Animal-source-component-free culture method suitable for Akk growth

Abstract

The invention relates to the technical field of bacterial culture, particularly discloses a culture method free of animal source components and suitable for Akk growth, and aims to solve the problem that viruses, toxins, allergens and the like are possibly carried in the culture process in the prior art. Obtaining Akk in a frozen state, and unfreezing at room temperature; inoculating into a target culture medium according to the inoculum size of 10%, standing and culturing for 48 hours in a preset air environment, and activating three generations to restore the vitality of thalli; the target culture medium comprises the following components: 11.38 g / L of yeast extract powder, 34.13 g / L of neutral soy peptone, 3.5 g / L of glucose, 4.2 g / L of disodium hydrogen phosphate, 3 g / L of galactooligosaccharide, 3 g / L of N-acetyl glucosamine, 1.5 g / L of threonine and 0.5 g / L of L-cysteine. The culture medium provided by the invention is suitable for Akk growth, has simple components, does not contain animal-derived components, does not carry viruses, toxins, allergens and the like, and meets the requirements of people.

Description

Technical Field

[0001] The invention relates to the technical field of bacterial culture, in particular to a culture method without animal-derived components suitable for Akk growth. Background Art

[0002] Akk bacteria are gram-negative anaerobic, non-motile, non-spore-forming oval bacteria belonging to the Planctomycetes Verrucomicrobia-Chlamydiae superphylum. The long axis of the single cell is about 0.6-1.0 μm. The organisms mostly grow as single cells or in pairs, and rarely grow in chains. Akk grows fastest at 37°C and pH 6.5. It does not grow at pH values ​​below 5.5 or above 8. It can use intestinal mucin as its only carbon and nitrogen source.

[0003] Studies have found that Akk bacteria are closely related to the host's health, metabolism, and immunity. Using animal models and human experiments, researchers have found that changes in the abundance of Akk bacteria are significantly associated with many diseases, including neurological diseases, hypertension, diabetes, and obesity. The regulatory role of Akk bacteria on host health has been widely recognized. Studies have shown that it has the functions of promoting intestinal mucosal barrier repair, regulating intestinal flora metabolism, and affecting the "gut-liver axis" and "brain-gut axis", indicating that Akk bacteria may be a potential next-generation probiotic that can improve host health.

[0004] Since Akk is a strict anaerobic bacterium, it is difficult to culture and isolate it, and the requirement of anaerobic culture limits people's further research on it. In addition, most of the known Akk bacterial culture media contain animal components, and the nutrients from animals may carry viruses, toxins, allergens, etc., which also limits the use of Akk bacteria in human food and medicine.

[0005] Based on this, a culture method suitable for Akk growth without animal-derived components is now provided, which can eliminate the disadvantages of existing devices. Summary of the invention

[0006] The purpose of the present invention is to provide a culture method suitable for the growth of Akk without animal-derived components, which solves the problem that the culture process in the prior art may carry viruses, toxins, allergens, etc.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A culture method suitable for Akk growth without animal-derived components comprises the following steps:

[0009] Step 1: Obtain the frozen Akk and thaw it at room temperature;

[0010] Step 2: Inoculate the target culture medium at a 10% inoculum volume, culture it in a predetermined air environment for 48 hours, and activate it for three generations to restore the vitality of the bacteria;

[0011] The composition of the target culture medium is: yeast extract powder 11.38g / L, neutral soy peptone 34.13g / L, glucose 3.5g / L, disodium hydrogen phosphate 4.2g / L, galacto-oligosaccharide 3g / L, N-acetylglucosamine 3g / L, threonine 1.5g / L, L-cysteine ​​0.5g / L.

[0012] On the basis of the above technical solution, the present invention also provides the following optional technical solution:

[0013] In an optional scheme: the specific operation of thawing is: carefully take out the frozen ampoule containing cells from the liquid nitrogen storage box, put it into a liquid nitrogen container or on dry ice, quickly transfer it to the laboratory, and check whether the lid is tightened to remove liquid nitrogen residue.

[0014] In an optional scheme: In a microbiological safety cabinet, wrap the cap of the frozen ampoule with a paper towel soaked in 70% alcohol, then turn the cap a quarter turn to release any residual liquid nitrogen, then re-tighten the cap, and quickly thaw in a water bath: quickly transfer the ampoule to a 37°C water bath and shake gently until only one or two small ice crystals remain. Be careful not to completely immerse the ampoule to avoid contamination. It takes about 1-2 minutes. Disinfection and transfer: Before opening the ampoule, wipe the ampoule with a paper towel soaked in 70% alcohol, then pipette the entire contents of the ampoule into a sterile tube, and then slowly add preheated culture medium.

[0015] In an optional embodiment: the floating accuracy of the components of the target culture medium is plus or minus 0.01-0.05 g / L.

[0016] In an optional embodiment: the floating accuracy of the components of the target culture medium is plus or minus 0.02 g / L.

[0017] In an optional solution: the predetermined air environment is: 5% H2, 10% CO2, 85% O2.

