消化酶的肠道益生菌克服了传统口服益生菌的缺点,显著提升了对结肠炎的治疗能力。(The present invention provides a method of increasing the survival rate of intestinal probiotics during oral delivery, comprising: the hydrophobic nano material containing transition metal is used, the transition metal and sugar and protein on the surface of the intestinal probiotics form coordination, and then the intestinal probiotics are wrapped through self-assembly, so that the contact between digestive enzymes with large molecular weight and hydrophilicity in digestive juice and the intestinal probiotics is blocked. The invention also provides intestinal probiotics capable of resisting digestive enzymes, which is formed by adsorbing hydrophobic nano materials containing transition metals on the surfaces of the intestinal probiotics through coordination, and the hydrophobic nano materials wrap the probiotics to form a hydrophobic protective layer. The invention also provides a method for preparing intestinal probiotics capable of resisting digestive enzymes. The intestinal probiotics capable of resisting digestive enzymes overcomes the defects of the traditional oral probiotics, and remarkably improves the treatment capacity of colitis.)

1. A method of increasing the survival rate of intestinal probiotics during oral delivery comprising: the preparation method comprises the steps of using a hydrophobic nano material containing transition metal, forming a coordination effect by utilizing the transition metal and sugar and protein on the surface of the intestinal probiotics, wrapping the intestinal probiotics through self-assembly, and blocking the contact of digestive enzymes with large molecular weight and hydrophilicity in digestive juice and the intestinal probiotics.

2. The method of claim 1, wherein: the hydrophobic nano material containing transition metal is a carbon-based nano material doped with transition metal elements and N elements or O elements; most preferred are carbon materials containing transition metal atom-N4 or transition metal atom-O, pyridine nitrogen, pyrrole nitrogen, and graphene structures. The transition metal element is preferably any one or a mixture of more than two of copper, iron, manganese, cobalt, zinc, cerium or gold; more preferably any one or a mixture of two or more of copper, iron, zinc, or gold; most preferably any of copper, iron or gold.

3. The method of claim 1, wherein: the hydrophobic nano material containing transition metal is obtained by high-temperature calcination of a precursor substance of transition metal wrapped by a metal/organic framework compound; the transition metal element is preferably any one or a mixture of more than two of copper, iron, manganese, cobalt, zinc, cerium or gold; more preferably any one or a mixture of two or more of copper, iron, zinc, or gold; most preferably any of copper, iron or gold; the high-temperature calcination temperature is 800-1800 ℃.

4. Intestinal probiotics capable of resisting digestive enzymes is formed by adsorbing hydrophobic nano materials containing transition metals on the surfaces of the intestinal probiotics through coordination, and the hydrophobic nano materials wrap the probiotics to form a hydrophobic protective layer; the intestinal probiotics is preferably selected from one or a mixture of more than two of bifidobacteria, lactobacillus, clostridium butyricum or yeast; the bifidobacterium is preferably one or a mixture of more than two of bifidobacterium longum, bifidobacterium adolescentis and bifidobacterium breve.

5. Intestinal probiotic bacteria according to claim 4, characterized in that: the hydrophobic nano material containing transition metal is a carbon-based nano material doped with transition metal elements and N elements or O elements; most preferred are carbon materials containing transition metal atom-N4 or transition metal atom-O, pyridine nitrogen, pyrrole nitrogen, and graphene structures; the transition metal element is preferably any one or a mixture of more than two of copper, iron, manganese, cobalt, zinc, cerium or gold; more preferably any one or a mixture of two or more of copper, iron, zinc, or gold; most preferably any of copper, iron or gold.

6. Intestinal probiotic bacteria according to claim 4, characterized in that: the hydrophobic nano material containing transition metal is obtained by high-temperature calcination of a precursor substance of transition metal wrapped by a metal/organic framework compound; preferably, the precursor material of the transition metal coated by the metal/organic framework compound comprises 94-99% of the main material of the metal/organic framework compound and 1-6% of the transition metal element by weight percent; the transition metal element is preferably any one or a mixture of more than two of copper, iron, manganese, cobalt, zinc, cerium or gold; more preferably any one or a mixture of two or more of copper, iron, zinc, or gold; most preferably any of copper, iron or gold.

