Method for constructing intestinal microorganism in-vitro simulation system and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly provides a method for constructing an intestinal microorganism in-vitro simulation system, which comprises the following steps: taking cecum contents, uniformly mixing, inoculating into an anaerobic fermentation tank for fermentation, and controlling the addition speed of a culture medium; the pH is controlled to be 6.0-7.0 during fermentation, and fermentation is performed for 1-7 days to obtain fermentation liquor containing intestinal microorganisms. According to the method for constructing the intestinal microorganism in-vitro simulation system provided by the invention, the dominant flora in the fermentation liquor is basically consistent with the original flora of cecum, the dominant flora of the original flora in the body can be reduced to a certain extent, the dynamic change of the intestinal microorganisms can be simulated in vitro, and the method is easy to standardize, simple to operate, low in cost and suitable for popularization and application. The method is suitable for evaluating the interaction effect of the exogenously added microecologics and the intestinal microorganisms, does not depend on hosts, is less influenced by individual differences of the hosts, and is an effective supplement for animal experiments.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to a method for constructing an in vitro simulation system of intestinal microorganisms and an application thereof. Background Art

[0002] Intestinal microorganisms are of great importance to animals. Microorganisms can participate in host metabolic pathways to regulate the physiological functions of animals by connecting the intestines, brain, liver and kidneys. The intestinal microbiome interacts and communicates with host cells in various ways, and together with the host genome, affects human health. Some substances produced by the intestinal flora, such as short-chain fatty acids (SCFAs), carotenoids, 5-hydroxytryptamine, etc., are released when the intestinal flora is disturbed, such as lipopolysaccharide.

[0003] Due to the huge number of intestinal microorganisms, the culture medium of microorganisms of different types and living habits is specific, difficult to separate, and more than 99% of bacterial species have not been fully recorded, making it difficult to fully study intestinal microorganisms through isolation and culture. Therefore, when studying the functions and mechanisms of intestinal microorganisms, animal or human experiments are often used to collect microbial samples in the intestine by collecting feces or slaughtering, but they face problems such as high cost, complex operation, low representativeness of feces, and potential ethical issues. Summary of the invention

[0004] The purpose of the present invention is to overcome the problems of high cost, complex operation, low representativeness and poor stability of existing intestinal microbial research.

[0005] To this end, the present invention provides a method for constructing an in vitro simulation system of intestinal microorganisms, comprising the following steps: taking the contents of the cecum, mixing them evenly and inoculating them into an anaerobic fermentation tank for fermentation, and controlling the rate of adding the culture medium; during the fermentation period, controlling the pH at 6.0-7.0, fermenting for 1-7 days, and obtaining a fermentation liquid containing intestinal microorganisms.

[0006] Specifically, the culture medium includes: 6-10 g / L tryptone, 5-9 g / L yeast extract, 4-8 g / L sodium chloride, 1.0-3.0 g / L glucose, 2.4-4.4 g / L beef extract, and 0.3-0.8 g / L L-cysteine ​​hydrochloride. ， Soluble starch 6-12g / L, agar powder 6-10g / L.

[0007] Specifically, the addition rate of the above culture medium is 3 to 10 mL / L / h.

[0008] Specifically, the above-mentioned inoculation amount of the cecum contents is 1 based on the volume of the culture medium in the fermenter. ～ 2g / L.

[0009] Specifically, sodium hydroxide is used to adjust the pH.

[0010] The method for constructing an in vitro simulation system of intestinal microorganisms provided by the present invention can be used to study the effects of exogenously added microecological preparations on intestinal microorganisms.

[0011] The present invention also provides a method for evaluating the effect of an exogenous additive, comprising the following steps:

[0012] (1) constructing an in vitro simulation system of intestinal microorganisms using the above method;

[0013] (2) Dividing the in vitro intestinal microbial simulation system into an experimental group and a control group, and adding the exogenous additive to be tested to the experimental group;

[0014] (3) The control group and the experimental group continued to ferment under the same conditions, and the growth of the bacterial flora in the fermentation liquid was dynamically monitored. Based on the differences in the bacterial flora between the experimental group and the control group, the effect of the exogenous additive was evaluated.

