WO2023061100A1 - Inactivated bacteroides fragilis powder and preparation method therefor - Google Patents

Abstract

Provided are an inactivated bacteroides fragilis powder and a preparation method therefor. The inactivated bacterial powder comprises inactivated bacteroides fragilis. The thallus of inactivated bacteroides fragilis comprises a complete cell morphology, and the number of bacteria reaches 1*10 11 Cell/g or above. The preparation method can guarantee the integrity of thallus in the inactivated bacteroides fragilis powder. The inactivated bacterial powder can effectively mitigate cancer-related gastrointestinal side effects.

Description

A kind of Bacteroides fragilis inactivated bacteria powder and preparation method thereof

Priority and related applications

This application claims to enjoy the priority of the previous application submitted to the State Intellectual Property Office of China on October 12, 2021, with the patent application number 202111188973.9, and the invention title "An inactivated Bacteroides fragilis bacteria powder and its preparation method". The entirety of said prior application is incorporated by reference into this application.

technical field

The invention relates to the preparation and application technology of inactivated Bacteroides fragilis powder, in particular to an inactivated Bacteroides fragilis powder and a preparation method thereof.

Background technique

Bacteroides fragilis (Bacteroides fragilis) is a member of the genus Bacteroides among Gram-negative anaerobic bacteria, belonging to the Bacteroides phylum, which is completely different from Bifidobacteria and lactic acid bacteria of the Firmicutes phylum. There are 25 species of Bacteroides, 10 species from humans only, 10 species from animals only, and 5 species from both humans and animals. Bacteroides fragilis is an obligate anaerobic bacterium, which can be divided into enterotoxigenic Bacteroides fragilis (ETBF) and non-enterotoxigenic Bacteroides fragilis (ETBF) according to whether it can synthesize and secrete B. Bacteroides fragilis (Nontoxigenic Bacteroides fragilis, NTBF). Bacteroides fragilis, as part of the normal intestinal flora of humans and animals, mainly exists in the colon. In addition, the mucous membranes of the respiratory tract, gastrointestinal tract, and genitourinary tract can also colonize and grow.

The possibility of NTBF as a probiotic has been reported in the literature (Sun F, Zhang Q, Zhao J, Zhang H, Zhai Q, Chen W., A potential species of next-generation probiotics? The dark and light sides of Bacteroides fragilis in health.Food Res Int.2019 Dec;126:108590.doi:10.1016/j.foodres.2019.108590.Epub 2019 Jul 27.PMID:31732047.). However, due to the live bacteria characteristics of probiotics, there are the following side effects in its use: 1) there is a risk of translocation to local tissues and blood in critically ill patients, the elderly and newborns, especially premature infants; Bacteria may acquire/transfer antibiotic resistance genes and/or virulence genes; 3) Live bacteria have strict requirements on environmental conditions, are easy to be inactivated during extraction, transportation and storage, and are not easy to standardize; 4) Product texture changes with the state of live bacteria , stability and palatability fluctuations.

In response to the above problems, paraprobiotics came into being. Co-probiotics are defined as "non-viable microbial cells which, when administered orally or topically in sufficient quantities, confer benefits to the user". This definition includes microbial cells with incomplete morphology and microbial cells with complete morphology. The mainstream research point of view believes that broken microbial cells can release functional molecules better, so they have better effects; but for intact microbial cells that have been inactivated, there is no A unified view can explain why it is superior to live bacteria in some applications. At present, the research on secondary probiotics is concentrated on the first-generation probiotics such as Lactobacillus and Bifidobacterium, and the research direction is relatively fixed. Therefore, it is necessary to develop new probiotics to expand the scope of indications of probiotics.

Contents of the invention

In view of the above background technology, the present invention provides the following technical solutions:

In the first aspect, the present invention provides a kind of Bacteroides fragilis inactivated bacteria powder, which includes Bacteroides fragilis inactivated bacteria (the preservation number of Bacteroides fragilis is B. fragilis ZY-312 of CGMCC NO.10685); The bacteria of the inactivated Bacteroides fragilis include a complete cell morphology structure; the number of bacteria reaches more than 1×10 ¹¹ Cell/g.

According to an embodiment of the present invention, the inactivated bacteria powder of Bacteroides fragilis further includes auxiliary materials.

According to an embodiment of the present invention, in the inactivated Bacteroides fragilis bacteria powder, the mass ratio of the inactivated Bacteroides fragilis bacteria to the auxiliary material is 1:(0.05-4).

According to an embodiment of the present invention, the adjuvants comprise excipients. Preferably, the excipients are solid at room temperature.

Preferably, the excipients include mannitol, sorbitol, maltodextrin, lactose, sodium chloride, maltose, sucrose, glucose, trehalose, dextran, proline, lysine, alanine, casein , skim milk at least one or a combination of two or more.

According to an embodiment of the present invention, sodium chloride is included in the excipient. Preferably, in the excipient, the mass fraction of sodium chloride is 0-25wt%.

In a second aspect, the present invention also provides a preparation method for inactivated Bacteroides fragilis powder, in particular, the inactivated Bacteroides fragilis powder prepared by the preparation method has a complete bacterial cell structure, and the preparation method Include the following steps:

(1) take Bacteroides fragilis fermentation culture;

(2) After the fermentation culture is finished, the fermentation liquid is centrifuged, and the thallus is collected, and the weight-to-volume ratio of the thallus to the sodium chloride aqueous solution is 1g: (10-30) mL, and the sodium chloride aqueous solution is added for washing and centrifugation to obtain the washed After the bacteria;

(3) adding the first excipient solution to the washed bacterial cells to mix and resuspend to obtain the bacterial cell solution, then perform inactivation treatment, centrifuge, and collect the inactivated bacteria sludge;

(4) adding the second excipient solution to the inactivated bacteria slime obtained in step (3) to obtain the inactivated bacteria stock solution;

(5) drying the stock solution of inactivated bacteria obtained in step (4) until the residual moisture is lower than 5 wt%, to obtain the inactivated bacteria powder of Bacteroides fragilis.

According to an embodiment of the present invention, in step (2), the number of bacteria in the fermentation broth reaches above 10 ⁸ CFU/mL.

According to an embodiment of the present invention, in step (2), the mass concentration of the aqueous sodium chloride solution is 0.6-1.5wt%, preferably 0.65-1.2wt%, more preferably 0.8-1.0wt%, most preferably 0.85- 0.95 wt%, eg 0.9 wt% sodium chloride aqueous solution.