7. A method for preparing a gut probiotic resistant to digestive enzymes comprising the steps of:

1) preparing a hydrophobic nano material containing a transition metal element, and dispersing the hydrophobic nano material into a buffer solution to prepare a solution A;

2) culturing intestinal probiotics, centrifugally collecting, and re-dispersing the intestinal probiotics into a buffer solution to prepare a solution B;

3) mixing and oscillating the solution A obtained in the step 1) and the solution B obtained in the step 2); and (4) centrifugally collecting the intestinal probiotics and washing to obtain the intestinal probiotics protected by the hydrophobic nano material, namely the intestinal probiotics capable of resisting digestive enzymes.

8. The method of claim 7, wherein: the hydrophobic nano material containing the transition metal element in the step 1) is a carbon-based nano material doped with the transition metal element and an N element or an O element; most preferred are carbon materials containing transition metal atom-N4 or transition metal atom-O, pyridine nitrogen, pyrrole nitrogen, and graphene structures; the transition metal element is preferably any one or a mixture of more than two of copper, iron, manganese, cobalt, zinc, cerium or gold; more preferably any one or a mixture of two or more of copper, iron, zinc, or gold; most preferably any of copper, iron or gold.

9. The method of claim 7, wherein: the hydrophobic nanomaterial containing transition metal in the step 1) is obtained by high-temperature calcination of a precursor substance of transition metal wrapped by a metal/organic framework compound; the preferable precursor substance is obtained by doping transition metal nanoparticles in a metal/organic framework compound; more preferably, the precursor comprises 94-99 wt% of metal/organic framework compound and 1-6 wt% of transition metal element. The preferable high-temperature calcination temperature is 800-1800 ℃; the transition metal element is preferably any one or a mixture of more than two of copper, iron, manganese, cobalt, zinc, cerium or gold; more preferably any one or a mixture of two or more of copper, iron, zinc, or gold; most preferably any of copper, iron or gold.

10. The method of claim 7, wherein: step 3) when the solution A obtained in the step 1) and the solution B obtained in the step 2) are mixed, the concentration of the intestinal probiotics in the mixed solution is controlled to be 0.5-50 multiplied by 10⁶CFU/mL, the concentration of the hydrophobic nano material is 0.1-5 mg/mL.

Technical Field

The present invention relates to a method for improving the survival rate of intestinal probiotics during oral delivery.

Background

Inflammatory Bowel Disease (IBD) affects the health of millions of people each year, severely affecting the quality of life of patients. IBD is a type of chronic inflammation of the intestinal tract, which is characterized by recurrent attacks, mainly including the two phenotypes of Ulcerative Colitis (UC) and Crohn's Disease (CD). The prevalence of IBD in developed areas such as North America and Europe is high, about 5 per mill of the general population; the prevalence of IBD in continental china is about 2.2% o and may be underestimated.

Both the direct and indirect costs associated with IBD are high. About 250 million people see a diagnosis due to IBD every year, and the medical cost generated directly exceeds $ 50 million. While the indirect costs associated with IBD are higher. IBD is very likely to cause a decrease in quality of life and efficiency of work, or even depression, due to its long course and frequent recurrent episodes, and these costs are difficult to measure in money, and even affect the patient's family members. Studies have shown that IBD patients have a higher prevalence of depression and anxiety than other patients with chronic diseases, and less than one third of IBD patients have both depression and anxiety.

At present, the pathogenesis of IBD is not clear, but more and more researches show that the pathogenesis of IBD is related to factors such as intestinal flora imbalance, immune response disorder, intestinal barrier damage and the like. The intestinal flora is an important part of the human body, and the interaction between intestinal microorganisms and the intestinal mucosal immunity influences the initiation and regulation of immune response. Disturbance of the intestinal flora may lead to excessive or inappropriate immune responses, which may result in damage to the intestinal mucosa. With the popularization of high-throughput sequencing technology and the development of bioinformatics analysis means, more and more researches show that the imbalance of intestinal microecological systems and the reduction of the number of beneficial intestinal flora are common in patients with inflammatory bowel diseases.

Bifidobacteria are gram-positive, anaerobic and mycobacterial bacteria. The bifidobacterium is used as a dominant bacterium in human intestinal flora, has a symbiotic relationship with a host, and has an important effect on maintaining normal intestinal flora and human health. Bifidobacteria ferment the sugars to lactic acid, which helps to lower the pH of the intestinal tract. Some strains have homologues of enzymes that repair oxidative damage, such as NADH oxidase and NADH peroxidase. Meanwhile, part of the strains also contain proteins and lipids for reversing oxidative damage, such as: thiol peroxidase, alkyl hydroperoxide reductase, peptide methionine sulfoxide reductase, and the like. Thus, certain bifidobacterium strains may be used as probiotics for the intervention treatment of certain intestinal diseases. As indicated in Chinese consensus of experts in clinical application of microecological modulators (2020 edition): the probiotics such as bifidobacterium, lactobacillus acidophilus, lactobacillus rhamnosus, clostridium butyricum and the like are used as adjuvant therapy of light-medium IBD to help to maintain and relieve the state of illness, and have good safety and tolerance. However, bifidobacteria are sensitive to heat, moisture and digestive enzymes and are susceptible to attack by gastric digestive enzymes during oral administration, leading to death or inactivation.