[0015] Specifically, the amount of the exogenous additive added is 0.1 to 0.4 g / L based on the volume of the fermentation liquid containing intestinal microorganisms.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0017] The method for constructing an in vitro simulation system of intestinal microorganisms provided by the present invention has the dominant flora in the fermentation broth being basically consistent with the original flora in the cecum, and can restore the dominant flora of the original flora in the body to a certain extent, and can simulate the dynamic changes of intestinal microorganisms in vitro, and is easy to standardize, simple to operate, and low in cost. It is suitable for evaluating the interaction effect between exogenously added microecological preparations and intestinal microorganisms, and because it does not depend on the host, it is less affected by individual differences in the host, and is an effective supplement to animal experiments.

[0018] The present invention will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the growth curve of the cecal flora in Example 1 of the present invention.

[0020] Figure 2 This is the result of detecting the number of Escherichia coli and Lactobacillus in Example 1 of the present invention.

[0021] Figure 3It is the analysis of intestinal microbial diversity in Example 1 of the present invention; A is the Sob value and Shannon value of the fermentation broth at different fermentation times; B is the Sob value and Shannon value of the fermentation broth of three batches of 168h; 1-168h is the fermentation broth of the first batch of 168h, 2-168h is the fermentation broth of the second batch of 168h, 3-168h is the fermentation broth of 168h, the same below; C is the PcoA analysis chart of microorganisms at different fermentation times and fermentation batches; D is the distribution of microorganisms at the phylum level in the fermentation broth at different fermentation times; E is the distribution of cecal microorganisms of broiler chickens cultured in vitro at different fermentation times at the genus level. .

[0022] Figure 4 This is the growth curve of the cecal flora in Example 2 of the present invention; wherein A is a 28-day-old in vitro fermentation system, and B is a 42-day-old in vitro fermentation system.

[0023] Figure 5 It is the detection result of the number of Escherichia coli and Lactobacillus in Example 2 of the present invention; wherein A is the number of Lactobacillus in the 28-day fermentation system, B is the number of Escherichia coli in the 28-day fermentation system, C is the number of Lactobacillus in the 42-day fermentation system, and D is the number of Escherichia coli in the 42-day fermentation system.

[0024] Figure 6 It is the intestinal microbial diversity analysis in Example 2 of the present invention; wherein A is the community composition at the phylum level of the in vitro fermentation system at 28 days old, B is the community composition at the genus level of the in vitro fermentation system at 28 days old, C is the community composition at the phylum level of the in vitro fermentation system at 42 days old, and D is the community composition at the genus level of the in vitro fermentation system at 42 days old; NC-28d: in vitro fermentation system without the addition of probiotics, C-28d in vitro fermentation system intervened by probiotics, 28d: 28-day-old original broiler cecal sample, NC-42d, C-42d, and 42d are the same. DETAILED DESCRIPTION

[0025] The technical scheme in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Although the representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be limited by the attached claims and their equivalents.

[0026] The present invention provides a method for constructing an in vitro simulation system of intestinal microorganisms, comprising the following steps: taking cecal contents, mixing them evenly, and then inoculating them into an anaerobic fermentation tank for fermentation, wherein the inoculation amount of the cecal contents is 1 to 2 g / L based on the volume of the culture medium in the fermentation tank, and the addition rate of the culture medium is controlled to be 3 to 10 mL / L / h, that is, based on the capacity of the fermentation tank, 3 to 10 ml of culture medium is added per liter of the fermentation tank per hour. During the fermentation, sodium hydroxide is used to control the pH to 6.0 to 7.0, and the fermentation is carried out for 1 to 7 days to obtain a fermentation liquid containing intestinal microorganisms.

[0027] The culture medium includes: tryptone 6-10 g / L, yeast extract powder 5-9 g / L, sodium chloride 4-8 g / L, glucose 1.0-3.0 g / L, beef extract powder 2.4-4.4 g / L, L-cysteine ​​hydrochloride 0.3-0.8 g / L ， Soluble starch 6-12g / L, agar powder 6-10g / L.

[0028] The present invention also provides a method for evaluating the effect of an exogenous additive, comprising the following steps:

[0029] (1) constructing an in vitro simulation system of intestinal microorganisms using the above method;

[0030] (2) The in vitro intestinal microbial simulation system is divided into an experimental group and a control group, and the exogenous additive to be tested is added to the experimental group; the amount of the exogenous additive added is 0.1-0.4 g / L.