According to an embodiment of the present invention, in step (3), the weight-to-volume ratio of the bacteria to the first excipient solution is 1 g:(5-40) mL.

According to an embodiment of the present invention, in step (3), in the first excipient solution, the mass fraction of the excipient is 4-30 wt%, and the excipient has the above-mentioned meanings.

According to an embodiment of the present invention, in step (3), the solvent of the first excipient solution is selected from aqueous sodium chloride solution, wherein the aqueous sodium chloride solution has the above-mentioned meanings. Further preferably, the solvent of the first excipient solution is selected from physiological saline, such as 0.9wt% sodium chloride aqueous solution.

According to an embodiment of the present invention, in step (3), the inactivation treatment method is selected from at least one of heat inactivation, freeze inactivation or chemical inactivation, preferably heat inactivation.

Preferably, the heat inactivation temperature is 60-100° C., and the heat inactivation time is 10-60 minutes.

According to an embodiment of the present invention, in step (4), a second excipient solution is added to make the total weight of the inactivated bacteria stock solution consistent with the weight of the bacterial cell solution before inactivation in step (3). Preferably, said second excipient solution is the same or different from said first excipient solution.

According to an embodiment of the present invention, in the second excipient solution, the mass fraction of the excipient is 4-30 wt%, and the excipient has the above-mentioned meanings.

Preferably, the solvent of the second excipient solution is selected from aqueous sodium chloride solution, wherein the aqueous sodium chloride solution has the above-mentioned meanings. Further preferably, the solvent of the first excipient solution is selected from physiological saline, such as 0.9 wt% sodium chloride aqueous solution.

Exemplarily, the second excipient solution is the same as the first excipient solution.

According to an embodiment of the present invention, in step (5), the drying method is selected from vacuum freeze drying and/or spray drying, preferably vacuum freeze drying.

Preferably, the vacuum freeze-drying conditions include: a freezing temperature of -20-40° C., a freezing time of 1-3 hours, and a vacuum degree of 0.20-0.25 mbar.

Exemplarily, the vacuum freeze-drying process includes: prefreezing at -40±2°C for 1 to 3 hours, then prefreezing at -20±2°C for 0.5 to 1 hour, and finally prefreezing at -40±2°C for 0.5 to 2 hours, Under 0.25mbar vacuum degree, the inactivated bacteria powder is prepared by drying once and analyzing and drying.

According to an embodiment of the present invention, in the preparation method, the conditions of the centrifugation are not specifically limited, as long as the desired centrifugation effect can be achieved, for example, the centrifugation speed is 10000-20000 rpm.

In the third aspect, the present invention also provides a pharmaceutical preparation, which contains the above-mentioned inactivated Bacteroides fragilis powder.

According to an embodiment of the present invention, the pharmaceutical preparation is selected from at least one or a combination of two or more of oral administration preparations, enemas or gels.

Those skilled in the art can understand that the features contained in various aspects and embodiments according to the present invention can be combined independently of each other, as long as there is no conflict between the combined features.

Beneficial effect:

The inventors of the present invention have developed a bacterial powder capable of ensuring the integrity of the inactivated Bacteroides fragilis thallus and a preparation method thereof, and the inactivated Bacteroides fragilis powder obtained by the preparation method of the present invention can more effectively ensure that it is compatible with live bacteria. Consistency of formulation-related properties, especially the ability to mitigate cancer-related gastrointestinal side effects when administered. In addition, compared with the living bacteria preparation, the storage method of the pharmaceutical preparation prepared by the inactivated bacterial powder of the present invention is simpler, and the validity period of the pharmaceutical preparation is also prolonged.

Description of drawings

Fig. 1 is the microscopic examination picture of the bacterial powder obtained as excipient with 5wt% glycine of embodiment 1;

Fig. 2 is the microscopic examination picture of the bacterial powder obtained as excipient with 5wt% mannitol of embodiment 2;

Fig. 3 is the microscopic examination picture of the bacterial powder obtained as excipient with 5wt% dextran of embodiment 3;

Fig. 4 is the microscopic examination picture of the bacterial powder obtained as excipient with 5wt% maltodextrin of embodiment 4;

Fig. 5 is the microscopic examination picture of the bacterial powder obtained as excipient with 5wt% maltodextrin+0.9wt% normal saline of embodiment 7;

Fig. 6 is the microscopic examination figure of the bacterial powder obtained as excipient with sodium chloride aqueous solution in comparative example 2;

Fig. 7 is respectively from left to right the transmission electron micrographs of the inactivated bacteria obtained by the inactivation temperature of Example 17, Example 19 and Comparative Example 4.

Detailed ways

The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following examples are only to illustrate and explain the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies realized based on the above contents of the present invention are covered within the scope of protection intended by the present invention.

Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

In the following examples and comparative examples, unless otherwise specified, methods known in the art were used to ferment and cultivate Bacteroides fragilis, wherein the preservation number of Bacteroides fragilis is Bacteroides fragilis ZY-312 of CGMCC NO.10685.

Example 1

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, then collect the wet bacteria, add 0.9wt% physiological saline according to the ratio of bacteria: normal saline = 1:15 (g: mL) to resuspend and wash the bacteria sludge, centrifuge again, and collect Washed bacteria;

(3) Add 5wt% glycine excipient solution (5wt% refers to the mass fraction of excipient glycine in the excipient solution, the same below) to the obtained thallus of step (2), according to the thallus: excipient Bacterial agent solution = 1:10 (g:mL) ratio to add, stir and disperse, heat inactivate at 80±5°C for 20±5 minutes to obtain inactivated bacteria liquid, centrifuge at 14000rpm to collect inactivated bacteria sludge ;

(4) Add 5wt% glycine excipient solution to the inactivated bacteria slime collected in step (3), make the total weight consistent with the weight of the bacteria liquid before inactivation, stir and dissolve completely, and obtain the inactivated bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 2

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, then collect the wet bacteria, add 0.9wt% physiological saline according to the ratio of bacteria: normal saline = 1:25 (g: mL) to resuspend and wash the bacteria sludge, centrifuge again, and collect Washed bacteria;

(3) Add 5wt% mannitol excipient solution to the bacteria obtained in step (2), add according to the ratio of bacteria: excipient solution = 1:15 (g:mL), stir and disperse at 80 ± 5 Heat inactivation at ℃ for 20±5 minutes to obtain the inactivated bacteria liquid and collect the inactivated bacteria sludge;