Therefore, there is a need to develop a method for improving the survival rate of intestinal probiotics during oral delivery, so as to exert the intervention treatment efficacy of intestinal diseases of the oral administration route of the intestinal probiotics.

Disclosure of Invention

The invention aims to provide a method for improving the survival rate of intestinal probiotics in an oral delivery process, which can prevent the intestinal probiotics of a human body from being attacked by digestive enzymes in gastrointestinal fluid and reduce the death or inactivation rate of the probiotics in the oral delivery process to the intestinal tract, thereby enhancing the intestinal colonization capacity and improving the health care and disease treatment effects of the probiotics.

It is another object of the present invention to provide a gut probiotic that is resistant to digestive enzymes, maintains a high survival rate during oral delivery, and treats IBD by modulating immune response and gut flora.

It is a further object of the present invention to provide a process for preparing said gut probiotic bacteria resistant to digestive enzymes, which is simple and efficient and has good versatility.

The above object of the present invention is achieved by the following technical solutions:

first, there is provided a method of improving survival of intestinal probiotics during oral delivery comprising: the hydrophobic nano material is used for wrapping the intestinal probiotics through self-assembly, so that the contact between digestive enzymes with large molecular weight and hydrophilicity in digestive juice and the intestinal probiotics is prevented.

The preferable method of the invention is to use a hydrophobic nano material containing transition metal, and utilize the coordination effect formed by the transition metal and sugar and protein on the surface of the intestinal probiotics, so as to wrap the intestinal probiotics through self-assembly and prevent digestive enzymes with large molecular weight and hydrophilicity in digestive juice from contacting the intestinal probiotics.

In a further preferred method of the present invention, the hydrophobic nanomaterial containing transition metal is a carbon-based nanomaterial doped with transition metal elements and N or O elements; most preferred are carbon materials containing transition metal atom-N4 or transition metal atom-O, pyridine nitrogen, pyrrole nitrogen, and graphene structures.

In a more preferred method of the present invention, the hydrophobic nanomaterial containing transition metal is obtained by high temperature calcination of a precursor material of transition metal coated by a metal/organic framework compound. The transition metal element can be selected from any one or a mixture of more than two of copper, iron, manganese, cobalt, zinc, cerium or gold; preferably any one or a mixture of more than two of copper, iron, zinc or gold; most preferably any of copper, iron or gold. The high-temperature calcination temperature is 800-1800 ℃.

In the preferable method, the wrapping of the intestinal probiotics by the hydrophobic nano material through self-assembly is realized by mixing the intestinal probiotics and the hydrophobic nano material in a buffer solution, and the concentration of the intestinal probiotics in the buffer solution is preferably 0.5-50 x 10⁶CFU/mL, the concentration of the hydrophobic nano material in the buffer solution is 0.1-5 mg/mL.

In the method of the present invention, the intestinal probiotic bacteria may be selected from bifidobacterium, lactobacillus, clostridium butyricum or yeast; the bifidobacterium may be selected from one or more of bifidobacterium longum, bifidobacterium adolescentis and bifidobacterium breve.

On the basis, the invention also provides intestinal probiotics capable of resisting digestive enzymes, which is formed by adsorbing hydrophobic nano materials containing transition metals on the surfaces of the intestinal probiotics through coordination, and the hydrophobic nano materials wrap the probiotics to form a hydrophobic protective layer.

In the intestinal probiotics resisting digestive enzymes, which is preferred by the invention, the intestinal probiotics can be selected from bifidobacteria, lactobacilli, clostridium butyricum or yeasts; the bifidobacterium may be selected from one or more of bifidobacterium longum, bifidobacterium adolescentis and bifidobacterium breve.

In the preferable intestinal probiotics for resisting digestive enzymes, the hydrophobic nano material containing transition metal is a carbon-based nano material doped with transition metal elements and N elements or O elements; most preferred are carbon materials containing transition metal atom-N4 or transition metal atom-O, pyridine nitrogen, pyrrole nitrogen, and graphene structures.

