Thermal inactivation method and application of ackermania muciniphila

Abstract

The invention relates to a thermal inactivation method of Akkermansia muciniphila and an application of the thermal inactivation method. The thermal inactivation method provided by the invention comprises the following steps: carrying out thermal inactivation on an AKK bacterium fermentation liquid; wherein the AKK bacterium fermentation liquor is mixed liquor containing AKK bacterium viable thalli and a fermentation culture medium; the AKK bacterium comprises one or more of an AM02 bacterium (the preservation number is CGMCC (China General Microbiological Culture Collection Center) No. 22794) and an AM06 bacterium (the preservation number is CGMCC No. 22793). The invention also relates to inactivated AKK bacteria prepared by adopting the thermal inactivation method. By adopting the thermal inactivation method, the thermal inactivation temperature is low, the inactivation efficiency is high, the AKK viable bacteria cannot be detected, and the prepared inactivated bacteria product is high in safety, good in thallus form integrity, free of breakage and capable of effectively protecting effective active ingredients in the thallus; the thallus yield is high, the process is simple, and the production is easy to operate and control.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and further to a method for heat inactivation of Akkermansia muciniphila and an application thereof, and further to a method for heat inactivation of Akkermansia muciniphila and a method for preparing inactivated Akkermansia muciniphila. Background Art

[0002] Akkermansia muciniphila (AKK or AKK bacteria for short) belongs to the phylum Verrucomicrobia, family Akkermansiaceae, and genus Akkermansia. AKK bacteria is a Gram-negative bacterium. AKK bacteria are widely distributed in the intestinal mucus layer of humans and animals and are one of the high-abundance symbiotic bacteria in the intestine. Studies have found that AKK bacteria are associated with a variety of systemic diseases such as obesity, diabetes, immune regulation, alcoholic fatty liver disease, and coronary artery sclerosis. AKK content is one of the important markers of intestinal microecological balance. Based on a series of important discoveries on the physiological functions of AKK bacteria in recent years, especially the important role of AKK bacteria in tumor immunotherapy, AKK bacteria have been hailed as an important candidate strain of "second-generation probiotics (NGP)" with great application and development value - living biological therapeutic drugs (LBP).

[0003] After heat inactivation, AKK bacteria can be developed into inanimate microorganisms and / or preparations containing AKK components that are beneficial to host health, namely postbiotics. Compared with live bacterial preparations, postbiotics have the advantages of good stability, easy storage and transportation, high safety, and good tolerance. According to literature reports, inactivated AKK bacteria can also play a probiotic and therapeutic role, such as: inactivated AKK bacteria can alleviate obesity in mice and reduce food energy efficiency. In the brown and white fat of mice that orally inactivated Akk bacteria, the expression of lipid droplet regulator perilipin2-related proteins is downregulated; inactivated AKK bacteria can alleviate periodontitis induced by Porphyromonas gingivalis; pasteurized AKK bacteria and live AKK bacteria can significantly reduce the fecal and systemic burden of Salmonella in the streptomycin-treated mouse infection model, and reduce inflammation during infection; inactivated AKK bacteria have anti-allodynia and neurosuppressive properties, which may help relieve pain and anxiety in IBS patients. Some literature reports that the effectiveness of inactivated AKK bacteria is better than that of live bacteria. For example, AKK bacteria can improve obesity and insulin resistance index in mice, reduce plasma triglyceride levels, etc., and pasteurized AKK bacteria are better than live bacteria; pasteurized AKK bacteria can increase the patient's insulin sensitivity island (about 30%), reduce insulinemia and plasma total cholesterol, while live AKK bacteria do not have this effect; AKK can effectively improve obesity by improving mouse food intake, reducing fat cell volume, and reducing blood sugar content, among which the effect of inactivated AKK bacteria and its extracellular vesicles is significantly better than that of live AKK bacteria; AKK can reduce the weight of adipose tissue, improve glucose homeostasis, and regulate inflammation levels, among which inactivated AKK bacteria are significantly better than live bacteria in improving glucose tolerance; pasteurized Akk bacteria are more effective than live Akk bacteria in increasing the concentrations of intestinal polyamines, short-chain fatty acids, 2-hydroxybutyric acid and various bile acids.

[0004] The inactivated AKK bacteria can exert probiotic and therapeutic effects, which is directly related to the integrity of the bacteria and the effectiveness of the active ingredients. Studies have reported that the probiotic and therapeutic effects of AKK bacteria are related to its outer membrane proteins to a certain extent. For example, in 2017, Patrice D Cani's team found in the journal Nature Medicine that the pilus-like outer membrane protein Amuc_1100 of AKK bacteria can independently play a role in partial fat reduction and maintenance of intestinal barrier function. The protein has a stable structure at 70°C. In 2021, the National University of Seoul in South Korea revealed the key to the bidirectional regulation of AKK bacteria, a protein P9 on the outer membrane, which can stimulate the increase of GLP-1 levels, and explained the mechanism of AKK bacteria to improve obesity from the perspective of brown adipose tissue (BAT) heat production. On April 6, 2024, Korean researchers published a blockbuster study in the journal Nature Communications: Amuc_1409, an outer membrane protein secreted by AKK bacteria, can significantly improve intestinal health, especially when the intestine is damaged or aged. In 2021, the pasteurized AKK bacteria (Akkermansia muciniphila ATCC BAA-835 strain) developed by A-Mansia Biotech was approved by the EU EFSA as a new food (NF 2019 / 1366, EFSA-Q-2019-00767). Its control standard for live AKK bacteria in the product is "less than 10 CFU / g", but live AKK bacteria can still be detected in the final product. The inactivation efficiency of its inactivation process is not high, resulting in incomplete inactivation, and there may also be safety hazards. However, if the inactivation temperature is too high, the AKK bacteria and its effective active ingredients (such as outer membrane proteins, etc.) may be damaged, ruptured or inactivated. Therefore, in the production process of inactivated AKK bacteria, bacterial inactivation technology is a key step in the production process, and the control of live bacterial residues and the integrity of inactivated bacteria are key technical control points and monitoring points for quality. How to improve the inactivation efficiency of AKK bacteria while maintaining the integrity of the inactivated AKK bacteria and the effectiveness of the active ingredients of AKK bacteria is an urgent problem to be solved in actual production. Summary of the invention

[0005] Based on this, the present application provides a method for thermal inactivation of Akkermansia muciniphila and its application, and further provides a method for thermal inactivation of Akkermansia muciniphila and a method for preparing inactivated Akkermansia muciniphila.

[0006] In a first aspect of the present application, a method for thermal inactivation of Akkermansia muciniphila is provided, comprising the following steps:

[0007] The AKK bacteria fermentation liquid is heat-inactivated; wherein the AKK bacteria fermentation liquid is a mixture containing live AKK bacteria and a fermentation medium; the AKK bacteria in the AKK bacteria fermentation liquid is Akkermansia muciniphila;

[0008] AKK bacteria include one or more of AM02 bacteria and AM06 bacteria;

[0009] AM02 is Akkermansia muciniphila AM02, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on June 28, 2021, with the deposit number CGMCC No.22794;

[0010] The AM06 bacterium is Akkermansia muciniphila AM06. Akkermansia muciniphila AM06 was deposited in the General Microbiology Center of China National Microbiological Culture Collection on June 28, 2021, with the deposit number CGMCC No.22793.

[0011] In a second aspect of the present application, a method for preparing inactivated Akkermansia muciniphila is provided, comprising the following steps:

[0012] Provide AKK bacteria fermentation liquid;

[0013] The AKK bacterial fermentation broth is heat-inactivated;

[0014] After the heat inactivation is completed, the bacteria are collected to obtain the inactivated AKK bacteria;

[0015] Among them, the AKK bacteria are Akkermansia muciniphila; the AKK bacteria include one or more of AM02 bacteria and AM06 bacteria.

[0016] The thermal inactivation method of Akkermansia muciniphila provided in the present application adopts low-temperature thermal inactivation treatment, which is different from traditional high-temperature sterilization, has high inactivation efficiency (can achieve that live AKK bacteria cannot be detected), good bacterial morphological integrity, can achieve no fragmentation, can retain the effective active ingredients in the inactivated bacteria to a high degree, and in addition, the thermal inactivation method has a high bacterial yield, a simple process, and easy operation and control in production. The prepared inactivated bacteria product has good stability and high safety. The thermal inactivation method can meet the requirements of industrial scale-up production.

[0017] The thermal inactivation method of Akkermansia muciniphila provided in the present application has a low thermal inactivation temperature and a high inactivation efficiency, and can achieve that live Akkermansia muciniphila cannot be detected, and the obtained inactivated bacteria product has high safety.

[0018] The present application provides a method for thermal inactivation of Akkermansia muciniphila. In the inactivated bacteria product prepared, the bacteria have good morphological integrity and can be free of breakage, thereby effectively protecting the effective active ingredients in the bacteria.

[0019] The thermal inactivation method of Akkermansia muciniphila provided in the present application has high bacterial yield, simple process, easy operation and control in production, and the prepared inactivated bacteria product can meet the industrial requirements of edible value and / or medicinal value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0021] Figure 1 These are transmission electron microscopy (TEM) images of the room temperature group of AM06, AM02 and ATCC BAA 835 strains in Experimental Example 1 (heat inactivation process A, 1L scale).

[0022] Figure 2 Transmission electron microscopy images of AM06, AM02 and ATCC BAA 835 strains heat-inactivated at 60°C for 60 min in Experimental Example 1 (heat-inactivation process A, 1 L scale).

[0023] Figure 3Transmission electron microscopy images of AM06, AM02 and ATCC BAA 835 strains heat-inactivated at 70°C for 60 min in Experimental Example 1 (heat-inactivation process A, 1 L scale).

[0024] Figure 4 This is a transmission electron microscopy image of AM06, AM02 and ATCC BAA 835 strains heat-inactivated at 80°C for 30 min in Experimental Example 1 (heat-inactivation process A, 1L scale).

[0025] Figure 5 Transmission electron microscopy images of AM06, AM02 and ATCC BAA 835 strains heat-inactivated at 100°C for 30 min in Experimental Example 1 (heat-inactivation process A, 1 L scale).

[0026] Figure 6 Transmission electron microscopy images of the room temperature group of AM06, AM02 and ATCC BAA 835 strains in Experimental Example 3 (heat inactivation process B, 1L scale).

[0027] Figure 7 This is a transmission electron microscopy image of AM06, AM02 and ATCC BAA 835 strains heat-inactivated at 60°C for 60 min in Experimental Example 3 (heat-inactivation process B, 1 L scale).

[0028] Figure 8 Transmission electron microscopy images of AM06, AM02 and ATCC BAA 835 strains heat-inactivated at 70°C for 60 min in Experimental Example 3 (heat-inactivation process B, 1 L scale).

[0029] Fig. 9 This is a transmission electron microscopy image of AM06, AM02 and ATCC BAA 835 strains heat-inactivated at 80°C for 30 min in Experimental Example 3 (heat-inactivation process B, 1L scale).

[0030] Fig.10 Transmission electron microscopy images of AM06, AM02 and ATCC BAA 835 strains heat-inactivated at 100°C for 30 min in Experimental Example 3 (heat-inactivation process B, 1 L scale).

[0031] Fig.11 Schematic diagram of the process of thermal inactivation of AKK bacteria in one embodiment of the present application.