(4) Add 5wt% mannitol excipient solution to the inactivated bacteria slime collected in step (3), make the total weight consistent with the weight of the bacteria liquid before inactivation, stir and dissolve completely, and obtain the inactivated bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 3

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, then collect the wet bacteria, add 0.9wt% physiological saline according to the ratio of bacteria: normal saline = 1:16 (g: mL) to resuspend and wash the bacteria sludge, centrifuge again, and collect Washed bacteria;

(3) Add 5wt% dextran excipient solution to the bacteria obtained in step (2), add according to the ratio of bacteria: excipient solution = 1:10 (g:mL), stir and disperse at 70±5°C Heat inactivation for 40±5 minutes to obtain the inactivated bacteria liquid and collect the inactivated bacteria sludge;

(4) Add 5wt% dextran excipient solution to the inactivated bacteria slime collected in step (3), make the total weight consistent with the weight of the bacteria liquid before inactivation, stir and dissolve completely, and obtain the inactivated bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 4

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, then collect wet bacteria, add 0.9wt% normal saline according to the ratio of bacteria: normal saline = 1:22 (g: mL) to resuspend and wash the bacteria sludge, centrifuge again, and collect Washed bacteria;

(3) Add 5wt% maltodextrin excipient solution to the cells obtained in step (2), add according to the ratio of cells: excipient solution = 1:8 (g: mL), stir and disperse at 65 ± Heat inactivation at 5°C for 20±5 minutes to obtain an inactivated bacterial liquid, which was centrifuged at 14,000 rpm to collect the inactivated bacterial sludge.

(4) Add 5wt% maltodextrin excipient solution to the inactivated bacteria slime collected in step (3), make the total weight consistent with the weight of the bacteria liquid before inactivation, stir and dissolve completely, and obtain the inactivated bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 5

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, then collect the wet bacteria, add 0.9wt% physiological saline according to the ratio of bacteria: normal saline = 1:15 (g: mL) to resuspend and wash the bacteria sludge, centrifuge again, and collect Washed bacteria;

(3) Add 5wt% lactose excipient solution to the bacterial cells obtained in step (2), add the bacterial cells: excipient solution = 1:10 (g:mL) ratio, stir and disperse at 85±5°C Heat inactivation for 35±5 minutes to obtain the inactivated bacteria liquid, and collect the inactivated bacteria sludge.

(4) Add 5wt% lactose excipient solution to the inactivated bacteria slime collected in step (3), make the total weight consistent with the weight of the bacteria liquid before inactivation, stir and dissolve completely, and obtain the inactivated bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 6

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, set the rotation speed to 14000rpm, then collect the wet bacteria, add 0.9wt% normal saline according to the ratio of bacteria: normal saline = 1:25 (g: mL) to resuspend and wash the bacteria sludge , and centrifuge again to collect the washed bacteria;

(2) Add 5wt% skim milk excipient solution to the cells obtained in step (2), add according to the ratio of cells: excipient solution = 1:9 (g:mL), stir and disperse at 85 ± 5 Heat inactivation at ℃ for 25±5 minutes to obtain the inactivated bacteria solution, which is centrifuged at 14000rpm to collect the inactivated bacteria sludge;

(4) Add 5wt% skim milk excipient solution to the inactivated bacteria slime collected in step (3), make the total weight consistent with the weight of the bacteria liquid before inactivation, stir and dissolve completely, and obtain the inactivated bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 7

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, then collect wet bacteria, add 0.9wt% physiological saline according to the ratio of bacteria: normal saline = 1:25 (g: mL) to resuspend and wash the bacteria sludge, centrifuge again, and collect Washed bacteria;

(3) Add 5wt% maltodextrin plus 0.9wt% physiological saline excipient solution to the bacteria obtained in step (2), and add according to the ratio of bacteria: excipient solution = 1:8 (g:mL), After stirring and dispersing, heat inactivate at 85±5°C for 40±5 minutes to obtain inactivated bacteria liquid and collect inactivated bacteria sludge;

(4) Add 5wt% maltodextrin to the inactivated bacterium sludge that step (3) collects and add 0.9wt% physiological saline excipient solution (wherein, solvent is 0.9wt% physiological saline, the same below), so that the total weight is the same as The weight of the bacterial solution before inactivation is the same, and it is completely dissolved by stirring to obtain the original solution of the inactivated bacteria;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 8

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, set the rotation speed to 14000rpm, then collect the wet bacteria, add 0.9wt% normal saline according to the ratio of bacteria: normal saline = 1:23 (g: mL) to resuspend and wash the bacteria sludge , and centrifuge again to collect the washed bacteria;

(3) Add 15wt% maltodextrin plus 0.9wt% physiological saline excipient solution to the bacteria obtained in step (2), and add according to the ratio of bacteria: excipient solution = 1:12 (g:mL), After stirring and dispersing, heat inactivate at 85±5°C for 40±5 minutes to obtain the inactivated bacteria liquid, which is centrifuged at 14000rpm to collect the inactivated bacteria sludge;

(4) Add 10% maltodextrin to the inactivated bacteria slime collected in step (3) and add 0.9wt% physiological saline excipient solution to make the total weight consistent with the weight of the bacteria liquid before inactivation, stir to dissolve completely, and obtain inactivated Live bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 9

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, then collect wet bacteria, add 0.9wt% physiological saline according to the ratio of bacteria: normal saline = 1:25 (g: mL) to resuspend and wash the bacteria sludge, centrifuge again, and collect Washed bacteria;

(3) Add 20% maltodextrin plus 0.9wt% physiological saline excipient solution to the bacteria obtained in step (2), and add according to the ratio of bacteria: excipient solution = 1:12 (g:mL), After stirring and dispersing, heat inactivate at 90±5°C for 15±5 minutes to obtain inactivated bacteria liquid, which is centrifuged to collect inactivated bacteria sludge;

(4) Add 20% maltodextrin to the inactivated bacteria slime collected in step (3) and add 0.9wt% physiological saline excipient solution to make the total weight consistent with the weight of the bacteria liquid before inactivation, stir to dissolve completely, and obtain inactivated Live bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 10

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, set the rotation speed to 14000rpm, then collect the wet bacteria, add 0.9wt% normal saline according to the ratio of bacteria: normal saline = 1:20 (g: mL) to resuspend and wash the bacteria sludge , and centrifuge again to collect the washed bacteria;