In a further preferable intestinal probiotic for resisting digestive enzymes, the hydrophobic nanomaterial containing transition metal is obtained by high-temperature calcination of a precursor substance of transition metal wrapped by a metal/organic framework compound.

In the still further preferable intestinal probiotics for resisting digestive enzymes, the metal/organic framework compound wraps the precursor of the transition metal, and the precursor comprises 94-99% of main material of the metal/organic framework compound and 1-6% of transition metal elements in percentage by weight.

In the intestinal probiotics for resisting digestive enzymes, the transition metal element can be selected from any one or a mixture of more than two of copper, iron, manganese, cobalt, zinc, cerium or gold; preferably any one or a mixture of more than two of copper, iron, zinc or gold; most preferably any of copper, iron or gold.

In addition, the present invention also provides a method for preparing intestinal probiotics capable of resisting digestive enzymes, comprising the following steps:

1) preparing a hydrophobic nano material containing a transition metal element, and dispersing the hydrophobic nano material into a buffer solution to prepare a solution A;

2) culturing intestinal probiotics, centrifugally collecting, and re-dispersing the intestinal probiotics into a buffer solution to prepare a solution B;

3) mixing and oscillating the solution A obtained in the step 1) and the solution B obtained in the step 2); and (4) centrifugally collecting the intestinal probiotics and washing to obtain the intestinal probiotics protected by the hydrophobic nano material, namely the intestinal probiotics capable of resisting digestive enzymes.

In the preferable method for preparing the intestinal probiotics capable of resisting digestive enzymes, the hydrophobic nano material containing the transition metal element in the step 1) is a carbon-based nano material doped with the transition metal element and N element or O element; most preferred are carbon materials containing transition metal atom-N4 or transition metal atom-O, pyridine nitrogen, pyrrole nitrogen, and graphene structures.

In the preferred method for preparing intestinal probiotics capable of resisting digestive enzymes, the transition metal element can be selected from any one or a mixture of more than two of copper, iron, manganese, cobalt, zinc, cerium or gold; preferably any one or a mixture of more than two of copper, iron, zinc or gold; most preferably any of copper, iron or gold.

In the more preferable method for preparing intestinal probiotics capable of resisting digestive enzymes, the hydrophobic nano material containing transition metal in the step 1) is obtained by high-temperature calcination of precursor materials of transition metal wrapped by metal/organic framework compounds. The precursor is obtained by doping transition metal nanoparticles in a metal/organic framework compound, the doping amount of the transition metal can influence the composition structure, the coordination mode and the shape and size of the final transition metal nanoparticles, and in order to better form coordination with the surface of intestinal probiotics and complete self-assembly, in the preferred precursor, the metal/organic framework compound accounts for 94-99% by weight percent, and the transition metal element accounts for 1-6% by weight percent. The high-temperature calcination temperature is 800-1800 ℃.

In order to be more beneficial to the formation of coordination between the hydrophobic nano material and the intestinal probiotics,in the preferable method for preparing the intestinal probiotics capable of resisting digestive enzymes, when the solution A obtained in the step 3) and the solution B obtained in the step 2) are mixed, the concentration of the intestinal probiotics in the mixed solution is controlled to be 0.5-50 multiplied by 10⁶CFU/mL, the concentration of the hydrophobic nano material is 0.1-5 mg/mL.

According to the method for preparing the intestinal probiotics capable of resisting digestive enzymes, sugar and protein on the surface of bacteria and transition metal on the hydrophobic nano material are utilized to form coordination, and the hydrophobic nano material is wrapped on the surface of the bacteria through self-assembly; the general structure and formation process of the bacterium protected by the nano material are shown in figure 1. The nano material wrapped on the surface of the bacteria has a compact and hydrophobic structure, and the digestive enzyme with large molecular weight and hydrophilicity cannot contact the bacteria, so that the attack of the digestive enzyme on the bacteria is blocked, the bacteria can be protected in digestive juice, and the death and inactivation of the bacteria are avoided; after reaching the intestinal tract, hydrophobic small molecular amino acid and fatty acid can permeate the hydrophobic nano material, compete with sugar and protein on the surface of bacteria, and are combined to transition metal, so that the coordination of the hydrophobic nano material and the bacteria is destroyed, and the hydrophobic nano material falls off from the surface of the bacteria. Based on the above principle, intestinal probiotics including bifidobacteria still maintain the original colonization ability and function in intestinal tract after oral delivery, and IBD can be treated by adjusting immune response and intestinal flora.