[0032] Fig.12 It is a schematic flow chart of a method for preparing inactivated AKK bacteria in one embodiment of the present application. DETAILED DESCRIPTION

[0033] The present application will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and are not used to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present application, and the equivalent form obtained also falls within the protection scope of the present application. For example, the features described or described as part of an embodiment can be combined in another embodiment in a suitable manner to produce a new embodiment. In addition, in the description below, a large amount of details are given in order to provide a more comprehensive understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing implementation modes and embodiments and are not intended to limit this application.

[0035] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0036] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items. "Any and all combinations" include any combination of two related listed items, any more related listed items, or all related listed items. For example, "A and / or B" includes three parallel solutions: A, B, and "a combination of A and B".

[0037] In the present application, "multiple", "multiple", "multiple times", "multiple", "several", etc., unless otherwise specified, refer to a quantity greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0038] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and the like, when used in other ways to mean "one or more", are also understood in the same way unless otherwise specified.

[0039] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0040] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0041] In this application, "preferred", "better", "more preferred", "suitable", "better", and "preferable" are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of this application. If multiple "preferred" items appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "preferred" item is independent.

[0042] In this application, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent different technical solutions are related in terms of the content covered, but should not be understood as a limitation on the previous technical solution, nor can it be understood as a limitation on the protection scope of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0043] In this application, "optionally", "optional", and "optional" refer to being optional, that is, to being selected from either of the two parallel schemes of "yes" or "no". If multiple "optional" items appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" item is independent of each other. Unless otherwise specified, descriptions such as "optionally include" and "optionally contain" in this application, taking "optionally include" as an example, mean "may include or not include". "Optional component X" means that component X exists or does not exist.

[0044] The terms "contain", "include" and "include" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0045] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0046] The temperature parameters in this application, unless otherwise specified, allow both constant temperature treatment and fluctuations within a certain temperature range. It should be understood that constant temperature treatment allows the temperature to fluctuate within the accuracy range of instrument control. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0047] In the present application, the term "room temperature" or "normal temperature" generally refers to 10°C to 30°C, for example, 20°C±5°C. In some embodiments of the present application, "room temperature" or "normal temperature" refers to 20°C to 30°C.

[0048] In this application, if the unit of the data range is followed only by the right endpoint, it means that the units of the left endpoint and the right endpoint are the same. For example, 3~5h means that the units of the left endpoint "3" and the right endpoint "5" are both h (hours), which have the same meaning as 3h~5h. In addition, similar descriptions involving other parameters such as temperature, concentration, speed, etc. are also applicable to the above understanding.

[0049] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0050] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0051] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form new technical solutions.

[0052] In this application, the terms "first", "second", etc. in "the first aspect", "the second aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0053] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0054] In this application, "value" refers to the number and its reasonable approximations. The definition of "value" can be applied to discrete numerical points or to the endpoints of a numerical range. Unless otherwise specified, "approximation" refers to a numerical interval based on the reasonable fluctuation range of the number. The reasonable fluctuation range may vary depending on the type and value of the number. The reasonable fluctuation range can be reasonably confirmed based on the accuracy of the test or measurement method.

[0055] When it comes to numerical values ​​or numerical ranges, unless otherwise specified, it should be understood that the numerical value covers its reasonable approximations, and the numerical range covers reasonable approximations of the two endpoints. Those skilled in the art can understand that the acceptable fluctuation range of the approximation can be included in the interval covered by the indicated numerical value or numerical range. That is, in this application, unless otherwise specified, "N1" can be reasonably understood as "about N1", and "N1-N2" can be reasonably understood as "about N1 to about N2", where N1 and N2 are two unequal numerical values. For example, in some cases, due to one or more factors such as reasonable deviations allowed in the art, instrument control accuracy, etc., the approximate values ​​within the approximate range should also be included in the range indicated by the numerical range; for example, "concentration is 5.8g / L~6.2g / L" can be understood as "about 5.8g / L to about 6.2g / L"; further, taking the left endpoint as "1g / L" and its approximate value of ±0.1g / L as an example, the approximate values ​​of 0.9g / L, 0.95g / L, etc. within the approximate range shown as "about 1g / L" should also be included in the range indicated by 1g / L~4g / L.

[0056] In this article, unless otherwise specified, "about" means within a certain range above or below the number, and the fluctuation range may vary depending on the type and value of the number. For example, it is allowed to be within a range of ±10%, ±5%, ±2%, ±1%, etc. For example, about 1g / L can be selected from 1±0.1g / L.

[0057] In the present application, unless otherwise specified, "Akkermansia muciniphila" and "Akkermansia muciniphila" have the same meaning and can be used interchangeably.

[0058] In the first aspect of the present application, a method for thermal inactivation of Akkermansia muciniphila is provided (see Fig.11 ), which comprises the following steps:

[0059] S200: heat-inactivating the AKK bacteria fermentation liquid; wherein the AKK bacteria fermentation liquid is a mixed liquid containing live AKK bacteria and a fermentation medium; the AKK bacteria in the AKK bacteria fermentation liquid is Akkermansia muciniphila;

[0060] AKK bacteria include one or more of AM02 bacteria and AM06 bacteria;

[0061] AM02 is Akkermansia muciniphila AM02, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on June 28, 2021, with the deposit number CGMCC No.22794;

[0062] The AM06 bacterium is Akkermansia muciniphila AM06. Akkermansia muciniphila AM06 was deposited in the General Microbiology Center of China National Microbiological Culture Collection on June 28, 2021, with the deposit number CGMCC No.22793.

[0063] The Akkermansia muciniphila AM06 involved in this application, whose classification name is Akkermansia muciniphila, was deposited in the General Microbiology Center of China Culture Collection Administration on June 28, 2021, with the address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 22793; the strain was received and registered by the collection center on June 28, 2021, and was detected as a viable strain by the collection center on June 28, 2021.

[0064] The Akkermansia muciniphila AM02 involved in this application, whose classification name is Akkermansia muciniphila, was deposited in the General Microbiology Center of China Culture Collection Administration on June 28, 2021, with the address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 22794; the strain was received and registered by the collection center on June 28, 2021, and was detected as a viable strain by the collection center on June 28, 2021.

[0065] In the present application, “Akkermansia muciniphila AM06” may be referred to as “AM06” or “AM06 bacteria”.

[0066] In the present application, “Akkermansia muciniphila AM02” may be referred to as “AM02” or “AM02 bacteria”.

[0067] For different AKK bacteria, fine-tuning of sterilization conditions will lead to huge differences in inactivation efficiency, bacterial yield, and bacterial morphology, as shown in the following Experimental Example 3. Different AKK bacteria require different heat inactivation conditions that match the characteristics of the bacteria.

[0068] The thermal inactivation method of Akkermansia muciniphila provided in the present application adopts low-temperature thermal inactivation treatment, which is different from traditional high-temperature sterilization, has high inactivation efficiency (can achieve that live AKK bacteria cannot be detected), good bacterial morphological integrity, can achieve no fragmentation, can retain the effective active ingredients in the inactivated bacteria to a high degree, and in addition, the thermal inactivation method has a high bacterial yield, a simple process, and easy operation and control in production. The prepared inactivated bacteria product has good stability and high safety. The thermal inactivation method can meet the requirements of industrial scale-up production.

[0069] The thermal inactivation method of Akkermansia muciniphila provided in the present application has a low thermal inactivation temperature and a high inactivation efficiency, and can achieve that live Akkermansia muciniphila cannot be detected, and the obtained inactivated bacteria product has high safety.

[0070] The thermal inactivation method of Akkermansia muciniphila provided in the present application has good bacterial morphological integrity, can be achieved without fragmentation, and can effectively protect the effective active ingredients in the bacterial body.

[0071] The thermal inactivation method of Akkermansia muciniphila provided in the present application has high bacterial yield, simple process, easy operation and control in production, and the prepared inactivated bacteria product can meet the industrial requirements of edible value and / or medicinal value.

[0072] In some embodiments, the sum of the percentages of the number of AM02 bacteria and AM06 bacteria in the AKK bacteria is 80% to 100%.

[0073] In some embodiments, the AKK bacteria are selected from one or more of AM02 bacteria and AM06 bacteria.

[0074] In some embodiments, the fermentation medium is a non-animal derived medium.

[0075] In the present application, the non-animal-derived culture medium can be used as a fermentation medium or a seed medium.

[0076] In some embodiments, the non-animal-derived culture medium contains N-acetyl-D-glucosamine, soy peptone, yeast extract powder, potassium dihydrogen phosphate, sodium carbonate, glucose, L-threonine, ammonium chloride, Tween 80, L-cysteine ​​hydrochloride, and water.

[0077] In this application, unless otherwise specified, "yeast extract powder" refers to a liquid, paste or powder product made from baker's yeast or brewer's yeast through a series of processing steps such as autolysis and enzymolysis, cell wall separation, concentration and drying. Yeast extract powder contains ingredients such as amino acids, polypeptides, proteins, vitamins, trace elements, etc., and can be used to provide nutritional factors such as nitrogen sources and growth factors for the growth of microorganisms. Yeast extract powder can be purchased commercially.

[0078] Without limitation, the concentration of N-acetyl-D-glucosamine in the non-animal derived culture medium can be 5 g / L to 12 g / L, can be 5 g / L to 8 g / L, can be further selected as 5.5 g / L to 6.5 g / L, can be further selected as 5.8 g / L to 6.2 g / L, and can also be any of the following concentrations or a range consisting of any two of the following concentrations: 5 g / L, 5.5 g / L, 5.6 g / L, 5.8 g / L, 6 g / L, 6.2 g / L, 6.4 g / L, 6.5 g / L, 7 g / L, 7.5 g / L, 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, 10 g / L, 11 g / L, 12 g / L, etc.

[0079] Without limitation, the concentration of soy peptone in the non-animal-derived culture medium can be 25 g / L to 40 g / L, optionally 26 g / L to 30 g / L, or any of the following concentrations or a range consisting of any two of the following concentrations: 25 g / L, 26 g / L, 28 g / L, 30 g / L, 32 g / L, 34 g / L, 35 g / L, 36 g / L, 38 g / L, 40 g / L, etc.

[0080] Without limitation, the concentration of yeast extract in the non-animal derived culture medium can be 12 g / L to 20 g / L, optionally 14 g / L to 18 g / L, further optionally 15.5 g / L to 16.5 g / L, and can also be any of the following concentrations or a range consisting of any two of the following concentrations: 12 g / L, 12.5 g / L, 13 g / L, 13.5 g / L, 14 g / L, 14.5 g / L, 15 g / L, 15.5 g / L, 16 g / L, 16.5 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, etc.

[0081] Without limitation, the concentration of potassium dihydrogen phosphate in the non-animal derived culture medium can be 2g / L to 5g / L, can be 3g / L to 4g / L, can be further selected as 3.2g / L to 3.6g / L, can be further selected as 3.3g / L to 3.5g / L, can also be any of the following concentrations or a range consisting of any two of the following concentrations: 2g / L, 2.5g / L, 3g / L, 3.1g / L, 3.2g / L, 3.3g / L, 3.4g / L, 3.5g / L, 3.6g / L, 3.8g / L, 4g / L, 4.5g / L, 5g / L, etc.