(3) Add 25% maltodextrin plus 0.9wt% physiological saline excipient solution to the bacteria obtained in step (2), and add according to the ratio of bacteria: excipient solution = 1:15 (g:mL), After stirring and dispersing, heat inactivate at 80±5°C for 20±5 minutes to obtain inactivated bacteria liquid, which is centrifuged to collect inactivated bacteria sludge;

(4) Add 25% maltodextrin to the inactivated bacteria slime collected in step (3) and add 0.9wt% physiological saline excipient solution to make the total weight consistent with the weight of the bacteria liquid before inactivation, stir to dissolve completely, and obtain inactivated Live bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 11

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, set the rotation speed to 14000rpm, then collect the wet bacteria, add 0.9wt% normal saline according to the ratio of bacteria: normal saline = 1:18 (g: mL) to resuspend and wash the bacteria sludge , and centrifuge again to collect the washed bacteria;

(3) Add 5wt% maltodextrin plus 0.6wt% sodium chloride aqueous excipient solution to the bacteria obtained in step (2), and proceed according to the ratio of bacteria: excipient solution = 1:12 (g:mL) Add, stir and disperse, heat inactivate at 80±5°C for 20±5 minutes to obtain inactivated bacteria liquid, centrifuge to collect inactivated bacteria sludge;

(4) add 5wt% maltodextrin to the inactivated bacterium slime collected in step (3) and add 0.6wt% sodium chloride aqueous solution excipient solution, make the total weight consistent with the bacterial liquid weight before inactivation, stir and dissolve completely, Obtain the stock solution of inactivated bacteria;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 12

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, then collect the wet bacteria, add 0.9wt% physiological saline according to the ratio of bacteria: normal saline = 1:20 (g: mL) to resuspend and wash the bacteria sludge, centrifuge again, and collect Washed bacteria;

(3) Add 5wt% maltodextrin plus 1.2wt% sodium chloride aqueous excipient solution to the bacteria obtained in step (2), and add according to the ratio of bacteria: excipient solution = 1:8 (g:mL) , after stirring and dispersing, heat inactivation at 90±5°C for 30±5 minutes to obtain the inactivated bacteria liquid, which is centrifuged at 14000rpm to collect the inactivated bacteria sludge;

(4) Add 5wt% maltodextrin to the inactivated bacterium slime collected in step (3) and add 1.2wt% sodium chloride aqueous excipient to make the total weight consistent with the weight of the bacteria liquid before inactivation, stir to dissolve completely, and obtain sterilized Live bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 13

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, set the rotation speed to 14000rpm, then collect the wet bacteria, add 0.9wt% normal saline according to the ratio of bacteria: normal saline = 1:18 (g: mL) to resuspend and wash the bacteria sludge , and centrifuge again to collect the washed bacteria;

(3) Add 5wt% maltodextrin plus 0.9wt% physiological saline excipient solution to the cells obtained in step (2), and add according to the ratio of cells: excipient solution = 1:6 (g:mL), After stirring and dispersing, heat inactivate at 70±5°C for 40±5 minutes to obtain inactivated bacteria liquid, centrifuge to collect inactivated bacteria sludge;

(4) Add 5wt% maltodextrin and 0.9wt% physiological saline excipient solution to the inactivated bacteria slime collected in step (3), make the total weight consistent with the weight of the bacteria liquid before inactivation, stir to dissolve completely, and obtain Live bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 14

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, set the rotation speed to 14000rpm, then collect the wet bacteria, add 0.9wt% normal saline according to the ratio of bacteria: normal saline = 1:15 (g: mL) to resuspend and wash the bacteria sludge , and centrifuge again to collect the washed bacteria;

(3) Add 5wt% maltodextrin plus 0.9wt% physiological saline excipient solution to the bacteria obtained in step (2), and add according to the ratio of bacteria: excipient solution = 1:18 (g:mL), After stirring and dispersing, heat inactivate at 70±5°C for 40±5 minutes to obtain inactivated bacterial liquid, which is centrifuged at 14000rpm to collect inactivated bacterial sludge;

(4) Add 5wt% maltodextrin and 0.9wt% physiological saline excipient solution to the inactivated bacteria slime collected in step (3), make the total weight consistent with the weight of the bacteria liquid before inactivation, stir to dissolve completely, and obtain Live bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 15

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, then collect the wet bacteria, add 0.9wt% physiological saline according to the ratio of bacteria: normal saline = 1:20 (g: mL) to resuspend and wash the bacteria sludge, centrifuge again, and collect Washed bacteria;

(3) Add 5wt% maltodextrin plus 0.9wt% physiological saline excipient solution to the bacteria obtained in step (2), add according to the ratio of bacteria: excipient solution = 1:30 (g:mL), and stir After dispersion, heat inactivate at 80±5°C for 20±5 minutes to obtain the inactivated bacteria liquid, which is centrifuged to collect the inactivated bacteria sludge;

(4) Add 5wt% maltodextrin and 0.9wt% physiological saline excipient solution to the inactivated bacteria slime collected in step (3), make the total weight consistent with the weight of the bacteria liquid before inactivation, stir to dissolve completely, and obtain Live bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 16

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, then collect the wet bacteria, add 0.9wt% physiological saline according to the ratio of bacteria: normal saline = 1:10 (g: mL) to resuspend and wash the bacteria sludge, centrifuge again, and collect Washed bacteria;

(3) Add 5wt% maltodextrin plus 0.9wt% physiological saline excipient solution to the cells obtained in step (2), and add according to the ratio of cells: excipient solution = 1:40 (g:mL), After stirring and dispersing, heat inactivate at 80±5°C for 20±5 minutes to obtain inactivated bacteria liquid, which is centrifuged to collect inactivated bacteria sludge;

(4) Add 5wt% maltodextrin and 0.9wt% physiological saline excipient solution to the inactivated bacteria slime collected in step (3), make the total weight consistent with the weight of the bacteria liquid before inactivation, stir to dissolve completely, and obtain Live bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 17

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, then collect wet bacteria, add 0.9wt% physiological saline according to the ratio of bacteria: normal saline = 1:25 (g: mL) to resuspend and wash the bacteria sludge, centrifuge again, and collect Washed bacteria;

(3) Add 5wt% maltodextrin plus 0.9wt% physiological saline excipient solution to the bacteria obtained in step (2), add according to the ratio of bacteria: excipient solution = 1:15 (g:mL), and stir After dispersion, heat inactivate at 60±5°C for 20±5 minutes to obtain the inactivated bacteria liquid, which is centrifuged to collect the inactivated bacteria sludge;