Compared with the prior art, the preparation method has the advantages of simple process, high preparation speed and good controllability; the adopted nano material has simple structure and high preparation efficiency, can conveniently adjust the size and obstruct the performance of digestive enzyme, and has excellent biocompatibility; the method does not depend on the special structure of the surface of the bacteria, and has good universality; the coating amount of the nano material on the surface of bacteria can be conveniently regulated and controlled, and the nano material is suitable for various probiotics; the nano material for protecting the probiotics has no toxic or side effect on a human body, and does not influence the field planting and the function of the bifidobacteria in the intestinal tract. The technology of using the nano material to protect the intestinal probiotics overcomes the defects of the traditional oral bifidobacteria, and obviously improves the treatment capability on colitis. The bifidobacterium protected by the nano material obtained by the method shows excellent biocompatibility and capability of treating IBD in vivo experiments of mice and beagle dogs, and has strong clinical transformation potential.

Drawings

Fig. 1 is a schematic diagram of the structure and formation process of the intestinal probiotics capable of resisting digestive enzymes prepared by the invention.

FIG. 2 is a TEM image of the protective nanomaterial of example 1.

FIG. 3 is a scanning electron micrograph of Bifidobacterium before and after being protected with nanomaterial in example 1, wherein (a) is Bifidobacterium pristine and (b) is Bifidobacterium protected with nanomaterial.

Fig. 4 is the survival rate of the nanomaterial-protected bifidobacterium obtained in example 1 after treatment in simulated gastric fluid for 1 hour.

Fig. 5 is the effect of the nanomaterial-protected bifidobacterium obtained in example 1 on treatment of DSS-induced UC in mice.

Fig. 6 shows the effect of the nanomaterial-protected bifidobacterium obtained in example 1 on the treatment of UC in beagle dogs caused by acetic acid.

FIG. 7 shows the effect of the nanomaterial-protected Bifidobacterium obtained in example 1 on CD in mice.

Detailed Description

The present invention provides a method of increasing the survival rate of intestinal probiotics during oral delivery, comprising: the hydrophobic nano material is used for wrapping the intestinal probiotics through self-assembly, so that the contact between digestive enzymes with large molecular weight and hydrophilicity in digestive juice and the intestinal probiotics is prevented. The preferable method for improving the survival rate of the intestinal probiotics in the oral delivery process is to use a hydrophobic nano material containing transition metal, utilize the coordination effect of the transition metal and sugar and protein on the surface of the intestinal probiotics, further wrap the intestinal probiotics through self-assembly, and prevent digestive enzymes with high molecular weight and hydrophilicity in digestive juice from contacting the intestinal probiotics.

The invention also provides intestinal probiotics capable of resisting digestive enzymes, which is formed by adsorbing hydrophobic nano materials containing transition metals on the surfaces of the intestinal probiotics through coordination, and the hydrophobic nano materials wrap the probiotics to form a hydrophobic protective layer.

The intestinal probiotics in the invention can be selected from bifidobacteria, lactobacilli, clostridium butyricum or yeasts; the bifidobacterium may be selected from one or more of bifidobacterium longum, bifidobacterium adolescentis and bifidobacterium breve.

The hydrophobic nano material containing transition metal is a carbon-based nano material doped with transition metal elements and N elements or O elements; most preferred are carbon materials containing transition metal atom-N4 or transition metal atom-O, pyridine nitrogen, pyrrole nitrogen, and graphene structures.

The transition metal element in the invention can be selected from any one or a mixture of more than two of copper, iron, manganese, cobalt, zinc, cerium or gold; preferably any one or a mixture of more than two of copper, iron, zinc or gold; most preferably any of copper, iron or gold.

The invention also provides a method for preparing the intestinal probiotics capable of resisting digestive enzymes, which comprises the following steps: preparing a hydrophobic nano material containing a transition metal element, and dispersing the hydrophobic nano material into a buffer solution to prepare a solution A; culturing intestinal probiotics, centrifugally collecting, and re-dispersing the intestinal probiotics into a buffer solution to prepare a solution B; mixing the obtained solution A and the obtained solution B, and shaking for 5-15 min; and (4) centrifugally collecting the intestinal probiotics and washing to obtain the intestinal probiotics protected by the hydrophobic nano material, namely the intestinal probiotics capable of resisting digestive enzymes.