[0082] Without limitation, the concentration of glucose in the non-animal derived culture medium can be 1g / L to 4g / L, can be 1g / L to 3g / L, can be further selected as 1.5g / L to 2.5g / L, can be further selected as 1.8g / L to 2.2g / L, and can also be any of the following concentrations or a range consisting of any two of the following concentrations: 1g / L, 1.2g / L, 1.4g / L, 1.5g / L, 1.6g / L, 1.8g / L, 2g / L, 2.2g / L, 2.4g / L, 2.5g / L, 2.6g / L, 2.8g / L, 3g / L, 3.2g / L, 3.4g / L, 3.5g / L, 3.6g / L, 3.8g / L, 4g / L, etc.

[0083] Without limitation, the concentration of sodium carbonate in the non-animal derived culture medium can be 1g / L to 4g / L, can be 2g / L to 4g / L, can be further selected as 2.5g / L to 3.5g / L, can be further selected as 2.8g / L to 3.2g / L, and can also be any of the following concentrations or a range consisting of any two of the following concentrations: 1g / L, 1.2g / L, 1.4g / L, 1.5g / L, 1.6g / L, 1.8g / L, 2g / L, 2.2g / L, 2.4g / L, 2.5g / L, 2.6g / L, 2.8g / L, 3g / L, 3.2g / L, 3.4g / L, 3.5g / L, 3.6g / L, 3.8g / L, 4g / L, etc.

[0084] Without limitation, the concentration of L-threonine in the non-animal derived culture medium can be 1 g / L to 4 g / L, can be 2 g / L to 4 g / L, can be further selected as 2.5 g / L to 3.5 g / L, can be further selected as 2.8 g / L to 3.2 g / L, and can also be any of the following concentrations or a range consisting of any two of the following concentrations: 1 g / L, 1.2 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.8 g / L, 2 g / L, 2.2 g / L, 2.4 g / L, 2.5 g / L, 2.6 g / L, 2.8 g / L, 3 g / L, 3.2 g / L, 3.4 g / L, 3.5 g / L, 3.6 g / L, 3.8 g / L, 4 g / L, etc.

[0085] Without limitation, the concentration of ammonium chloride in the non-animal derived culture medium can be 0.1g / L to 1g / L, can be 0.2g / L to 0.8g / L, can be further 0.4g / L to 0.6g / L, can be further 0.45g / L to 0.55g / L, can also be any of the following concentrations or a range consisting of any two of the following concentrations: 0.1g / L, 0.2g / L, 0.3g / L, 0.4g / L, 0.45g / L, 0.5g / L, 0.55g / L, 0.6g / L, 0.7g / L, 0.8g / L, 0.9g / L, 1g / L, etc.

[0086] Without limitation, the concentration of Tween 80 in the non-animal derived culture medium can be 0.5g / L to 2g / L, can be 0.5g / L to 1.5g / L, can be further 0.8g / L to 1.2g / L, can be further 0.9g / L to 1.1g / L, can be any of the following concentrations or a range consisting of any two of the following concentrations: 0.5g / L, 0.6g / L, 0.7g / L, 0.8g / L, 0.9g / L, 1g / L, 1.1g / L, 1.2g / L, 1.3g / L, 1.4g / L, 1.5g / L, 1.6g / L, 1.8g / L, 2g / L, etc.

[0087] Without limitation, the concentration of L-cysteine ​​hydrochloride in the non-animal derived culture medium can be 0.1 g / L to 1 g / L, can be 0.2 g / L to 0.8 g / L, can be further 0.4 g / L to 0.6 g / L, can be further 0.45 g / L to 0.55 g / L, can be any of the following concentrations or a range consisting of any two of the following concentrations: 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.45 g / L, 0.5 g / L, 0.55 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, etc.

[0088] Without limitation, the non-animal-derived culture medium may comprise 5 g / L to 12 g / L of N-acetyl-D-glucosamine, 25 g / L to 40 g / L of soy peptone, 12 g / L to 20 g / L of yeast extract, 2 g / L to 5 g / L of potassium dihydrogen phosphate, 1 g / L to 4 g / L of glucose, 1 g / L to 4 g / L of sodium carbonate, 1 g / L to 4 g / L of L-threonine, 0.1 g / L to 1 g / L of ammonium chloride, 0.5 g / L to 2 g / L of Tween 80, 0.1 g / L to 1 g / L of L-cysteine ​​hydrochloride, and water; optionally, the water is the balance.

[0089] In some embodiments, the non-animal-derived culture medium may include 5.8 g / L to 6.2 g / L of N-acetyl-D-glucosamine, 26 g / L to 30 g / L of soy peptone, 15.5 g / L to 16.5 g / L of yeast extract, 3.3 g / L to 3.5 g / L of potassium dihydrogen phosphate, 1.8 g / L to 2.2 g / L of glucose, 2.8 g / L to 3.2 g / L of sodium carbonate, 2.8 g / L to 3.2 g / L of L-threonine, 0.45 g / L to 0.55 g / L of ammonium chloride, 0.9 g / L to 1.1 g / L of Tween 80, 0.45 g / L to 0.55 g / L of L-cysteine ​​hydrochloride, and water, optionally, the water is the balance.

[0090] In some embodiments, the non-animal-derived culture medium may include 6 g / L of N-acetyl-D-glucosamine, 28 g / L of soy peptone, 16 g / L of yeast extract, 3.4 g / L of potassium dihydrogen phosphate, 2 g / L of glucose, 3 g / L of sodium carbonate, 3 g / L of L-threonine, 0.5 g / L of ammonium chloride, 1 g / L of Tween 80, 0.5 g / L of L-cysteine ​​hydrochloride and water; optionally, water is the balance.

[0091] In some embodiments, the steps of preparing the AKK bacteria fermentation broth include:

[0092] S110: inoculating AKK bacteria into a seed culture medium for activation culture to prepare a seed solution;

[0093] S120: inoculating the seed liquid into the fermentation medium for fermentation culture.

[0094] In some embodiments, the heat inactivation method meets one or more of the following characteristics:

[0095] (a1) After the fermentation culture is completed and before heat inactivation, the pH value of the fermentation liquid is adjusted to 6.5-9.0, and can also be any of the following values ​​or a range consisting of any two of the following values: 6.5, 6.6, 6.8, 7.0, 7.2, 7.4, 7.5, 7.6, 7.8, 8.0, 8.2, 8.4, 8.5, 8.6, 8.8, 9.0, etc.;

[0096] (a2) the seed culture medium is a non-animal-derived culture medium, and the composition of the non-animal-derived culture medium can refer to the context of the present application; without limitation, the seed culture medium can contain 5 g / L to 12 g / L of N-acetyl-D-glucosamine, 25 g / L to 40 g / L of soy peptone, 12 g / L to 20 g / L of yeast extract powder, 2 g / L to 5 g / L of potassium dihydrogen phosphate, 1 g / L to 4 g / L of glucose, 1 g / L to 4 g / L of sodium carbonate, 1 g / L to 4 g / L of L-threonine, 0.1 g / L to 1 g / L of ammonium chloride, 0.5 g / L to 2 g / L of Tween 80, 0.1 g / L to 1 g / L of L-cysteine ​​hydrochloride and water;

[0097] (a3) The conditions for performing activation culture include: performing activation culture under anaerobic conditions, the temperature of activation culture is 36° C. to 38° C., and the activation culture is static culture; without limitation, the temperature of activation culture can also be any of the following temperatures or ranges: 37±0.2° C., 37±0.4° C., 37±0.5° C., 37±0.1° C., 36.5° C. to 37.5° C., etc.;

[0098] (a4) The conditions for performing activation culture include: the number of activation culture is 1 to 3 times (e.g., 1, 2 or 3 times), and the duration of each activation culture is 20 hours to 30 hours; without limitation, the duration of each activation culture can also be any of the following durations or a range consisting of any two of the following durations: 20 hours, 22 hours, 24 hours, 25 hours, 26 hours, 28 hours, 30 hours, etc.;

[0099] (a5) The conditions for fermentation culture include: fermentation culture under anaerobic conditions, the fermentation culture temperature is 36°C to 38°C, the fermentation culture is carried out under stirring conditions, the stirring speed is 50rpm to 100rpm, and the fermentation culture duration is 9h to 24h; without limitation, the fermentation culture temperature can also be any of the following temperatures or ranges: 37±0.2°C, 37±0.4°C, 37±0.5°C, 37±0.1°C, 36.5°C to 37.5°C, etc.; without limitation, the stirring speed can also be any of the following speeds or a range selected from any two of the following speeds: 50rpm, 60rpm, 70rpm, 80rpm, 90rpm, 100rpm, etc.; without limitation, the fermentation culture duration can also be any of the following durations or a range selected from any two of the following durations: 9h, 10h, 12h, 14h, 15h, 16h, 18h, 20h, 22h, 24h, etc.;

[0100] (a6) The volume of the fermentation system for fermentation culture is 1L to 100L, and can also be any of the following volumes or a range consisting of any two of the following volumes: 1L, 5L, 10L, 15L, 20L, 25L, 30L, 40L, 50L, 60L, 70L, 75L, 80L, 90L, 100L, etc.

[0101] In some embodiments, the heat inactivation method meets one or more of the following characteristics:

[0102] (b1) The temperature for heat inactivation is 60°C to 80°C, and can also be any one of the following temperatures or a range consisting of any two of the following temperatures: 60°C, 62°C, 64°C, 65°C, 66°C, 68°C, 70°C, 72°C, 74°C, 75°C, 76°C, 78°C, 80°C, etc.;

[0103] (b2) thermal inactivation is carried out under stirring conditions, and the stirring speed is 100 rpm to 500 rpm, and can also be any one of the following speeds or a range consisting of any two of the following speeds: 100 rpm, 150 rpm, 200 rpm, 250 rpm, 300 rpm, 350 rpm, 350 rpm, 400 rpm, 450 rpm, 500 rpm, etc.;

[0104] (b3) The duration of heat inactivation is 10 min to 60 min, and may be any of the following durations or a range consisting of any two of the following durations: 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, etc.;

[0105] (b4) The pH condition for heat inactivation is 6.5-9.0, and can also be any of the following values ​​or a range consisting of any two of the following values: 6.5, 6.6, 6.8, 7.0, 7.2, 7.4, 7.5, 7.6, 7.8, 8.0, 8.2, 8.4, 8.5, 8.6, 8.8, 9.0, etc.