(4) Add 5wt% maltodextrin and 0.9wt% physiological saline excipient solution to the inactivated bacteria slime collected in step (3), make the total weight consistent with the weight of the bacteria liquid before inactivation, stir to dissolve completely, and obtain Live bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 18

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, then collect wet bacteria, add 0.9wt% physiological saline according to the ratio of bacteria: normal saline = 1:18 (g: mL) to resuspend and wash the bacteria sludge, centrifuge again, and collect Washed bacteria;

(3) Add 5wt% maltodextrin and 0.9wt% physiological saline to the cells obtained in step (2) as an excipient, add the cells according to the ratio of cells: excipient solution = 1:12 (g:mL), and stir After dispersion, heat inactivate at 90±5°C for 20±5 minutes to obtain inactivated bacteria liquid, centrifuge to collect inactivated bacteria sludge;

(4) Add 5wt% maltodextrin and 0.9wt% physiological saline excipient solution to the inactivated bacteria slime collected in step (3), make the total weight consistent with the weight of the bacteria liquid before inactivation, stir to dissolve completely, and obtain Live bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally freeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 19

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, then collect wet bacteria, add 0.9wt% physiological saline according to the ratio of bacteria: normal saline = 1:25 (g: mL) to resuspend and wash the bacteria sludge, centrifuge again, and collect Washed bacteria;

(3) Add 5wt% maltodextrin plus 0.9wt% physiological saline excipient solution to the bacteria obtained in step (2), add according to the ratio of bacteria: excipient solution = 1:8 (g:mL), and stir After dispersion, heat inactivate at 100°C for 10±5 minutes to obtain the inactivated bacteria liquid, which is centrifuged to collect the inactivated bacteria sludge;

(4) Add 5wt% maltodextrin and 0.9wt% physiological saline excipient solution to the inactivated bacteria slime collected in step (3), make the total weight consistent with the weight of the bacteria liquid before inactivation, stir to dissolve completely, and obtain Live bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 20

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, then collect the wet bacteria, add 0.9wt% physiological saline according to the ratio of bacteria: normal saline = 1:12 (g: mL) to resuspend and wash the bacteria sludge, centrifuge again, and collect Washed bacteria;

(3) Add 5wt% maltodextrin and 0.9wt% physiological saline excipient to the cells obtained in step (2), and add them according to the ratio of cells: excipient solution = 1:15 (g:mL), After stirring and dispersing, heat inactivate at 80°C for 30±5 minutes to obtain the inactivated bacteria liquid, which is centrifuged to collect the inactivated bacteria sludge;

(4) Add 5wt% maltodextrin and 0.9wt% physiological saline excipient solution to the inactivated bacteria slime collected in step (3) to make the total weight consistent with the weight of the bacteria liquid before inactivation, stir and dissolve completely, and obtain inactivated Bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 21

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, then collect the wet bacteria, add 0.9wt% physiological saline according to the ratio of bacteria: normal saline = 1:20 (g: mL) to resuspend and wash the bacteria sludge, centrifuge again, and collect Washed bacteria;

(3) Add 5wt% maltodextrin plus 0.9wt% physiological saline excipient solution to the bacteria obtained in step (2), add according to the ratio of bacteria: excipient solution = 1:10 (g:mL), and stir After dispersion, heat inactivate at 80°C for 40±5 minutes to obtain inactivated bacteria liquid, centrifuge to collect inactivated bacteria sludge;

(4) Add 5wt% maltodextrin and 0.9wt% physiological saline excipient solution to the inactivated bacteria slime collected in step (3), make the total weight consistent with the weight of the bacteria liquid before inactivation, stir to dissolve completely, and obtain Live bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Example 22

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, then collect the wet bacteria, add 0.9wt% physiological saline according to the ratio of bacteria: normal saline = 1:20 (g: mL) to resuspend and wash the bacteria sludge, centrifuge again, and collect Washed bacteria;

(3) Add 5wt% maltodextrin plus 0.9wt% physiological saline excipient solution to the cells obtained in step (2), and add according to the ratio of cells: excipient solution = 1:10 (g:mL), After stirring and dispersing, heat inactivate at 80°C for 50±5 minutes to obtain the inactivated bacteria liquid, which is centrifuged to collect the inactivated bacteria sludge;

(4) Add 5wt% maltodextrin and 0.9wt% physiological saline excipient solution to the inactivated bacteria slime collected in step (3), make the total weight consistent with the weight of the bacteria liquid before inactivation, stir to dissolve completely, and obtain Live bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Comparative example 1

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermented liquid, then collect the wet bacteria, add purified water according to the ratio of bacteria: pure water = 1:20 (g:mL) to resuspend and wash the bacteria sludge, centrifuge again, and collect the washed bacteria bacteria;

(3) Add 0.9wt% physiological saline to the bacteria obtained in step (2), add according to the ratio of bacteria: normal saline = 1:10 (g:mL), stir and disperse, heat inactivate at 80±5°C for 20 ±5 minutes, get the inactivated bacteria solution, centrifuge to collect the inactivated bacteria sludge;

(4) Add purified water to the inactivated bacteria slime collected in step (3) to make the total weight consistent with the weight of the bacteria liquid before inactivation, and stir to dissolve completely to obtain the inactivated bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Comparative example 2

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, then collect the wet bacteria, add 0.9wt% physiological saline according to the ratio of bacteria: normal saline = 1:20 (g: mL) to resuspend and wash the bacteria sludge, centrifuge again, and collect Washed bacteria;

(3) Add 0.9wt% normal saline to the bacteria obtained in step (2), add according to the ratio of bacteria: normal saline = 1:10 (g:mL), stir and disperse, heat inactivate at 80±5°C for 20 ±5 minutes to obtain the inactivated bacteria liquid, centrifuge to collect the inactivated bacteria sludge;

(4) Add physiological saline to the inactivated bacteria slime collected in step (3) to make the total weight consistent with the weight of the bacteria liquid before inactivation, and stir to dissolve completely to obtain the inactivated bacteria stock solution;

5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally freeze at -40±2°C Freeze for 0.5-2 hours, and prepare inactivated bacterial powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Comparative example 3

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, then collect wet bacteria, add 0.9wt% physiological saline according to the ratio of bacteria: normal saline = 1:25 (g: mL) to resuspend and wash the bacteria sludge, centrifuge again, and collect Washed bacteria;