[0018] A culture medium without animal-derived ingredients suitable for the growth of Akk comprises the following components: 11.38 g / L yeast extract powder, 34.13 g / L neutral soy peptone, 3.5 g / L glucose, 4.2 g / L disodium hydrogen phosphate, 3 g / L galacto-oligosaccharide, 3 g / L N-acetylglucosamine, 1.5 g / L threonine and 0.5 g / L L-cysteine.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides a culture medium which is suitable for the growth of Akk and has simple ingredients and does not contain animal-derived ingredients. The Akk cultured in the culture medium can be used for humans, such as food, feed, and medicine, thus meeting people's needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effect of different carbon sources on AkkOD value.

[0022] Figure 2 The effect of different nitrogen sources on AkkOD value.

[0023] Figure 3 The effects of different potential Akk growth-promoting substances on Akk OD value.

[0024] Figure 4 This is the growth curve of Akk in the culture medium before and after optimization.

[0025] Figure 5 The effect of different carbon sources on the number of viable Akk cells.

[0026] Figure 6 The effect of different nitrogen sources on the number of viable Akk bacteria. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Akkermansia muciniphila ATCC BAA-835 used in the experiment was purchased from Beijing Biobo Biotechnology Co., Ltd.; brain heart infusion broth, brain heart infusion agar, and porcine gastric mucin were purchased from Qingdao Haibo Biotechnology Co., Ltd.; Basicl anaerobic / microaerobic workstation was purchased from Chongqing Jiangxue Technology Co., Ltd.; LDZM-LI vertical high-pressure steam sterilizer was purchased from Shanghai Xiren Scientific Instrument Co., Ltd.; SW-CJ-2D double-person single-sided vertical clean bench was purchased from Shanghai Hujing Medical Equipment Co., Ltd.; UNIC-7200 visible spectrophotometer was purchased from Jinan Boxin Biotechnology Co., Ltd.;

[0029] See also Figure 1-Figure 6 , the embodiment of the present invention discloses a culture method suitable for Akk growth without animal-derived components:

[0030] Finding a carbon source suitable for Akk growth:

[0031] ① Take out the Akk preserved in glycerol from the -80℃ refrigerator and thaw it at room temperature. Inoculate it into BHI broth + mucin medium at a 10% inoculum size, culture it statically at 5% H2, 10% CO2, 85% O2, and 37℃ for 48 hours, and activate it for three generations to restore the vitality of the bacteria.

[0032] ② Replace the glucose in the initial culture medium (BHI+mucin culture medium) with different carbon sources in equal amounts, sterilize the culture medium at 121℃ for 10 minutes and immediately transfer it to an anaerobic incubator. Inoculate the activated Akk bacteria solution of the third generation into each group at a 10% inoculum amount, shake well and place it in a static culture at 5% H2, 10% CO2, 85% O2, and 37℃ for 48 hours. Perform OD600nm determination and dilution plate viable bacteria count. The results are shown in the figure. It can be clearly seen from OD600nm that there are significant differences in the utilization effects of Akk on different carbon sources (P<0.05), among which glucose has the best utilization effect (0.39), followed by oligogalactose (0.31), and arabinogalactan has the worst utilization effect (0.11); there are also significant differences in the number of viable cells, with glucose reaching the highest (1.32±0.24)×108CFU / mL, and arabinogalactan having the worst effect (1.08±0.16)×107CFU / mL. Both OD600nm and the number of viable cells indicate that Akk has the best utilization effect on glucose among these carbon sources.

[0033] Example 2

[0034] Finding a nitrogen source suitable for Akk growth:

[0035] ① Take out the Akk preserved in glycerol from the -80℃ refrigerator and thaw it at room temperature. Inoculate it into BHI broth + mucin medium at a 10% inoculum size, culture it statically at 5% H2, 10% CO2, 85% O2, and 37℃ for 48 hours, and activate it for three generations to restore the vitality of the bacteria.

[0036] ② Based on Example 1, the tryptone and beef heart extract powder of the initial culture medium (BHI+mucin culture medium) were replaced with different nitrogen sources in equal amounts. The culture medium was sterilized at 121°C for 10 minutes and then immediately transferred to an anaerobic incubator. The Akk bacterial solution of the third generation was inoculated into each group at a 10% inoculum, and after being shaken well, it was placed in a static culture at 5% H2, 10% CO2, 85% O2, and 37°C for 48 hours. OD600nm determination and viable count of dilution plate were performed. The OD600nm values ​​of each test group were 0.04, 0.50, 0.46, 0.52, 0.55, and 0.43, respectively. There were significant differences in the OD600nm values ​​among the groups (P<0.05); and the viable counts of each group were 0, (2.36±0.11)×108, (2.56±0.24)×108, (3.78±0.16)×108, (5.14±0.26)×108, and (2.83±0.13)×108, respectively. From the results of OD600nm values ​​and viable counts, it can be seen that Akk has the best utilization effect on the composite nitrogen source (yeast extract powder + neutral soy peptone).