[0106] In some embodiments, the heat inactivation method meets one or more of the following characteristics:

[0107] (c1) the fermentation medium and the seed medium each independently contain 6 g / L of N-acetyl-D-glucosamine, 28 g / L of soy peptone, 16 g / L of yeast extract, 3.4 g / L of potassium dihydrogen phosphate, 2 g / L of glucose, 3 g / L of sodium carbonate, 3 g / L of L-threonine, 0.5 g / L of ammonium chloride, 1 g / L of Tween 80, 0.5 g / L of L-cysteine ​​hydrochloride and water; optionally, the water is the balance;

[0108] (c2) the fermentation medium and the seed medium each independently contain 5.8 g / L to 6.2 g / L of N-acetyl-D-glucosamine, 26 g / L to 30 g / L of soy peptone, 15.5 g / L to 16.5 g / L of yeast extract, 3.3 g / L to 3.5 g / L of potassium dihydrogen phosphate, 1.8 g / L to 2.2 g / L of glucose, 2.8 g / L to 3.2 g / L of sodium carbonate, 2.8 g / L to 3.2 g / L of L-threonine, 0.45 g / L to 0.55 g / L of ammonium chloride, 0.9 g / L to 1.1 g / L of Tween 80, 0.45 g / L to 0.55 g / L of L-cysteine ​​hydrochloride, and the balance of water;

[0109] (c3) the fermentation medium and the seed medium are each independently a non-animal-derived medium, and further, the non-animal-derived medium may be a medium made of food-grade raw materials or a medium made of pharmaceutical-grade raw materials;

[0110] (c4) After the fermentation culture is completed and before the heat inactivation is performed, the pH value of the fermentation liquid is adjusted to 8.0-8.5, and can also be any of the following values ​​or a range consisting of any two of the following values: 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, etc.;

[0111] (c5) The activation culture temperature is 37±0.5°C, and any suitable activation culture temperature may also be referred to in the context of this application;

[0112] (c6) The conditions for performing activation culture include: the number of activation culture is 2 to 3 times (such as 2 or 3 times), and the duration of each activation culture is 20 hours to 30 hours; without limitation, the duration of each activation culture can also be any of the following durations or a range consisting of any two of the following durations: 20 hours, 22 hours, 24 hours, 25 hours, 26 hours, 28 hours, 30 hours, etc.;

[0113] (c7) The activation culture is performed 2 or 3 times, the duration of the first activation culture is 22 hours to 24 hours, and the duration of the second activation culture is 20 hours to 22 hours; without limitation, the duration of the first activation culture can also be any of the following durations or a range consisting of any two of the following durations: 22 hours, 22.5 hours, 23 hours, 23.5 hours, 24 hours, etc.; without limitation, the duration of the second activation culture can also be any of the following durations or a range consisting of any two of the following durations: 20 hours, 20.5 hours, 21 hours, 21.5 hours, 22 hours, etc.;

[0114] (c8) the fermentation temperature is 37±0.5°C, and any suitable fermentation temperature in the context of this application may also be referred to;

[0115] (c9) The fermentation culture duration is 10 h to 14 h, and can also be any of the following durations or a range consisting of any two of the following durations: 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, etc.;

[0116] (c10) The volume of the fermentation system for fermentation culture is 1 L to 100 L, and any suitable volume of the fermentation system in the context of this application may also be referred to;

[0117] (c11) The temperature for heat inactivation is 70±2°C or 70±1°C. Please refer to any suitable heat inactivation temperature in the context of this application.

[0118] In the second aspect of the present application, a method for preparing inactivated Akkermansia muciniphila is provided, that is, a method for preparing inactivated AKK bacteria is provided, which comprises the following steps (see Fig.12 ):

[0119] S100: providing AKK bacteria fermentation liquid;

[0120] S200: heat-inactivating the AKK bacterial fermentation broth;

[0121] S300: collecting the bacteria after the heat inactivation is completed to obtain the inactivated AKK bacteria;

[0122] Among them, the AKK bacteria are Akkermansia muciniphila; the AKK bacteria include one or more of AM02 bacteria and AM06 bacteria.

[0123] In some embodiments, the method for preparing inactivated AKK bacteria satisfies one or more of the following characteristics:

[0124] (d1) AKK bacteria fermentation broth is the AKK bacteria fermentation broth used in the aforementioned heat inactivation method;

[0125] (d2) heat-inactivating the AKK bacteria fermentation broth using the aforementioned heat-inactivation method;

[0126] (d3) collecting the cells by centrifugation after the heat inactivation is completed;

[0127] (d4) The step of collecting the bacterial cells after the heat inactivation comprises: adding a flocculant to the mixed solution obtained after the heat inactivation, and then collecting the bacterial cells by centrifugation; optionally, the flocculant comprises chitosan and γ-polyglutamic acid.

[0128] In this application, the "flocculant" used can make some particles or grains with negative (positive) charges in water that are difficult to separate approach each other, reduce their electric potential, make them unstable, and use their aggregation properties to concentrate these particles, and further separate them by physical or chemical methods. Generally, different substances to be separated need to select matching flocculants according to their material characteristics.

[0129] In some embodiments, the flocculant includes chitosan and γ-polyglutamic acid. Chitosan and γ-polyglutamic acid can play a synergistic role in the following ways: chitosan is a natural cationic polymer with poor water solubility and a narrow pH application range; γ-polyglutamic acid is a natural anionic polymer with good water solubility, biodegradability, and non-toxicity to the human body and the environment. By using chitosan and γ-polyglutamic acid together, the flocculation effect of AKK bacteria can be enhanced and the bacterial yield can be improved.

[0130] In some embodiments, the method for preparing inactivated AKK bacteria satisfies one or more of the following characteristics:

[0131] (e1) the step of collecting the bacterial cells after the heat inactivation comprises: adding a flocculant mother solution to the mixed solution obtained after the heat inactivation, and then collecting the bacterial cells by centrifugation; the flocculant mother solution comprises a flocculant, and the concentration of the flocculant in the flocculant mother solution can be 2 g / L to 10 g / L, or any one of the following concentrations or a range consisting of any two of the following concentrations: 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, etc.;

[0132] (e2) the flocculant comprises chitosan and γ-polyglutamic acid in a mass ratio of 1:3 to 3:1; without limitation, the mass ratio of chitosan to γ-polyglutamic acid in the flocculant can also be any of the following ratios or values ​​or a range consisting of any two of the following ratios or values: 1:3, 1:2.8, 1:2.6, 1:2.5, 1:2.4, 1:2.25, 1:2.2, 1:2, 1:2. 1.8, 1:1.75, 1:1.6, 1:1.5, 1:1.25, 1:1.2, 1:1.1, 1:1, 1.1:1, 1.2:1, 1.25:1, 1.5:1, 1.6:1, 1.75:1, 1.8:1, 2:1, 2.2:1, 2.25:1, 2.4:1, 2.5:1, 2.75:1, 2.8:1, 3:1, etc.;

[0133] (e3) The method of collecting the bacteria by centrifugation is cup centrifugation or tube centrifugation.

[0134] In some embodiments, after the heat inactivation is completed, the bacteria are collected by cup centrifugation, the centrifugal force is 10000×g to 16000×g, and the centrifugation time is 20min to 40min; or, after the heat inactivation is completed, the bacteria are collected by tube centrifugation, the centrifugal force is 10000×g to 16000×g, and the flow rate is 0.5L / min to 2.0L / min. Without limitation, the centrifugal force of the cup centrifugation can also be any of the following values ​​or a range selected from any two of the following values: 10000×g, 11000×g, 12000×g, 13000×g, 14000×g, 15000×g, 16000×g, etc. Without limitation, the centrifugation time of the cup centrifugation can be any of the following durations or a range selected from any two of the following durations: 20min, 25min, 30min, 35min, 40min, etc. Without limitation, the centrifugal force of the tubular centrifuge can also be any of the following values ​​or a range consisting of any two of the following values: 10000×g, 11000×g, 12000×g, 13000×g, 14000×g, 15000×g, 16000×g, etc. Without limitation, the flow rate of the tubular centrifuge can be any of the following flow rates or a range consisting of any two of the following flow rates: 0.5L / min, 0.6L / min, 0.8L / min, 1L / min, 1.2L / min, 1.4L / min, 1.5L / min, 1.6L / min, 1.8L / min, 2.0L / min, etc.

[0135] The embodiments of the present application will be described in detail below in conjunction with some experimental examples. It should be understood that these experimental examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following experimental examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or conventional conditions in the art, or can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0136] In the following experimental examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operating accuracy are allowed.

[0137] In the following examples, "normal temperature" means 10°C to 30°C.

[0138] In the following examples, "room temperature" means 10°C to 30°C.

[0139] The following examples involve the value aEb described in scientific notation, which is equivalent to a×10 b For example, 1×10 4 It can be expressed as 1E4, and 7.9E+09 means 7.9×109 .

[0140] The yeast extract powder used in the following examples was purchased from Angel Yeast, model FM888, and its main components were nutritional factors such as amino acids, polypeptides, proteins, vitamins, and trace elements.

[0141] In the following examples, the percentage ratios of gases, if no gas is specified, refer to the molar ratio, for example, 5% CO2 / 10% H2 / 85% N2, 7% CO2 / 93% N2, etc.

[0142] In the following examples, "the percentage of free DNA in the supernatant to inactivated bacterial DNA" refers to the mass percentage unless otherwise specified.

[0143] The thermal inactivation process used in the following examples, unless otherwise specified, is operated as follows: heating is performed by passing high-temperature steam through the jacket.

[0144] Experimental Example 1. Effect of different heat inactivation temperatures on heat inactivation of AKK bacteria (heat inactivation process A: resuspending AKK bacteria in saline for heat inactivation, 1L scale)

[0145] (1) Culture medium formulation

[0146] The non-animal derived medium contains 6 g / L N-acetyl-D-glucosamine, 28 g / L soy peptone, 16 g / L yeast extract, 3.4 g / L potassium dihydrogen phosphate, 2 g / L glucose, 3 g / L sodium carbonate, 3 g / L L-threonine, 0.5 g / L ammonium chloride, 1 g / L Tween 80, 0.5 g / L L-cysteine ​​hydrochloride and water. After dissolution, sterilize at high temperature (118°C to 120°C) for 20 min.

[0147] (2) AKK bacteria culture

[0148] The AKK bacteria used were strain AM06 with a preservation number of CGMCC No. 22793, strain AM02 with a preservation number of CGMCC No. 22794, and strain ATCC BAA-835.

[0149] The AKK bacteria glycerol tube seeds were inoculated into 10 mL of non-animal-derived culture medium at a ratio of 2% (v / v) with an inoculation concentration of 2.0E+08 CFU / mL. The culture was performed anaerobically and statically at 37±1°C for 24 hours using ternary gas (5% CO2 / 10% H2 / 85% N2, molar ratio) as anaerobic protective gas to obtain a primary seed solution.

[0150] The first-level seed liquid that passed the microscopic examination was inoculated into 1000mL of non-animal-derived culture medium at a ratio of 2% (v / v) with an inoculation concentration of 5.0E+08CFU / mL. The culture was carried out anaerobically at 37±1°C for 20 hours using a ternary gas (5% CO2 / 10% H2 / 85% N2, molar ratio) as the anaerobic protective gas. After the culture, the second-level seed liquid was obtained. Samples were taken for detection of the number of viable bacteria (plate method) and the bacterial content (flow cytometry).

[0151] (3) Harvesting of bacterial cells

[0152] A floor-standing vacuum refrigerated centrifuge (LYNX6000: 4°C, 16000×g, 30 min) was used for centrifugation, and after centrifugation, the AKK bacterial sludge was harvested.

[0153] (4) Heat inactivation

[0154] Take AKK bacterial sludge, add physiological saline at a mass volume ratio of bacterial sludge: physiological saline (m:v, unit: g / mL) = 1:10, stir with a glass rod to disperse it in the physiological saline, use a stirrer at 1000 rpm for 5min to 20min to fully disperse the bacteria until there are no visible particles. Take samples for viable bacterial count (plate method) and bacterial content (flow cytometry) detection.