(3) Add 5wt% maltodextrin and 0.9wt% physiological saline excipient solution to the obtained bacteria in step (2), according to the mass ratio of bacteria: excipient solution=1:8 (g:mL) Add, stir and disperse, heat inactivate at 50±5°C for 20±5 minutes to obtain inactivated bacteria liquid, centrifuge to collect inactivated bacteria sludge;

(4) Add 5wt% maltodextrin and 0.9wt% physiological saline excipient solution to the inactivated bacteria slime collected in step (3), make the total weight consistent with the weight of the bacteria liquid before inactivation, stir to dissolve completely, and obtain Live bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

Comparative example 4

(1) Fermentation and cultivation of Bacteroides fragilis, so that the number of bacteria in the fermentation broth reaches more than 10 ⁸ CFU/mL;

(2) Centrifuge the fermentation broth, then collect the wet bacteria, add 0.9wt% physiological saline according to the ratio of bacteria: normal saline = 1:20 (g: mL) to resuspend and wash the bacteria sludge, centrifuge again, and collect Washed bacteria;

(3) Add 5wt% maltodextrin plus 0.9wt% physiological saline excipient solution to the cells obtained in step (2), and add according to the ratio of cells: excipient solution = 1:10 (g:mL), After stirring and dispersing, heat inactivate at 120±5°C for 20±5 minutes to obtain the inactivated bacteria liquid, which is centrifuged to collect the inactivated bacteria sludge;

(4) Add 5wt% maltodextrin and 0.9wt% physiological saline excipient solution to the inactivated bacteria slime collected in step (3), make the total weight consistent with the weight of the bacteria liquid before inactivation, stir to dissolve completely, and obtain Live bacteria stock solution;

(5) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40±2°C for 1 to 3 hours, then pre-freeze at -20±2°C for 0.5 to 1 hour, and finally refreeze at -40±2°C Pre-freeze for 0.5 to 2 hours, and prepare inactivated bacteria powder by drying once (-5±2°C and 0±2°C) and analyzing and drying (35±2°C) under 0.25mbar vacuum.

test case

The inactivated bacteria powder prepared by above-mentioned embodiment 1-22 and comparative example 1-4 is tested as follows:

(1) Performance test of inactivated bacteria powder

1. Supernatant DNA detection

1) Test method:

a) Wash the bacteria sludge obtained after centrifugation in step (2) of Example 7 and Comparative Example 1 three times to obtain the centrifugal supernatant after washing once, the centrifugal supernatant after washing twice, and the centrifugal supernatant after washing three times, and measure the centrifugal supernatant respectively. The DNA content of the final supernatant; and centrifuge the inactivated bacteria stock solution obtained after the treatment of step (4), and measure the DNA content of the supernatant. The test results are shown in Table 1.

b) Centrifuge the bacteria sludge obtained after the initial centrifugation in step (2) of Example 7 and Examples 11-12, and measure the DNA content of the supernatant and the DNA content of the bacteria precipitate. The test results are shown in Table 2.

2) The test results are as follows:

Table 1

It can be seen from Table 1 that in Comparative Example 1, purified water was used as the washing solvent, and the DNA content of the supernatant was much higher than that of the excipient solution using 0.9% normal saline as the solvent. Wt% normal saline as a washing solvent can keep the shape and structure of bacteria intact.

Table 2

It can be seen from Table 2 that using 0.9wt% physiological saline as the washing solvent can better keep the morphology of the bacteria intact.

2. Effect of different excipients on the structure and morphology of inactivated bacterial powder

Fig. 1-6 is respectively the Gram's staining microscopic examination picture of the inactivated bacterial powder of embodiment 1-4, 7 and comparative example 2, can find out through the contrast between Fig. 1-5 and Fig. 6, in preparation method The use of the excipient solution of the present invention can ensure the integrity of the bacterium in the inactivated bacterial powder, especially the use of 5wt% maltodextrin+0.9wt% saline as the freeze-drying excipient in Example 7 has the best effect.

3. The influence of different concentrations of maltodextrin (ie excipient) on the preparation of inactivated bacterial powder

Visually observe the inactivated bacterium powder that embodiment 7,8,9,10 makes, and record observation result in table 3.

table 3

group	Maltodextrin Concentration	character
Example 7	5%	fluffy, pale yellow
Example 8	10%	firmer, pale yellow
Example 9	20%	firm, yellow
Example 10	30%	firm, yellow

By observing the appearance of the inactivated bacteria powder prepared in Examples 7-10, the appearance of the inactivated bacteria powder in Example 7 is relatively fluffy and light yellow. On the premise that the integrity of the bacteria in the inactivated bacterial powder is the same, the properties of the inactivated bacterial powder prepared with 5wt% maltodextrin (ie excipient) are more conducive to large-scale production.

4. Bacteria: the influence of excipient solution concentration and inactivation temperature on the preparation of inactivated bacterial powder

The living bacteria of Example 7, Example 19, and Comparative Example 3 were detected by the dilution plate coating method, and the detection results were recorded in Table 4.

Table 4

It can be seen from Table 4 that at 50°C, the inactivation is not complete; at 85°C and above, it can be effectively inactivated. It can be seen from the drawings from left to right in Figure 7 that in the range of 60-100°C, the bacteria can maintain a complete shape; when the temperature is above 100°C (120°C), the bacteria are damaged and cannot maintain a complete shape.

5. Influence of inactivation time on the preparation of inactivated bacteria powder

The live bacteria were measured by the dilution plate coating method, and the test results were recorded in Table 5.

table 5

group

Sterilization effect

Example 7	no live bacteria detected
Example 20	no live bacteria detected
Example 21	no live bacteria detected
Example 22	no live bacteria detected

As shown in Table 5, the inactivation time of 15-55 minutes can effectively inactivate. Considering large-scale production and saving costs, 20±5 minutes is used as the inactivation time.