Taking bifidobacterium as an example, the preparation method specifically comprises the following steps:

1) preparing a Metal/organic framework precursor (MOF/Metal) containing transition Metal elements, wherein the content of Metal/organic framework compounds is 94-99% and the content of transition Metal elements is 1-6% in percentage by weight;

the process for preparing MOF/Metal precursors can generally be carried out in a manner that transition Metal nanoparticles are doped in the Metal/organic framework compound. The metal/organic framework compound can be selected from any one of ZIF-7, ZIF-8, ZIF-67, ZIF-68, ZIF-90, MIL-100, MIL-101, UiO-66, UiO-67, UiO-68, PCN-128, PCN-224, PCN-333, HKUST-1 or MOF-74, and the transition metal species can be selected from any one or a mixture of more than two of copper, iron, manganese, cobalt, zinc, cerium or gold.

The specific steps are preferably as follows: 50mL of a DMF solution of zinc acetate (10mM) and 50mL of a DMF solution of imidazole-2-carbaldehyde (20mM) were added dropwise simultaneously to 50mL of a DMF solution of imidazole-2-carbaldehyde (20mM) and a transition metal salt (2mM-10mM) at 30 ℃ at a rate of 30mL/h, and reacted for 5 min. Centrifuging at 12000rpm for 10min, and washing with methanol for three times to obtain MOF/Metal precursor;

2) calcining 500mg of MOF/Metal precursor substance obtained in the step 1) at high temperature for 3 hours at 800-1800 ℃ to obtain a protective nano material; dispersing the obtained protective nano material into a PBS buffer solution, and controlling the concentration of the protective nano material to be 0.2-10 mg/mL to obtain a nano material solution;

3) culturing Bifidobacterium, centrifuging, collecting, and dispersing in PBS buffer solution to control its concentration to 1-100 × 10⁶CFU/mL to obtain a bifidobacterium solution;

4) mixing the nano material solution obtained in the step 2) and the bifidobacterium solution obtained in the step 3) according to the volume ratio of 1:1, and shaking for 10 min; and centrifuging to collect the reacted bifidobacteria, and washing to obtain the bifidobacteria protected by the nano material.

The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention.

Example 1 preparation of nanomaterial protected Bifidobacterium

1)50mL of a DMF solution of zinc acetate (10mM) and 50mL of a DMF solution of imidazole-2-carbaldehyde (20mM) were added dropwise simultaneously to 50mL of a DMF solution of imidazole-2-carbaldehyde (20mM) and copper acetylacetonate (4mM) at 30 ℃ for 5 min. Centrifuging at 12000rpm for 10min, and washing with methanol for three times to obtain the MOF/Cu precursor.

2) The resulting MOF/Cu precursor is in N₂Calcining at 800 deg.C for 3 hr under protection to obtain protective nanometer material, and observing with transmission electron microscopeThe protective nanomaterial micro-topography is shown in figure 2. Dispersing the obtained protective nano material into PBS buffer solution, and controlling the concentration of the protective nano material to be 0.2mg/mL to obtain nano material solution;

3) culturing Bifidobacterium, centrifuging, collecting, and dispersing into PBS buffer solution to control concentration to 1 × 10⁶CFU/mL to obtain a bifidobacterium solution;

4) mixing the obtained 0.2mg/mL protective nanomaterial with 1 × 10⁶Mixing the CFU/mL bifidobacterium solution in the same volume, and oscillating for 10min to finish coordination and self-assembly; centrifuging to collect reacted Bacillus bifidus, and washing to obtain Bacillus bifidus protected by nanometer material, i.e. Bacillus bifidus capable of resisting digestive enzyme. The microscopic morphology of bifidobacteria before and after the reaction was observed by scanning electron microscopy, and the observation results are shown in fig. 3, in which (a) is the original bifidobacteria and (b) is the bifidobacteria protected by the nanomaterial. And (b) the prepared product is formed by wrapping a hydrophobic nano material on the surface of the intestinal probiotics, and the hydrophobic nano material forms a hydrophobic protective layer on the surface of the probiotics.

The bifidobacterium protected by the nano material prepared by the embodiment can be re-dispersed in a buffer solution to prepare liquid oral medicaments with different concentrations for treating IBD of a human body or an animal body.