[0155] As shown in Table 1, the heat inactivation effect of AKK strains at different temperatures and treatment times was investigated in groups, and the inactivation volume was 1L, among which: Group 1 was the normal temperature control group; Groups 2, 4, 6 and 7 were inactivated at 60℃, 70℃, 80℃ and 100℃, respectively, and samples were taken every 10 minutes, and the total inactivation was 30 minutes; Groups 3 and 5 were inactivated at 60℃ and 70℃ for 60 minutes, respectively. A stirring bar and a temperature probe were placed in a beaker, and a magnetic stirrer with a heating function was placed. The temperature was set to start heating. When the temperature of the bacterial solution reached the set temperature, the inactivation timing began. After each inactivation time, samples were taken for viable bacterial count, bacterial content and Gram staining microscopy. Scanning electron microscope (SEM) observations were performed on the bacterial solutions of the normal temperature group, 60℃ heat inactivation for 30min / 60min, 70℃ heat inactivation for 30min / 60min, 80℃ heat inactivation for 30min and 100℃ heat inactivation for 30min.

[0156] Table 1. Grouping of heat inactivation conditions

[0157] Group No. Heat inactivation temperature Inactivation time Stirring speed Bacterial liquid volume 1 normal temperature 60min 400rpm 1000mL 2 60℃ 10min, 20min, 30min 400rpm 1000mL 3 60℃ 60min 400rpm 1000mL 4 70℃ 10min, 20min, 30min 400rpm 1000mL 5 70℃ 60min 400rpm 1000mL 6 80℃ 10min, 20min, 30min 400rpm 1000mL 7 100℃ 10min, 20min, 30min 400rpm 1000mL

[0158] Take AKK bacterial sludge, add physiological saline at the ratio of bacterial sludge: physiological saline mass volume ratio (m:v, unit g / mL) = 1:2, 1:10 and 1:100, stir with a glass rod to disperse it in the physiological saline, use a stirrer at 1000rpm for 5min to 20min to fully disperse the bacteria until there are no visible particles. The heat inactivation conditions are 70℃, 30min, and the inactivation volume is 1L. Samples are taken for detection of viable bacteria count (plate method) and bacterial content (flow cytometry). Investigate the heat inactivation effect of different bacterial concentrations.

[0159] (5) Harvesting of inactivated bacteria

[0160] A floor-standing vacuum refrigerated centrifuge (LYNX6000: 4°C, 16000×g, 30 min) was used for centrifugation. After centrifugation of each group, the bacterial sludge was harvested to obtain the AKK inactivated bacterial sludge.

[0161] (6) Detection method

[0162] Viable bacteria count detection (before heat inactivation): Take the above test samples and dilute them 10 times in series with BHI according to the dilution system of 0.5mL+4.5mL. Accurately take 100μl of the bacterial solution of the final dilution and evenly spread it on the N-acetyl-D-glucosamine hemin chloride BHA plate. Make 3 plates for each dilution, and make a negative control at the same time. Place them in an anaerobic glove box (5% CO2 / 10% H2 / 85% N2) at 37℃ for cultivation. After about 48-96h of cultivation, observe the colony growth of each plate and count them. Calculate the viable bacteria count according to the sum of the colony counts of the 3 plates according to the following formula.

[0163] Viable bacteria count (CFU / mL) = sum of colony counts / 3×10×final dilution

[0164] AKK live bacteria test (after heat inactivation): Take 100μl of the above test sample and evenly spread it on the N-acetyl-D-glucosamine hemin chloride BHA plate. Make 3 plates for each dilution and 1 negative control at the same time. Place it in an anaerobic glove box (5% CO2 / 10% H2 / 85% N2) at 37℃ for cultivation. After about 48-96h of cultivation, observe the growth of colonies on each plate and count them. Calculate the number of live bacteria according to the following formula based on the sum of the colony counts on the 3 plates.

[0165] Viable bacteria count (CFU / mL) = sum of colony counts / 3×10×final dilution

[0166] The “final dilution” refers to the multiple of the test sample after 10-fold serial dilution.

[0167] Bacterial content detection (flow cytometry): Preparation of inactivated bacterial solution: Dilute the sample with purified water to an appropriate multiple to obtain the test solution. Take 0.5 mL of the test solution, add 25 μL of SYBR Green I dye solution, incubate in the dark for 5 minutes, and detect the bacterial content by flow cytometry.

[0168] Bacterial yield % = inactivated bacterial mud bacterial content × weight / inactivated bacterial liquid bacterial content × volume × 100%

[0169] Microscopic examination: Gram staining and scanning electron microscopy.

[0170] Gram microscopy method: (1) Smear: Take a clean glass slide, apply an appropriate amount of the test sample in the center of the slide, spread it evenly, make a thin slice, and mark it;

[0171] (2) Fixation: Allow the slide to air dry or quickly pass it over an alcohol lamp (so that the slide does not burn the back of your hand) to fix the bacteria.

[0172] (3) Primary staining: Add an appropriate amount of crystal violet staining solution to cover the bacterial surface. After staining for 1 minute, pour off the staining solution, rinse carefully with water, and absorb water with absorbent paper;

[0173] (4) Mordanting: Add an appropriate amount of iodine solution to cover the bacterial coating surface, mordant for 1 minute, pour off the dye solution, rinse carefully with water, and absorb the water with absorbent paper;

[0174] (5) Decolorization: Tilt the slide and continuously add 95% ethanol for 20-25 seconds until the effluent is colorless, then wash with water immediately;

[0175] (6) Re-staining: Add safranin and re-stain for 1 minute, pour off the staining solution, rinse carefully with water, absorb water with absorbent paper, and dry;

[0176] (7) Microscopic examination: Place the slide under an oil microscope to observe the Gram staining characteristics of the bacteria and the shape of the bacteria. Dark purple indicates Gram-positive bacteria, and red indicates Gram-negative bacteria.

[0177] The instrument and test parameters for transmission electron microscopy (TEM) microscopy were: manufacturer hitachi, model HT7700, acceleration voltage 80 kV, magnification ×25.0k.

[0178] (7) Experimental results

[0179] The fermentation broth of AKK bacteria (AM06, AM02 and ATCC BAA-835 strains) was centrifuged and the bacterial sludge was collected. After resuspending with physiological saline, the sludge was heat-inactivated and centrifuged again to collect the inactivated bacterial sludge. The results are shown in Table 2A, Table 2B, Table 2C, Table 3 and Table 4. The transmission electron microscopy (TEM) test results of Experimental Example 1 can be found in Figure 1-5 .

[0180] Inactivation efficiency: Inactivation efficiency of the inactivated bacterial solution of three AKK strains (AM06, AM02 and ATCC BAA-835 strains) was incompletely inactivated in some batches at 60°C for 30 min and 1 h, 70°C for 30 min and 1 h, and 80°C and 100°C for 30 min.

[0181] Bacterial yield: After the inactivated bacterial liquid of three AKK strains (AM06, AM02 and ATCC BAA-835 strains) was heat-inactivated at 60-100°C, the AKK bacterial yields of AM06, AM02 and ATCC BAA-835 inactivated bacterial slurry were only about 20.2%-40.8%, 38.8%-37.9% and 19.7%-30.8%, respectively, and the bacterial yields were all low.

[0182] Bacterial morphology: The external contours of the three AKK strains in the normal temperature group were clear and the biofilm was smooth; after 30 minutes of heat inactivation at 60℃, the external contours of the three AKK strains in the bacterial solution were clear, the biofilm was basically intact, and a small amount of wrinkles appeared. As the inactivation time increased, after 60 minutes of heat inactivation at 60℃, the external contours of the bacteria were clearer, the biofilm was relatively intact, and a blister-like morphology appeared at the border; after 30 minutes of heat inactivation at 70℃, the external contours of the three AKK strains in the bacterial solution were clearer, the biofilm was slightly wrinkled, and a small number of bacteria were incomplete. After 60 minutes of heat inactivation at 70℃, the external contours of the bacteria were clearer, the biofilm was wrinkled, a large number of fragments were visible, and there were fewer intact bacteria. As time went on, the damage to the biofilm worsened; after 30 minutes of heat inactivation at 80℃ and 100℃, the external contours of the three AKK strains in the bacterial solution became blurred, the biofilm was wrinkled, and the membrane fragments increased significantly as the temperature increased.

[0183] Furthermore, considering the bacterial morphology and bacterial yield, the heat inactivation condition of 70℃ for 30min was further selected, and the heat inactivation process was further explored with different ratios of bacterial mud: physiological saline. The number of viable bacteria in the inactivated bacterial solution of the three AKK strains ranged from 4.2E+08 to 3.8E+10 CFU / mL. In the heat inactivation at 70℃ for 30min, some batches were incomplete.

[0184] Table 2A. Detection results of heat-inactivated AKK bacteria at different temperatures (heat-inactivation process A: resuspending AKK bacteria in physiological saline for heat inactivation, 1L scale), AM06 strain.

[0185]

[0186]

[0187]

[0188] Table 2B. Results of heat-inactivated AKK bacteria at different temperatures (heat-inactivation process A: resuspending AKK bacteria in saline for heat inactivation, 1L scale), AM02 strain

[0189]

[0190]

[0191]

[0192] Table 2C. Results of heat-inactivated AKK bacteria at different temperatures (heat-inactivation process A: resuspending AKK bacteria in saline for heat inactivation, 1L scale), ATCC BAA-835 strain

[0193]

[0194]

[0195]

[0196] Table 3. Yield of heat-inactivated AKK cells at different temperatures (heat-inactivation process A: resuspending AKK cells in saline for heat inactivation, 1L scale)

[0197]

[0198] Table 4. Results of heat inactivation test at different AKK bacterial concentrations (heat inactivation process A: resuspending AKK bacterial cells in saline for heat inactivation, 1L scale)

[0199]

[0200] Experimental Example 2: Heat inactivation of AKK bacteria (heat inactivation process A: resuspending bacteria in physiological saline for heat inactivation, 30L scale)

[0201] (1) Culture medium preparation

[0202] Same as Experimental Example 1.

[0203] (2) AKK bacteria culture

[0204] The AKK bacteria include the AM06 strain with a preservation number of CGMCC No. 22793, the AM02 strain with a preservation number of CGMCC No. 22794, and the ATCC BAA-835 strain.

[0205] AKK seeds were inoculated into 10 mL of non-animal-derived culture medium at a ratio of 2% (v / v) to a concentration of 2.0E+08 CFU / mL, and cultured anaerobically at 37±1°C for 24 h under ternary gas (5% CO2 / 10% H2 / 85% N2, molar ratio) as anaerobic protective gas to obtain a primary seed solution;

[0206] The first-level seed liquid that passed the microscopic examination was inoculated into 1000mL of non-animal-derived culture medium at a ratio of 2% (v / v) with an inoculation concentration of 5.0E+08CFU / mL. The culture was carried out anaerobically at 37±1℃ for 20h with ternary gas (5% CO2 / 10% H2 / 85% N2, molar ratio) as anaerobic protective gas. After the culture, the second-level seed liquid was obtained.

[0207] Take 2 bottles of secondary seed liquid that have passed the microscopic examination and inoculate them into 30L non-animal culture medium at a ratio of 6.3% (v / v). The inoculation concentration is 1.0E+09CFU / mL. Culture anaerobically at 37℃±1℃ (7% CO2 / 93% N2, pressure maintained at 0.05±0.01MPa, rotation speed 100±10rpm, after the pH drops below 6.5, control the pH to 6.5±0.2 with 20% sodium hydroxide) for 10-14h to obtain AKK fermentation liquid.

[0208] (3) Harvesting of bacterial cells

[0209] Use GQ105 tubular centrifuge for centrifugation at 16000 rpm (15050×g) and 50 Hz. After centrifugation, collect the bacterial sludge.