(2) Drug efficacy experiment of Bacteroides fragilis inactivated bacterium powder of the present invention for treating side effects of cancer

1. Test method

Experimental design: select 80 BALB/c mice, half male and half male, and divide them into 8 groups at random according to animal sex and body weight interval, i.e. blank group, model group, positive group (loperamide 4mg/kg), comparison group ( Use B. fragilis NCTC9343 inactivated bacteria powder), and the B. fragilis inactivated bacteria powder group ( ¹⁰¹⁰ CFU/mL) that embodiment 2, 7, 9, 17 makes, 10 in every group, half male and half male. Except for the blank group, the animals in other groups were intraperitoneally injected with irinotecan 70mg/kg (0.14mL/10g) once a day for 5 consecutive days to prepare the diarrhea model; from 3 days before the model establishment, the animals in the corresponding groups were intragastrically administered (0.1mL/10g body weight) given imodium suspension and Bacteroides fragilis inactivated bacterial powder suspension, once a day in the morning and afternoon, for 11 consecutive days, the blank group and the model group were given the same amount of normal saline, and the last time Euthanasia after drug administration. After administration, the animals were generally observed and recorded every day, including the appearance and signs of the animals, behavioral activities, respiration, gland secretion, and feces, etc. The diarrhea of each animal was observed and recorded, and scored. At the end of modeling and experimentation, the feces of each animal were collected for weighing and determination of water content; about 0.5 mL of blood was collected from the orbital venous plexus of all surviving animals after isoflurane anesthesia, and the serum was collected after centrifugation and used for liquid chip reagents respectively. IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, TNF-α, INF-γ and other cytokines were detected by kits, and ICAM-1 was measured by ELISA kits; After euthanasia, the whole intestinal tract was collected, fixed with 10% formaldehyde solution, and stained with HE for pathological examination.

Detection method and frequency:

Food intake: Use an electronic balance to carry out the weighing operation of feed addition and remaining amount, record the added amount before administration (D0) and D4 respectively, and weigh the remaining amount once at D4, D8, and D11 (test end) respectively, and record , calculated according to the formula: average daily food intake (g) = (added amount - remaining amount)/number of animals/number of days.

Diarrhea record and scoring standard: Record the diarrhea situation of each experimental mouse every day, and the scoring standard of diarrhea refers to the diarrhea scoring method in the study of Kurita A et al. 0 points: normal or no stool; 1 point: mild diarrhea, slightly wet and soft stool; 2 points: moderate diarrhea, wet and shapeless stool, and mild perianal coloring; 3 points: severe diarrhea, Watery stools with heavy perianal pigmentation.

Intestinal histopathological examination: After euthanasia of all surviving animals, the entire intestinal tract was taken, fixed with 10% formaldehyde solution, and pathological tissue sections were prepared, stained with HE, and examined under a microscope for intestinal histopathological examination.

Data statistics and analysis: The data of body weight, food intake, diarrhea score and pathological examination results were expressed as mean ± standard deviation (Mean ± SD), and statistical analysis was performed using SPSS statistical software 25.0.

2. Test results

1) Food intake: The test results of food intake of animals in each group are shown in the table below.

Table 6 Statistical results of food intake of animals in each group (mean ± standard deviation, n=10)

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

As can be seen from Table 6, at D4 (i.e. before modeling), the animals in the blank group, the model group and each administration group (i.e. the positive group and the inactivated bacterial powder group of Examples 2, 7, 9, 17, the same below) There was no significant difference in average daily food intake (p>0.05). Compared with the model group, on D8 and D11 (i.e. after modeling), the average daily food intake of male and female animals in the blank group was significantly increased (p<0.01); at D8, the difference in food intake between each administration group and the model group Little; During D11, the food intake of positive group declines compared with model group; The model group was similar; the food intake of B. fragilis inactivated bacterial powder was similar to that of the model group, and the inactivated bacterial powder of NCTC 9343 was slightly lower than that of ZY-312 bacterial powder. There was no significant difference in drug efficacy in the Bacteroides fragilis inactivated bacterial powder group.

2) Stool wet weight, water content and diarrhea score:

The test results of wet weight and water content of animal feces in each group are shown in Table 7 below.

Table 7

Feces wet weight and water content statistical results of animals in each group (mean ± standard deviation, n = 10)

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

It can be seen from Table 7 that there is no significant difference in the wet weight of feces and the water content of feces from the end of modeling in the blank group to the end of the test. After modeling, the wet weight of feces in the model group and each administration group decreased compared with the blank group, and there was a significant difference in the positive group. At the end of the test, there was little difference in the wet weight of feces among the administration groups. However, after modeling, the water content of feces in the model group and each administration group was higher than that of the blank group. At the end of the test, the water content of feces in the model group was significantly different from that of the blank group. The positive group did not curb the rising trend of fecal water content, and even made this trend worse; the inactivated Bacteroides fragilis bacteria powder groups effectively curbed the rising trend of fecal water content, and the curbing effect of NCTC 9343 inactivated bacterial powder was worse than that of ZY- 312 bacteria powder. There is no significant difference in the efficacy of the inactivated bacteria powder in each group.

The diarrhea scores of animals in each group are shown in Table 8 below.

Table 8

The statistical result of the diarrhea score of each group of animals (mean ± standard deviation, n=5)

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

It can be seen from Table 8 that the animals in the model group and each administration group began to have diarrhea on the 4th day (D7). Compared with the blank group, the diarrhea score during the test period was significantly increased, and the diarrhea score increased with time.

For male animals, the positive group showed the same diarrhea score trend as the model group, but showed the effect of curbing the increase of diarrhea score; different from the positive group, each group of inactivated Bacteroides fragilis powder showed the effect of maintaining the stability of diarrhea score , although the scores of diarrhea in each dose group showed an increasing trend, the increase was less than that of the model group and the low dose group; the scores of D7 and D8 of each dose group were similar to those of the model group and the positive group, and they showed a restraining effect from D9. There was a big difference in the time of day. Shows a superior effect of curbing score rises. The effect of NCTC9343 inactivated bacteria powder was slightly worse than that of ZY-312 bacteria powder.

For female animals, the above pharmacodynamic results are basically similar.

There was no significant difference in the efficacy of Bacteroides fragilis inactivated bacteria powder within the group.

3) Inflammatory factor

The detection results of inflammatory factors in animals in each group are shown in Table 9 below.

Table 9

Statistical results of inflammatory factors in each group of animals (mean ± standard deviation, n=10)

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

It can be seen from Table 9 that compared with the blank group, the inflammatory factors TNF-α, IL-1β, IL-5, IL-6, IL-17, INF-γ and ICAM-1 proteins in the model group were significantly increased; the anti-inflammatory factors IL-4, IL-10, IL-13 decreased significantly. The positive group has a down-regulation effect on the level of inflammatory factors and an up-regulation effect on the level of anti-inflammatory factors. The Bacteroides fragilis inactivated bacterial powder group also showed similar regulatory effects on cytokines; the regulatory effect of NCTC 9343 inactivated bacterial powder was slightly worse than that of ZY-312 bacterial powder. There was no significant difference in drug efficacy within the group.