The survival rate of the bifidobacterium capable of resisting digestive enzymes prepared in the embodiment in simulated gastric juice is detected through in vitro experiments, and the result is shown in fig. 4, and the survival rate of the original bifidobacterium is only about 18% (left column in the figure) after the bifidobacterium is treated in the simulated gastric juice for 1 hour, while the survival rate of the bifidobacterium protected by the nano material can reach about 88% (right column in the figure), so that the bifidobacterium can be effectively resisted by the digestive enzymes in the gastric juice under the protection of the hydrophobic nano material, the high survival rate and the high activity can be maintained for a long time, and the bifidobacterium can be completely used for oral administration.

Example 2 preparation of nanomaterial protected Bifidobacterium

The overall scheme is similar to example 1, except that step 1) is specifically: 50mL of a DMF solution of zinc acetate (10mM) and 50mL of a DMF solution of imidazole-2-carbaldehyde (20mM) were added dropwise simultaneously to 50mL of a DMF solution of imidazole-2-carbaldehyde (20mM) and copper acetylacetonate (4mM) at 30 ℃ for 5 min. Centrifuging at 12000rpm for 10min, and washing with methanol for three times to obtain the MOF/Cu precursor.

Example 3 preparation of nanomaterial protected Bifidobacterium

The overall scheme is similar to example 1, except that: in the step 2), the high-temperature calcination condition is 1500 ℃ for 3 hours.

Example 4 preparation of nanomaterial protected Bifidobacterium

The overall scheme is similar to example 1, except that: in step 3), the concentration of the bifidobacterium solution is controlled to be 100 multiplied by 10⁶CFU/mL。

Example 5 preparation of nanomaterial protected Bifidobacterium

The overall scheme is similar to example 1, except that: in the step 2), the concentration of the protective nano material is controlled to be 10 mg/mL.

Example 6 preparation of nanomaterial protected Bifidobacterium

The overall scheme is similar to example 1, except that: in step 1), copper acetylacetonate is replaced by manganese chloride in an equimolar amount to obtain a MOF/Mn precursor.

Example 7 preparation of nanomaterial protected Bifidobacterium

The overall scheme is similar to that of example 1, except that step 1) is specifically: 50mL of a DMF solution of zinc acetate (10mM) and 50mL of a DMF solution of imidazole-2-carbaldehyde (20mM) were added dropwise simultaneously to 50mL of a DMF solution of imidazole-2-carbaldehyde (20mM) and platinum acetylacetonate (4mM) at 30 ℃ at a rate of 30mL/h, and reacted for 5 min. Centrifuging at 12000rpm for 10min, and washing with methanol for three times to obtain the MOF/Pt precursor.

Application example 1 mouse UC model experiment

A UC model was constructed using C57/L mice as experimental animals and bifidobacteria resistant to digestive enzymes prepared according to example 1 as therapeutic agents (i.e., the bifidobacteria protected by the nanomaterial obtained in example 1 were redispersed in buffer to give a protective nanomaterial concentration of 200. mu.g/mL and a bifidobacteria concentration of 1X 10%⁶CFU/mL), the therapeutic effect of the oral route of administration was observed. The specific experimental scheme is as follows:

when C57/L mice drink 3% DSS, UC model can be constructed in 3-5 days, bloody stool appears, and body weight is obviously reduced. The UC animal models obtained were randomly divided into 3 groups (experimental, control and blank), each of which contained 5 mice. The administration was continued for 5 days, and 100. mu.L of Bifidobacterium (1X 10) per mouse was gavaged in the control group⁶CFU/mL), experimental groups each mouse gavage 100 μ L of the above therapeutic drugs, blank groups each mouse gavage 100 μ L PBS.

After 5 days of continuous administration, the colon tissue of the mice was removed, and the average length of the colon was measured for each group of mice.

The results show that, as shown in fig. 5, compared to the healthy group of mice, the average colon length of the blank group without drug administration in the UC model is only about 80% of that of the healthy mice, demonstrating efficient establishment of the model; the increase in mean colon length of the control group administered with bifidobacteria compared to the blank group was very limited, less than about 4%, indicating that administration of unprotected bifidobacteria alone does not guarantee sufficient colonization in the gut to function; compared with a blank group and a control group, the average colon length of an experimental group which is administered with the bifidobacterium protected by the nano material is obviously improved, the average colon length is respectively improved by about 20 percent and 15 percent compared with the blank group and the control group, and the average colon length of healthy mice is more than 95 percent, which shows that the bifidobacterium protected by the nano material in the embodiment 1 can avoid the attack of digestive enzymes under the protection of the nano material, avoid the inactivation before entering the intestinal tract, and can be fixedly planted in the intestinal tract to fully exert the treatment effect on the UC caused by the DSS.