[0210] (4) Heat inactivation

[0211] Place AKK bacterial sludge in a 5L beaker, add physiological saline at a ratio of bacterial sludge: physiological saline (m:v; unit is g / mL) = 1:20, stir at a speed of 500-1000 rpm for 5-10 min until it is evenly dispersed, and obtain the pre-inactivation bacterial solution, and record the volume of the pre-inactivation bacterial solution.

[0212] Transfer the pre-inactivation bacterial solution to a 5L beaker (put in a magnetic stirrer in advance), place it in a magnetic stirrer with a heating function, put in a temperature probe, set the temperature to 70°C and the stirring speed to 400rpm, and start the inactivation timing after the bacterial solution temperature reaches 70±2°C. Keep it warm for 30±2min to perform heat inactivation treatment to obtain the inactivated bacterial solution, and record the volume of the inactivated bacterial solution.

[0213] (5) Harvesting of inactivated bacteria

[0214] Use GQ105 tubular centrifuge for centrifugation at 16000 rpm (15050 × g) and 50 Hz. After centrifugation, collect the inactivated bacterial sludge.

[0215] (6) Detection method

[0216] The viable bacterial count, AKK viable bacterial test, bacterial content, and microscopic examination were the same as those in Experimental Example 1.

[0217] DNA detection: Take the inactivated bacteria (centrifugal precipitation) and the centrifugal supernatant, use the bacterial DNA extraction kit (Guangzhou Jiebei Biotechnology Co., Ltd., product number: D3106) to extract DNA, detect the DNA concentration with an ultra-micro UV spectrophotometer (detection wavelength is 260nm), and calculate the percentage of free DNA in the supernatant of the inactivated bacterial solution to the inactivated bacterial DNA.

[0218] (7) Experimental results

[0219] Three strains of AKK bacteria (AM06, AM02 and ATCC BAA-835) were cultured step by step to 30 L, and the bacterial sludge was harvested after centrifugation. After adding physiological saline to resuspend evenly, heat inactivation (70°C for 30 min) was performed, and the inactivated bacterial sludge was harvested by centrifugation. The results are shown in Tables 5A, 5B, 5C and 6.

[0220] Inactivation efficiency: Three AKK strains (AM06, AM02 and ATCC BAA-835) were heat-inactivated at 70°C for 30 min, and three experiments were conducted. The bacterial content before and after inactivation decreased by about 10%-25%, and the percentage of free DNA in the supernatant to inactivated bacterial DNA increased from 1.8%-2.8% to 8.5%-14.8%. Some bacteria ruptured, and inactivation was incomplete in some cases.

[0221] Bacterial yield: The three AKK bacterial liquids were heat-inactivated at 70°C for 30 min, and three experiments were carried out. The average AKK bacterial yields of the inactivated bacterial slurry were 22.67%, 32.63% and 25.83%, respectively, which were all low.

[0222] Bacterial morphology: The three AKK bacterial cultures were heat-inactivated at 70°C for 30 min. The external contours of the AKK bacteria were clear, the bacterial membrane was slightly wrinkled, and a few bacteria were incomplete.

[0223] Table 5A. AKK bacteria heat inactivation test results (heat inactivation process A: resuspend AKK bacteria in saline for heat inactivation, 30L scale), AM06 strain

[0224]

[0225] Table 5B. AKK bacteria heat inactivation test results (heat inactivation process A: resuspend AKK bacteria in saline for heat inactivation, 30L scale), AM02 strain

[0226]

[0227] Table 5C. AKK bacteria heat inactivation test results (heat inactivation process A: resuspend AKK bacteria in saline for heat inactivation, 30L scale), ATCC BAA-835 strain

[0228]

[0229] Table 6. AKK bacteria heat inactivation cell yield results (heat inactivation process A: resuspending AM06 cells in physiological saline for heat inactivation, 30L scale)

[0230]

[0231] The effects of heat inactivation of three AKK bacterial solutions of AM06, AM02 and ATCC BAA-835 by replacing physiological saline with PBS solution in heat inactivation process A (including Experimental Examples 1 and 2) were similar to those of Experimental Examples 1 and 2, and similar to the experimental results of Experimental Examples 1 and 2. Taking Experimental Example 2 as an example, the three AKK bacterial solutions were heat inactivated at 70°C for 30 minutes, and some of the bacteria were ruptured, and inactivation was incomplete in some cases. The yield of inactivated bacterial sludge AKK bacteria was low, the external contour of the AKK bacteria was clear, the bacterial film was slightly wrinkled, and a small number of bacteria were incomplete.

[0232] Experimental Example 3: Effect of different heat inactivation temperatures on heat inactivation of AKK bacteria (heat inactivation process B: direct heat inactivation of fermentation broth, 1L scale)

[0233] (1) AKK bacteria culture

[0234] The AKK bacteria include the AM06 strain with a preservation number of CGMCC No. 22793, the AM02 strain with a preservation number of CGMCC No. 22794, and the ATCC BAA-835 strain.

[0235] According to the process of Experimental Example 2, 1 L of fermentation was carried out to obtain AKK bacteria fermentation liquid, and samples were taken for detection of viable bacteria count and bacterial content.

[0236] (2) Heat inactivation

[0237] As shown in Table 1, the heat inactivation effect of AM06 strain at different temperatures and treatment times was investigated in groups, and the inactivation volume was 1L, among which: Group 1 was the normal temperature control group; Groups 2, 4, 6 and 7 were inactivated at 60℃, 70℃, 80℃ and 100℃, respectively, and samples were taken every 10 minutes, and the total inactivation was 30 minutes; Groups 3 and 5 were inactivated at 60℃ and 70℃ for 60 minutes, respectively. A stirring bar and a temperature probe were placed in a beaker, and placed on a magnetic stirrer with a heating function. The temperature was set to start heating. When the temperature of the bacterial solution reached the set temperature, the inactivation timing began. After each inactivation time, 20mL of samples were taken for viable bacterial count detection and Gram staining microscopy. Scanning electron microscope observation was performed on the bacterial solutions of the normal temperature group, 60℃ heat inactivation for 30min / 60min, 70℃ heat inactivation for 30min / 60min, 80℃ heat inactivation for 30min and 100℃ heat inactivation for 30min.

[0238] (3) Harvesting of bacterial cells

[0239] A floor-standing vacuum centrifuge (Thermo LYNX6000) was used for 6×1000 mL cup centrifugation, and each group of inactivated fermentation broth was poured into a 1 L centrifuge cup. The centrifugation conditions were 16000×g and 30 min. After the centrifugation, the inactivated bacterial sludge was collected and weighed.

[0240] (4) Detection method

[0241] Same as Experimental Example 1.

[0242] (5) Experimental results

[0243] The fermentation broth of three strains of AKK bacteria (AM06, AM02 and ATCC BAA-835 strains) was directly heat-inactivated, and the bacteria were collected after heat inactivation. The results are shown in Table 7A, Table 7B, Table 7C and Table 8. The transmission electron microscopy (TEM) test results of Experimental Example 3 can be found in Figure 6-10 .

[0244] Inactivation efficiency: No live AKK bacteria were detected in all experimental groups (except the positive control group) of the three experiments on AM06 and AM02 strains, and the AM06 strain could be completely inactivated under the conditions of the lowest temperature and the shortest inactivation time (60℃, 10 min). Live AKK bacteria were detected in all experimental groups (except the positive control group) of the three experiments on ATCC BAA-835 strain after heat inactivation at 60℃ for 10 min, 20 min, 30 min and 60 min, while heat inactivation at 70℃ had incomplete inactivation in some cases, and ATCC BAA-835 strain could be completely inactivated at 80℃ and 100℃.

[0245] Bacterial yield: After the AM06 and AM02 fermentation broths are heat-inactivated at 60-100°C, the bacterial yield can reach more than 90%; after the ATCC BAA-835 fermentation broth is heat-inactivated at 60-100°C, the bacterial yield is about 50%.

[0246] Bacterial morphology: In the normal temperature group, 60℃ heat inactivation for 30min and 60min, 70℃ heat inactivation for 30min and 60min, and 80℃ heat inactivation for 30min, the external contours of the AM06 and AM02 strains in the inactivated bacterial solution were clear and the bacterial film was smooth; after 100℃ heat inactivation for 30min, the external contours of the AM06 and AM02 strains in the bacterial solution became blurred, the bacterial film shrank, and the membrane fragments increased significantly. In the normal temperature group, 60℃ heat inactivation for 30min and 60min, 70℃ heat inactivation for 30min, the external contours of the ATCC BAA-835 fermentation liquid in the inactivated bacterial solution were clear and the bacterial film was smooth; after 70℃ heat inactivation for 60min, 80℃ heat inactivation for 30min, and 100℃ heat inactivation for 30min, the external contours of the ATCC BAA-835 fermentation liquid in the bacterial solution became blurred, the bacterial film shrank, and the membrane fragments increased significantly.

[0247] It can be seen that for different AKK bacteria, fine-tuning of sterilization conditions will lead to huge differences in inactivation efficiency, bacterial yield, and bacterial morphology. Different AKK bacteria require different thermal inactivation conditions that match the characteristics of the strain.

[0248] Table 7A. Results of heat-inactivated AKK bacteria at different temperatures (heat-inactivation process B: direct heat inactivation of fermentation broth, 1L system), AM06 strain

[0249]

[0250]

[0251]

[0252] Table 7B. Results of heat-inactivated AKK bacteria at different temperatures (heat-inactivation process B: direct heat inactivation of fermentation broth, 1L system), AM02 strain

[0253]

[0254]

[0255] Table 7C. Detection results of AKK bacteria inactivated by heat at different temperatures (heat inactivation process B: direct heat inactivation of fermentation broth, 1L system), ATCC BAA-835 strain

[0256]

[0257]

[0258] Table 8. AKK bacteria heat inactivation cell yield (heat inactivation process B: direct heat inactivation of fermentation broth, 1L system)

[0259]

[0260] Experimental Example 4, AKK thermal inactivation (thermal inactivation process B: direct thermal inactivation of fermentation broth, 30L scale)

[0261] (1) AKK bacteria culture

[0262] The AKK bacteria adopt the AM06 strain with the preservation number of CGMCC No.22793 and the AM02 strain with the preservation number of CGMCC No.22794.

[0263] According to the process of Experimental Example 2, 30 L of fermentation was carried out to obtain AKK strain fermentation liquid, and samples were taken for detection of viable bacteria count and bacterial content.

[0264] (2) Heat inactivation

[0265] The fermentation liquid was directly heat-inactivated in a 50L fermenter. The heat inactivation process was carried out by heating the jacket. Under the condition of maintaining the stirring speed in the tank at 100rpm, the AKK strain fermentation liquid was heated to 70℃±3℃ and maintained for 30min±2min for heat inactivation. Samples were taken at 10min, 20min, and 30min for live bacteria detection, Gram staining microscopy, and bacterial content detection. After the heat inactivation and insulation was completed, the inactivated fermentation liquid was cooled by running tap water into the jacket of the fermenter. When the temperature was lower than 40℃, the tank was placed for centrifugation.