4) Pathological examination: the tissues of jejunum, ileum and colorectum were selected, and HE staining was used to evaluate the degree of pathological damage. The results are shown in Table 10.

Table 10

Intestinal pathological damage score of animals in each group (mean ± standard deviation, n = 10)

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

It can be seen from Table 10 that, compared with the blank group, multiple indicators (glandular expansion, intestinal mucosal injury, inflammatory cell infiltration, and mucosal hyperemia) in the model group showed malignant transformation. In some indicators, such as inflammatory cell infiltration, mucosal hyperemia, the positive drug has the ability to treat and contain the damage; in other indicators, such as glandular dilatation, the positive drug worsens the situation. Each group of the Bacteroides fragilis inactivated bacteria powder group showed a certain protective and therapeutic effect on intestinal damage. In intestinal mucosal injury, Bacteroides fragilis played a significant protective role; in mucosal congestion, although the curative effect was not as good as positive drugs, Bacteroides fragilis still had a therapeutic effect; in gland expansion, both ZY-312 and NCTC 9343 Played a therapeutic effect better than positive drugs. Among the above effects, the effect of ZY-312 is slightly higher than that of NCTC 9343.

The above experimental results show that the inactivated Bacteroides fragilis powder of the present invention can effectively prevent and treat side effects related to cancer treatment, especially diarrhea.

Exemplary embodiments of the present invention have been described above. However, the protection scope of the present application is not limited to the above-mentioned embodiments. Any modifications, equivalent replacements, improvements, etc. made by those skilled in the art within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. An inactivated Bacteroides fragilis bacteria powder, characterized in that it includes inactivated Bacteroides fragilis bacteria; the cells of the inactivated Bacteroides fragilis bacteria include a complete cell shape structure; the number of bacteria reaches 1×10 ¹¹ Cell/ more than g.
2. The inactivated bacteria powder of Bacteroides fragilis according to claim 1, characterized in that the inactivated bacteria powder of Bacteroides fragilis also includes auxiliary materials.

   Preferably, in the inactivated Bacteroides fragilis powder, the mass ratio of the inactivated Bacteroides fragilis to the auxiliary material is 1:(0.05-4).
3. The inactivated bacterium powder of Bacteroides fragilis according to claim 1 or 2, wherein the adjuvant comprises an excipient. Preferably, the excipients are solid at room temperature.

   Preferably, the excipients include mannitol, sorbitol, maltodextrin, lactose, sodium chloride, maltose, sucrose, glucose, trehalose, dextran, proline, lysine, alanine, casein , at least one or a combination of two or more of skim milk.

   Preferably, sodium chloride is included in the excipient.

   Preferably, in the excipient, the mass fraction of sodium chloride is 0-25wt%.
4. The preparation method of the inactivated Bacteroides fragilis powder according to any one of claims 1-3, characterized in that, in particular, the inactivated Bacteroides fragilis powder obtained by the preparation method has a cell structure of Completely, the preparation method comprises the following steps:

   (1) take Bacteroides fragilis fermentation culture;

   (2) After the fermentation culture is finished, the fermented liquid is centrifuged, and the thallus is collected, and the weight-to-volume ratio of the thallus and the sodium chloride aqueous solution is 1g: (10～30) mL, and the sodium chloride aqueous solution is added for washing and centrifugation to obtain the washed After the bacteria;

   (3) adding the first excipient solution to the washed bacterial cells to mix and resuspend to obtain the bacterial cell solution, then perform inactivation treatment, centrifuge, and collect the inactivated bacteria sludge;

   (4) adding the second excipient solution to the inactivated bacteria slime obtained in step (3) to obtain the inactivated bacteria stock solution;

   (5) drying the stock solution of inactivated bacteria obtained in step (4) until the residual moisture is lower than 5 wt%, to obtain the inactivated bacteria powder of Bacteroides fragilis.
5. The preparation method according to claim 4, characterized in that, in step (2), the number of bacteria in the fermentation broth reaches above 10 ⁸ CFU/mL.

   Preferably, in step (2), the mass concentration of the aqueous sodium chloride solution is 0.6-1.5wt%, preferably 0.65-1.2wt%, more preferably 0.8-1.0wt%, most preferably 0.85-0.95wt%, For example, 0.9 wt% sodium chloride aqueous solution.
6. The preparation method according to claim 4 or 5, characterized in that, in step (3), the weight-to-volume ratio of the bacteria to the first excipient solution is 1 g:(5-40) mL.

   Preferably, in step (3), in the first excipient solution, the mass fraction of the excipient is 4-30wt%.

   Preferably, in step (3), the solvent of the first excipient solution is selected from the sodium chloride aqueous solution described in claim 5. Further preferably, the solvent of the first excipient solution is selected from physiological saline, such as 0.9 wt% sodium chloride aqueous solution.
7. According to the preparation method according to any one of claims 4-6, it is characterized in that, in step (3), the method of the inactivation treatment is selected from at least one of heat inactivation, freezing inactivation or chemical inactivation , preferably heat inactivated.

   Preferably, the heat inactivation temperature is 60-100° C., and the heat inactivation time is 10-60 minutes.
8. According to the preparation method described in any one of claims 4-7, it is characterized in that, in step (4), the second excipient solution is added so that the total weight of the inactivated bacteria stock solution is the same as that of the bacteria before the inactivation in step (3). The weight of the body solution is the same. Preferably, said second excipient solution is the same or different from said first excipient solution.

   Preferably, in the second excipient solution, the mass fraction of the excipient is 4-30wt%.

   Preferably, the solvent of the second excipient solution is selected from the aqueous sodium chloride solution described in claim 5. Further preferably, the solvent of the second excipient solution is selected from physiological saline, such as 0.9 wt% sodium chloride aqueous solution.
9. The preparation method according to any one of claims 4-8, characterized in that, in step (5), the drying method is selected from vacuum freeze-drying and/or spray drying, preferably vacuum freeze-drying.

   Preferably, the vacuum freeze-drying conditions include: a freezing temperature of -20-40° C., a freezing time of 1-3 hours, and a vacuum degree of 0.20-0.25 mbar.
10. A pharmaceutical preparation, characterized in that the pharmaceutical preparation contains the inactivated Bacteroides fragilis powder according to any one of claims 1-3.

    Preferably, the pharmaceutical preparation is selected from at least one or a combination of two or more of oral administration preparations, enemas or gels.

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