Application example 2 beagle UC model experiment

A UC model was established using beagle dogs as the experimental animals and bifidobacteria resistant to digestive enzymes prepared according to the method of example 1 as the therapeutic agent (i.e., the bifidobacteria protected by the nanomaterial obtained in example 1 were redispersed in buffer to give a protective nanomaterial concentration of 500. mu.g/mL and a bifidobacteria concentration of 2.5X 10%⁶CFU/mL), the therapeutic effect of the oral route of administration was observed. The specific experimental scheme is as follows:

the beagle dog is clystered for 1min by 7% acetic acid and 10mL/kg, and the next day of colonoscopy shows that bleeding ulcer appears, which indicates that the UC model is successfully made. The UC model is randomly divided into a UC administration group and a UC non-administration group, and healthy dogs fed under the same condition are used as blank control groups, wherein each group comprises 3 dogs. Then continuously administering for 6 days, and performing intragastric gavage on each beagle dog by 15mL of the therapeutic drug in the UC administration group; UC dose not administer each beagle dog gavage 15mL PBS; blank control group (healthy group) each beagle dog was gavaged with 15mL PBS. The administration was continued for 6 days, and on day 1 after the molding and 2 and 7 days after the administration, each beagle dog was subjected to enema, feces were evacuated, and anesthesia was performed, and enteroscopy analysis was performed to observe whether the colon tissue of each group of beagle dogs was smooth, bleeding, ulcerated and scab.

As shown in fig. 6, the colon bleeding and redness of the beagle dogs of each group of the UC model on day 1 after molding, compared to the healthy dogs of the blank control group (healthy group), proved effective establishment of the model. After 2 days of gavage, the UC-dosed group had significant wound healing after treatment with the nanomaterial-protected bifidobacterium prepared in example 1, compared to the UC-free group with significant ulceration, and after 7 days of gavage, no symptoms remained with the UC-dosed group, which had substantially returned to a level consistent with that of the healthy group. The bifidobacterium in the medicament of the example 1 can avoid the attack of digestive enzymes under the protection of the nano material, avoid the inactivation before entering the intestinal tract and can be planted in the intestinal tract to fully exert the treatment effect on the UC caused by acetic acid.

Application example 3 mouse CD model experiment

A CD model was established using BALB/C mice as experimental animals, and bifidobacteria resistant to digestive enzymes prepared in example 1 were used as therapeutic agents (i.e., the bifidobacteria protected by the nanomaterial obtained in example 1 were redispersed in buffer so that the protective nanomaterial concentration was 200. mu.g/mL and the bifidobacteria concentration was 1X 10%⁶CFU/mL), the therapeutic effect of the oral route of administration was observed. The specific experimental scheme is as follows:

1% TNBS is firstly used for sensitizing for 7 days on the back of a BALB/C mouse, then 2% TNBS is filled into the colon part of the mouse, inversion is carried out for 30s, bloody stool immediately appears, the weight is obviously reduced, and the CD model is successful. The resulting CD animal models were randomly divided into 3 groups (experimental, control and blank) of 5 animals eachA mouse. The administration was continued for 4 days, and 100. mu.L of Bifidobacterium (1X 10) per mouse was gavaged in the control group⁶CFU/mL), experimental groups each mouse gavage 100 μ L of the above therapeutic drugs, blank groups each mouse gavage 100 μ L PBS.

After 4 days of continuous administration, the colon tissue of the mice was removed and the average length of the colon was measured for each group of mice.

Results as shown in fig. 7, the average colon length of the non-administered blank group was only about 90% of that of the healthy mice in the CD model, compared to the healthy mice, demonstrating efficient establishment of the model; the increase in mean colon length of the control group administered with bifidobacteria compared to the blank group was less than about 5%, indicating that administration of unprotected bifidobacteria alone does not ensure their colonization with sufficient activity in the gut to function; compared with a blank group and a control group, the average colon length of an experimental group which is administered with the bifidobacterium protected by the nano material is obviously improved, the average colon length is respectively improved by about 9 percent and 4 percent compared with the blank group and the control group, and the average colon length of healthy mice is more than 98 percent, which shows that the bifidobacterium protected by the nano material in the embodiment 1 can avoid the attack of digestive enzymes under the protection of the nano material, avoid the inactivation before entering the intestinal tract, and can be fixedly planted in the intestinal tract to fully exert the treatment effect on the CD caused by TNBS.