[0266] (3) Thermal inactivation curve study

[0267] During the fermentation liquid heat inactivation and temperature-raising stage and the heat preservation stage, sampling is carried out according to the sampling plan in Table 9:

[0268] ① During the inactivation process, the actual temperature is recorded in both the heating stage and the constant temperature stage and the temperature-time change curve is drawn;

[0269] ② Determine the number of live bacteria in the bacterial solution before inactivation (N0) and the number of live bacteria in the sample at different time points during the heating and constant temperature stages (Nt), and draw a thermal inactivation curve with lgNt / N0 as the ordinate and time as the abscissa.

[0270] Table 9. Thermal Inactivation Curve Study - Sampling Plan

[0271]

[0272] (4) Harvesting of bacterial cells

[0273] Use GQ105 tubular centrifuge for centrifugation at 16000 rpm (15050×g) and 50 Hz. Collect the bacterial sludge after centrifugation.

[0274] (5) Detection method

[0275] Same as Experimental Examples 1 and 2.

[0276] (6) Experimental results

[0277] AM06 and AM02 bacteria were cultured step by step to 30 L, 30 L of fermentation broth was directly heat-inactivated (70°C for 30 min), and the inactivated bacterial sludge was harvested by centrifugation. The results are shown in Tables 10A, 10B, 11 and 12.

[0278] Inactivation efficiency: After the fermentation broth of AM06 and AM02 strains was heat-inactivated at 70°C for 10-30 minutes, there was basically no loss in the bacterial content before and after heat inactivation in three experiments. There was no obvious change in the percentage of free DNA and bacterial DNA in the inactivated bacterial broth, and the bacteria were intact.

[0279] Bacterial yield: After the fermentation broth of AM06 and AM02 strains was heat-inactivated at 70°C for 30 minutes, the bacterial yield of the inactivated bacterial sludge was about 80% after three experiments.

[0280] Bacterial morphology: After heat inactivation at 70°C for 30 minutes, the external contours of the AM06 and AM02 strains in the bacterial solution were clearer and the bacterial film was more complete.

[0281] Thermal inactivation curve: AM06 strain, heating stage, heating for 20 minutes, when the temperature rises to 68.2℃, no AKK live bacteria can be detected in the bacterial solution (detection limit is 10CFU / mL), and it is completely inactivated; compared with the bacterial solution before inactivation, the number of AKK live bacteria decreased by more than 8.7lg. In the temperature maintenance stage, 70±2℃ was maintained for 30 minutes, and no AKK live bacteria were detected in the bacterial solution (detection limit is 10CFU / mL).

[0282] In this application, "lg" means "log10". The number of viable AKK cells is recorded as Nx. Taking "Nx>8.7lg" as an example, it means that the decrease value of lgNx is greater than 8.7.

[0283] For the AM02 strain, during the heating stage, the temperature was heated for 20 minutes. When the temperature rose to 66.8°C, no live AKK bacteria were detected in the bacterial solution (the detection limit was 10 CFU / mL), indicating complete inactivation. Compared with the bacterial solution before inactivation, the number of live AKK bacteria decreased by more than 8.8 lg. During the temperature maintenance stage, 70±2°C was maintained for 30 minutes, and no live AKK bacteria were detected in the bacterial solution (the detection limit was 10 CFU / mL).

[0284] Table 10A. AKK bacteria heat inactivation test results (fermentation broth direct heat inactivation, 30L scale), AM06 strain

[0285]

[0286] Table 10B. AKK bacteria heat inactivation test results (fermentation broth direct heat inactivation, 30L scale), AM02 strain

[0287]

[0288]

[0289] Table 11. AKK bacteria heat inactivation bacterial cell yield results (heat inactivation process B: direct heat inactivation of fermentation broth, 30L scale)

[0290]

[0291] Table 12. Thermal inactivation curve results (thermal inactivation process B: direct thermal inactivation of fermentation broth, 30L scale)

[0292]

[0293] Experimental Example 5, AKK thermal inactivation (thermal inactivation process C: direct thermal inactivation of fermentation broth, pH adjustment and addition of flocculant - 30L scale)

[0294] (1) AKK bacteria culture

[0295] The AKK bacteria adopt the AM06 strain with the preservation number of CGMCC No.22793 and the AM02 strain with the preservation number of CGMCC No.22794.

[0296] According to the process of Experimental Example 2, 30 L of fermentation was carried out to obtain AKK bacteria fermentation liquid, and samples were taken for detection of viable bacteria count and bacterial content.

[0297] (2) Heat inactivation

[0298] After the fermentation, the pH of the fermentation liquid was adjusted to 8.5 with 20% sodium hydroxide, and the fermentation liquid was directly heat-inactivated in a 50L fermenter. The heat inactivation process was carried out by heating the jacket. Under the condition of maintaining the stirring speed in the tank at 100rpm, the AKK strain fermentation liquid was heated to 60°C ± 3°C and maintained for 30min ± 2min for heat inactivation. Samples were taken at 10min, 20min, and 30min for live bacteria detection, Gram staining microscopy, and bacterial content detection. After the heat inactivation and heat preservation was completed, the inactivated fermentation liquid was cooled by running tap water into the jacket of the fermenter. When the temperature was lower than 40°C, the tank was placed for centrifugation.

[0299] (3) Thermal inactivation curve study

[0300] During the fermentation liquid heat inactivation and temperature-raising stage and the heat preservation stage, sampling is carried out according to the sampling plan in Table 9:

[0301] ① During the inactivation process, the actual temperature is recorded in both the heating stage and the constant temperature stage and the temperature-time change curve is drawn;

[0302] ② Determine the number of live bacteria in the bacterial solution before inactivation (N0) and the number of live bacteria in the sample at different time points during the heating and constant temperature stages (Nt), and draw a thermal inactivation curve with lgNt / N0 as the ordinate and time as the abscissa.

[0303] (4) Harvesting of bacterial cells

[0304] After the heat inactivation, add 5g / L flocculant mother solution (mass ratio of chitosan: γ-polyglutamic acid is 3:1) to make the flocculant working concentration in 30L inactivated fermentation broth 50ppm. Stir for 10min at 100rpm, then stop stirring and let stand for 30min-40min. Centrifuge using GQ105 tubular centrifuge, centrifugation conditions: 16000rpm (15050×g), frequency 50Hz, collect bacterial sludge after centrifugation.

[0305] (5) Detection method

[0306] Same as Experimental Examples 1 and 2.

[0307] (6) Experimental results

[0308] AM06 and AM02 bacteria were cultured step by step to 30 L, the pH of the fermentation broth was adjusted to 8.5, and the 30 L fermentation broth was directly heat-inactivated (60°C for 30 min). A flocculant solution with a final concentration of 20 ppm was added, and after stirring and standing, the inactivated bacterial sludge was harvested by centrifugation. The results are shown in Tables 13A, 13B, 14 and 15.

[0309] Inactivation efficiency: After the fermentation broth of strain AM06 and AM02 was heat-inactivated at 60°C for 10-30 minutes, there was basically no loss of bacterial content before and after heat inactivation in three experiments. There was no significant change in the percentage of free DNA and bacterial DNA in the inactivated bacterial broth, and the bacteria were intact without rupture.

[0310] Bacterial yield: After the fermentation broth of AM06 and AM02 strains was heat-inactivated at 60°C for 30 minutes, the bacterial yield of AM06 inactivated bacterial sludge was about 90% or more after three experiments.

[0311] Bacterial morphology: After heat inactivation at 60°C for 30 minutes, the external contours of the AM06 and AM02 strains in the bacterial solution were clearer and the bacterial film was more complete.

[0312] Thermal inactivation curve: AM06 strain, heating stage, heating 15min, when the temperature rises to 58.6℃, no AKK live bacteria can be detected in the bacterial solution (detection limit is 10CFU / mL), and it is completely inactivated; compared with the bacterial solution before inactivation, the number of AKK live bacteria decreased by >8.8lg. In the temperature holding stage, 60±2℃ for 30min, no AKK live bacteria were detected in the bacterial solution (detection limit is 10CFU / mL).

[0313] For the AM02 strain, during the heating stage, the temperature was heated for 15 minutes. When the temperature rose to 57.8°C, no live AKK bacteria were detected in the bacterial solution (the detection limit was 10 CFU / mL), indicating complete inactivation. Compared with the bacterial solution before inactivation, the number of live AKK bacteria decreased by more than 8.8 lg. During the temperature maintenance stage, the temperature was kept at 60±2°C for 30 minutes, and no live AKK bacteria were detected in the bacterial solution (the detection limit was 10 CFU / mL).

[0314] Table 13A. AKK bacteria heat inactivation test results (heat inactivation process C: direct heat inactivation of fermentation broth, 30L scale), AM06 strain

[0315]

[0316] Table 13B. AKK bacteria heat inactivation test results (heat inactivation process C: direct heat inactivation of fermentation broth, 30L scale), AM02 strain

[0317]

[0318] Table 14 AKK bacteria heat inactivation cell yield results (heat inactivation process C: direct heat inactivation of fermentation broth, 30L scale)

[0319]

[0320] Table 15 Thermal inactivation curve results (thermal inactivation process C: direct thermal inactivation of fermentation broth, 30L scale)

[0321]

[0322]

[0323] Experimental Example 5B. Changing the mass ratio of chitosan to γ-polyglutamic acid in the flocculant.

[0324] In Experimental Example 5B, based on Experimental Example 5, the mass ratio of chitosan:γ-polyglutamic acid in the flocculant mother liquor was changed to 3:1 and 1:1, and the AM06 and AM02 strains were heat-inactivated using the same operation method, which could achieve similar heat inactivation effects: the heat inactivation efficiency was high, and no live AKK bacteria were detected in the bacterial solution after heat inactivation (the detection limit was 10 CFU / mL); there was basically no loss of bacterial content before and after heat inactivation, and there was no obvious change in the percentage of free DNA to bacterial DNA in the inactivated bacterial solution, and the bacteria were intact and not broken; the bacterial yield was high; after heat inactivation, the external contours of the AM06 and AM02 strains in the bacterial solution were clearer, and the bacterial film was more complete.

[0325] Experimental Example 5C: Changing the type of flocculant.

[0326] Based on Experimental Example 5, the flocculants were replaced with dextran (Experimental Example 5C1) and sodium alginate (Experimental Example 5C2), respectively, and there was a certain flocculation effect, but the effect was significantly lower than the scheme using the combination of chitosan and γ-polyglutamic acid in Experimental Example 5 and Experimental Example 5A. The bacterial cell yield of Experimental Example 5C1 and Experimental Example 5C2 was significantly lower than the scheme using the combination of chitosan and γ-polyglutamic acid (Experimental Example 5 and Experimental Example 5A).

[0327] Experimental Example 6: Investigation of the activity of inactivating AKK bacteria

[0328] (1) Preparation of bacterial cells

[0329] According to the methods of Experimental Examples 4 and 5, live bacterial slurries and inactivated bacterial slurries of AM06 and AM02 strains were prepared and temporarily stored in a -20°C refrigerator.

[0330] (2) Detection method

[0331] AM06 and AM02 cells were lysed using lysozyme and an ultrasonic disruptor, and the supernatant was collected after centrifugation. Amuc_1100 protein was purified by metal affinity using Ni-16NTA His·Bind resin under natural conditions. After buffer exchange using a Zeba spin column, the Amuc_1100 protein content was determined by the BCA method.

[0332] The pilus-like outer membrane protein Amuc_1100 of AKK bacteria can independently play a role in partially reducing fat and maintaining intestinal barrier function, and the protein has a stable structure at 70°C. Since Amuc_1100 protein is considered to be one of the main components for the activity of inactivated AKK bacteria, Amuc_1100 protein can be used as an indicator to characterize the activity of AKK bacteria.