[0037] Example 3

[0038] Explore Akk growth promoting substances:

[0039] ① Take out the Akk preserved in glycerol from the -80℃ refrigerator and thaw it at room temperature. Inoculate it into BHI broth + mucin medium at a 10% inoculum size, culture it statically at 5% H2, 10% CO2, 85% O2, and 37℃ for 48 hours, and activate it for three generations to restore the vitality of the bacteria.

[0040] ②Based on the previous experiments, the literature was consulted to select substances that may promote the growth of Akk to replace mucin in the culture medium. The culture medium was sterilized at 121℃ for 10 minutes and then immediately transferred to an anaerobic incubator. The activated Akk bacterial solution of the third generation was inoculated into each group at a 10% inoculation rate, and then placed in 5% H2, 10% CO2, 85% O2, and 37℃ for 48 hours. OD600nm determination and dilution plate live bacteria count were performed. The OD600nm values ​​of each experimental group were 0.22, 0.44, 0.24, 0.34, 0.05, 0.24, 0.23, 0.28, 0.28, 0.23, and 0.24, respectively. Among them, N-acetylglucosamine, galacto-oligosaccharide, lysine, and threonine were significantly different from the blank group, and the OD600nm values ​​were significantly improved (P<0.05). The growth rates of Akk increased by 100%, 54.55%, 27.27% and 27.27% respectively. It may be a potential Akk growth-promoting substance, and the effect of the addition amount of each substance on the growth of Akk will be explored in the future.

[0041] Example 4

[0042] Growth curve of Akk cultured before and after optimization:

[0043] ① Take out the Akk preserved in glycerol from the -80℃ refrigerator and thaw it at room temperature. Inoculate it into BHI broth + mucin medium at a 10% inoculum size, culture it statically at 5% H2, 10% CO2, 85% O2, and 37℃ for 48 hours, and activate it for three generations to restore the vitality of the bacteria.

[0044] ② The activated Akk was inoculated in the optimized culture medium and BHI+mucin culture medium at a 10% inoculation rate, and 200ul of bacterial solution was injected into a 96-well plate at 0, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 hours, respectively. The absorbance values ​​of Akk cultured in the two culture media were measured at 600nm using an automatic microplate reader. The growth curve of Akk was drawn with the culture time as the horizontal axis and the absorbance value as the vertical axis.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A culture method suitable for the growth of Akk without animal-derived components, characterized in that: The following steps are involved: Step 1: Obtain the frozen Akk and thaw it at room temperature; Step 2: Inoculate the target culture medium at a 10% inoculum volume, culture it in a predetermined air environment for 24-72 hours, and activate it for three generations to restore the vitality of the bacteria; The composition of the target culture medium is: yeast extract powder 11.38g / L, neutral soy peptone 34.13g / L, glucose 3.5g / L, disodium hydrogen phosphate 4.2g / L, galacto-oligosaccharide 3g / L, N-acetylglucosamine 3g / L, threonine 1.5g / L, L-cysteine ​​0.5g / L.

2. The animal-free culture method suitable for Akk growth according to claim 1, characterized in that: The floating accuracy of the components of the target culture medium is plus or minus 0.01-0.05 g / L.

3. The animal-free culture method suitable for Akk growth according to claim 1, characterized in that: The floating accuracy of the components of the target culture medium is plus or minus 0.0 g / L.

4. The animal-free culture method suitable for Akk growth according to claim 1, characterized in that: The specific operation of thawing is: carefully take out the frozen ampoule containing cells from the liquid nitrogen storage box, put it into a liquid nitrogen container or on dry ice, quickly transfer it to the laboratory, check whether the lid is tightened, and remove liquid nitrogen residue.

5. The animal-free culture method suitable for Akk growth according to claim 4, characterized in that: In the microbiological safety cabinet, wrap the frozen ampoule cap with a paper towel soaked in 70% alcohol, then turn the cap a quarter turn to release any residual liquid nitrogen, then re-tighten the cap. Rapid water bath thawing: quickly transfer the ampoule to a 37°C water bath and shake gently until only one or two small ice crystals remain. Be careful not to completely immerse the ampoule to avoid contamination. It takes about 1-2 minutes. Disinfection and transfer: Before opening the ampoule, wipe the ampoule with a paper towel soaked in 70% alcohol, then pipette the entire contents of the ampoule into a sterile tube, and then slowly add preheated culture medium.

6. The animal-free culture method suitable for Akk growth according to claim 1, characterized in that: The cells were incubated in a predetermined air environment for 48 hours.

7. The animal-free culture method suitable for Akk growth according to claim 1, characterized in that: The predetermined air environment is: 5% H2, 10% CO2, 85% O2.

8. A medium without animal-derived components suitable for the growth of Akk, characterized in that: The invention comprises the following components: 11.38 g / L yeast extract powder, 34.13 g / L neutral soy peptone, 3.5 g / L glucose, 4.2 g / L disodium hydrogen phosphate, 3 g / L galacto-oligosaccharide, 3 g / L N-acetylglucosamine, 1.5 g / L threonine and 0.5 g / L L-cysteine.

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