[0333] (3) Experimental results

[0334] Please refer to Table 16.

[0335] Activity of inactivating AKK bacteria (using outer membrane protein Amuc_1100 as activity indicator): There was no significant change in the content of outer membrane protein Amuc_1100 before and after the inactivation of AM06 and AM02 strains.

[0336] Table 16. Detection results of AKK outer membrane protein Amuc_1100

[0337]

[0338] Experimental Example 7: Stability Study of Inactivated AKK Bacteria

[0339] (1) Preparation of AKK inactivated bacteria sludge

[0340] According to Experimental Example 4, AM06 and AM02 inactivated bacterial sludge were prepared.

[0341] (2) Preparation of AKK inactivated bacteria powder

[0342] The freeze-dried excipient solution (10% maltodextrin) was added in a ratio of inactivated bacterial slurry: freeze-dried excipient solution = 1:7 to 1:9 (mass ratio, 1:8 was used in this example), and a magnetic stirrer or blender was used to stir for 30-60 min at 900-1200 rpm until the bacteria were completely dispersed, and freeze-dried. The inactivated bacterial powder was prepared by crushing and sieving.

[0343] (3) Stability investigation

[0344] The inactivated bacteria powder was placed at 40°C ± 2°C and 75% RH ± 5% RH for accelerated stability testing, and at 25°C ± 2°C and 60% RH ± 5% RH for long-term stability testing.

[0345] (4) Detection method

[0346] Same as Experimental Example 1.

[0347] (5) Experimental results

[0348] Please refer to Table 17 and Table 18.

[0349] Accelerated test at 40℃±2℃75%RH±5%RH: The inactivated AKK powder was stored at 40℃±2℃75%RH±5%RH for 6 months. There was no significant difference in the bacterial content compared with that on day 0, and the live AKK bacteria were all <10CFU / g (the detection limit was 10CFU / g).

[0350] Long-term test at 25℃±2℃60%RH±5%RH: The AKK inactivated bacteria powder was stored at 25℃±2℃60%RH±5%RH for 12 months. There was no significant difference in the bacterial content compared with that on day 0, and the live AKK bacteria were all <10CFU / g (the detection limit was 10CFU / g).

[0351] Table 17. AKK inactivated bacteria powder stability test results (accelerated test 40℃±2℃75%RH±5%RH)

[0352]

[0353] Table 18. AKK inactivated bacteria powder stability test results (long-term test 25 ℃ ± 2 ℃ 60% RH ± 5% RH)

[0354]

[0355] The technical features of the above-mentioned embodiments and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0356] The above embodiments and examples only express several embodiments of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as limiting the scope of protection of the present application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the scope of protection of the present application. In addition, it should be understood that after reading the above teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent of this application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A method for thermal inactivation of Akkermansia muciniphila, characterized in that: The steps include: The AKK bacteria fermentation liquid is heat-inactivated; wherein the AKK bacteria fermentation liquid is a mixture containing live AKK bacteria and a fermentation medium; the AKK bacteria in the AKK bacteria fermentation liquid is Akkermansia muciniphila; AKK bacteria include one or more of AM02 bacteria and AM06 bacteria; AM02 is Akkermansia muciniphila AM02, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on June 28, 2021, with the deposit number CGMCC No.22794; The AM06 bacterium is Akkermansia muciniphila AM06. Akkermansia muciniphila AM06 was deposited in the General Microbiology Center of China National Microbiological Culture Collection on June 28, 2021, with the deposit number CGMCC No.22793.

2. The thermal inactivation method according to claim 1, characterized in that The sum of the percentages of the number of AM02 and AM06 bacteria in AKK bacteria was 80% to 100%; Optionally, the AKK bacteria is selected from one or more of AM02 bacteria and AM06 bacteria.

3. The thermal inactivation method according to claim 1, characterized in that The fermentation medium is a non-animal-derived medium; the non-animal-derived medium contains 5g / L to 12g / L N-acetyl-D-glucosamine, 25g / L to 40g / L soy peptone, 12g / L to 20g / L yeast extract powder, 2g / L to 5g / L potassium dihydrogen phosphate, 1g / L to 4g / L glucose, 1g / L to 4g / L sodium carbonate, 1g / L to 4g / L L-threonine, 0.1g / L to 1g / L ammonium chloride, 0.5g / L to 2g / L Tween 80, 0.1g / L to 1g / L L-cysteine ​​hydrochloride and water.

4. The heat inactivation method according to any one of claims 1 to 3, characterized in that: The preparation steps of AKK bacteria fermentation broth include: Inoculating AKK bacteria into a seed culture medium for activation culture to prepare a seed solution; The seed liquid is inoculated into the fermentation medium for fermentation culture.

5. The thermal inactivation method according to claim 4, characterized in that Meet one or more of the following characteristics: (a1) After the fermentation culture is completed and before heat inactivation, the pH value of the fermentation broth is adjusted to 6.5 to 9.0; (a2) the seed culture medium is a non-animal-derived culture medium, comprising 5 g / L to 12 g / L of N-acetyl-D-glucosamine, 25 g / L to 40 g / L of soy peptone, 12 g / L to 20 g / L of yeast extract powder, 2 g / L to 5 g / L of potassium dihydrogen phosphate, 1 g / L to 4 g / L of glucose, 1 g / L to 4 g / L of sodium carbonate, 1 g / L to 4 g / L of L-threonine, 0.1 g / L to 1 g / L of ammonium chloride, 0.5 g / L to 2 g / L of Tween 80, 0.1 g / L to 1 g / L of L-cysteine ​​hydrochloride, and water; (a3) The conditions for performing activation culture include: performing activation culture under anaerobic conditions, the temperature of activation culture is 36° C. to 38° C., and the activation culture is static culture; (a4) The conditions for performing activation culture include: the number of activation culture is 1 to 3 times, and the duration of each activation culture is 20 hours to 30 hours; (a5) The conditions for fermentation culture include: fermentation culture under anaerobic conditions, the fermentation culture temperature is 36° C. to 38° C., the fermentation culture is carried out under stirring conditions, the stirring speed is 50 rpm to 100 rpm, and the fermentation culture time is 9 h to 24 h; (a6) The volume of the fermentation system for fermentation culture is 1L to 100L.

6. The heat inactivation method according to any one of claims 1 to 3, characterized in that: Meet one or more of the following characteristics: (b1) the temperature for heat inactivation is 60° C. to 80° C.; (b2) thermal inactivation is carried out under stirring conditions at a stirring speed of 100 rpm to 500 rpm; (b3) The duration of heat inactivation is 10 min to 60 min; (b4) The pH condition for heat inactivation is 6.5 to 9.

0.

7. The heat inactivation method according to claim 5, characterized in that: Meet one or more of the following characteristics: (c1) the fermentation medium and the seed medium each independently contain 6 g / L of N-acetyl-D-glucosamine, 28 g / L of soy peptone, 16 g / L of yeast extract, 3.4 g / L of potassium dihydrogen phosphate, 2 g / L of glucose, 3 g / L of sodium carbonate, 3 g / L of L-threonine, 0.5 g / L of ammonium chloride, 1 g / L of Tween 80, 0.5 g / L of L-cysteine ​​hydrochloride and water; (c2) the fermentation medium and the seed medium each independently contain 5.8 g / L to 6.2 g / L of N-acetyl-D-glucosamine, 26 g / L to 30 g / L of soy peptone, 15.5 g / L to 16.5 g / L of yeast extract, 3.3 g / L to 3.5 g / L of potassium dihydrogen phosphate, 1.8 g / L to 2.2 g / L of glucose, 2.8 g / L to 3.2 g / L of sodium carbonate, 2.8 g / L to 3.2 g / L of L-threonine, 0.45 g / L to 0.55 g / L of ammonium chloride, 0.9 g / L to 1.1 g / L of Tween 80, 0.45 g / L to 0.55 g / L of L-cysteine ​​hydrochloride, and the balance of water; (c3) the fermentation medium and the seed medium are each independently a non-animal-derived medium, and the non-animal-derived medium is a medium made of food-grade raw materials or a medium made of pharmaceutical-grade raw materials; (c4) after the fermentation culture is completed and before heat inactivation, the pH value of the fermentation broth is adjusted to 8.0 to 8.5; (c5) The activation culture temperature is 37±0.5°C; (c6) The conditions for performing activation culture include: the number of activation culture is 2 to 3 times, and the duration of each activation culture is 20 hours to 30 hours; (c7) The activation culture is performed 2 or 3 times, the first activation culture lasts 22 to 24 hours, and the second activation culture lasts 20 to 22 hours; (c8) the fermentation temperature is 37±0.5°C; (c9) The fermentation culture duration is 10 h to 14 h; (c10) The volume of the fermentation system for fermentation culture is 1 L to 100 L; (c11) The temperature for heat inactivation is 70±2°C or 70±1°C.

8. A method for preparing inactivated Akkermansia muciniphila, characterized in that: The steps include: Provide AKK bacteria fermentation liquid; The AKK bacterial fermentation broth is heat-inactivated; After the heat inactivation is completed, the bacteria are collected to obtain the inactivated AKK bacteria; Wherein, the AKK bacteria is Akkermansia muciniphila; the AKK bacteria include one or more of AM02 bacteria and AM06 bacteria; AM02 is Akkermansia muciniphila AM02, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on June 28, 2021, with the deposit number CGMCC No.22794; The AM06 bacterium is Akkermansia muciniphila AM06. Akkermansia muciniphila AM06 was deposited in the General Microbiology Center of China National Microbiological Culture Collection on June 28, 2021, with the deposit number CGMCC No.22793.

9. The preparation method according to claim 8, characterized in that: Meet one or more of the following characteristics: (d1) the AKK bacteria fermentation broth is the AKK bacteria fermentation broth used in the heat inactivation method according to any one of claims 1 to 7; (d2) heat-inactivating the AKK bacteria fermentation broth using the heat inactivation method of claim 6 or 7; (d3) collecting the cells by centrifugation after the heat inactivation is completed; (d4) the step of collecting the bacterial cells after the heat inactivation is completed comprises: adding a flocculant to the mixed solution obtained after the heat inactivation is completed, and then collecting the bacterial cells by centrifugation; Optionally, the flocculant comprises chitosan and gamma-polyglutamic acid.

10. The preparation method according to claim 9, characterized in that: Meet one or more of the following characteristics: (e1) the step of collecting the bacterial cells after the heat inactivation is completed comprises: adding a flocculant mother solution to the mixed solution obtained after the heat inactivation is completed, and then collecting the bacterial cells by centrifugation; the flocculant mother solution comprises a flocculant, and the concentration of the flocculant in the flocculant mother solution is 2 g / L to 10 g / L; (e2) the flocculant comprises chitosan and γ-polyglutamic acid in a mass ratio of 1:3 to 3:1; (e3) The method of collecting the bacterial cells by centrifugation is cup centrifugation or tube centrifugation; Optionally, after the heat inactivation is completed, the bacteria are collected by cup centrifugation, the centrifugal force is 10000×g to 16000×g, and the centrifugation time is 20min to 40min; or, after the heat inactivation is completed, the bacteria are collected by tube centrifugation, the centrifugal force is 10000×g to 16000×g, and the flow rate is 0.5L / min to 2.0L / min.

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