[0031] (3) The control group and the experimental group continued to ferment under the same conditions, and the growth of the bacterial flora in the fermentation liquid was dynamically monitored. Based on the differences in the bacterial flora between the experimental group and the control group, the effect of the exogenous additive was evaluated.

[0032] The method for constructing an in vitro simulation system of intestinal microorganisms and the effect of its application are studied below through specific examples.

[0033] Embodiment 1:

[0034] 1. Culture medium preparation and sterilization

[0035] Prepare 5 L fermentation medium and adjust the pH to 6.5. The medium includes: 8 g / L tryptone, 7 g / L yeast extract powder, 6 g / L sodium chloride, 1.5 g / L glucose, 3.4 g / L beef extract powder, 0.6 g / L L-cysteine ​​hydrochloride, 10 g / L soluble starch, and 8 g / L agar powder.

[0036] Add the prepared culture medium to the feed tank. Wrap the temperature probe and pH electrode of the anaerobic fermentation tank with tin foil, put the entire fermentation tank in the autoclave for sterilization, and sterilize the feed tank separately. The sterilization conditions are: 115℃, 30min.

[0037] 2. Construction of in vitro simulation system of intestinal microorganisms

[0038] 2.1 Preparing the fermentation tank

[0039] Connect all parts of the anaerobic fermentation tank, check whether the temperature probe and pH electrode can work properly, pump about 1L of fresh culture medium into the fermentation tank, set the temperature to 37℃, and the pH to 6.5.

[0040] 2.2 Pretreatment of cecal contents of broiler chickens

[0041] At the Tuanfeng test base, 10 AA broilers were taken and fed with a basic diet until 28 and 42 days of age. The broilers were then slaughtered, and their cecal contents were taken out and mixed evenly, put into a sampling tube, and the samples were stored in a liquid nitrogen tank. When used, 2 g of the preserved cecal contents of the broilers were taken out on the clean bench, 100 mL of sterile VL culture medium was added to a sterilized 250 mL triangular flask, and paraffin oil was poured in to seal it. The broilers were cultured at a constant temperature for 16 hours at a culture temperature of 37°C.

[0042] 2.3 Inoculation and simulated fermentation

[0043] The cecal contents after about 16 hours of culture were inoculated into the anaerobic fermentation tank in three batches for fermentation. After 4 hours, the fermentation system was operated and the addition rate of the culture medium was controlled to be 5 mL / L / h. During the fermentation, the pH was adjusted with sodium hydroxide solution to keep the pH value at 6.5. The fermentation time was 7 days, and the fermentation broth was collected every day during the fermentation period for the determination of microorganisms in the fermentation broth.

[0044] 2.4 Sampling

[0045] Use 10 mL centrifuge tubes to collect the fermentation broth at 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h, and measure the OD 600 The number of Escherichia coli and Lactobacillus in the fermentation broth was determined and the collected fermentation broth samples were stored in a -20°C refrigerator. Subsequently, the microbial diversity of the fermentation broth was detected and analyzed at 24h, 48h, 72h, 96h, 120h, 144h and 168h.

[0046] 2.5 Results of in vitro anaerobic culture test of cecal flora of broiler chickens

[0047] The dynamic monitoring results of the cecal flora growth curve are as follows: Figure 1 As shown, the growth curves of the three batches are not completely consistent, but the trends are basically the same; from 8 to 24 hours, they are in a rapid growth period, from 32 to 68 hours, the overall growth slows down, and the OD curve begins to show a downward trend from 68 hours.

[0048] The results of the Escherichia coli and Lactobacillus test are as follows: Figure 2 As shown, the number of lactic acid bacteria basically maintained at a certain level for 24-72 hours, and then decreased with the extension of fermentation time; the number of Escherichia coli reached the highest level at 48 hours, and then decreased with the extension of fermentation time; at 48 hours, the number of Escherichia coli was higher than that of Lactobacillus, and was lower than that of Lactobacillus at other fermentation times.

[0049] The results of intestinal microbial diversity analysis are as follows Figure 3 As shown, Figure 3 D and Figure 3 "28d" in E is the detection result of the original flora of the cecum. The dominant flora in the cecum microorganisms after fermentation is basically consistent with the original flora of the cecum. The in vitro simulation system of broiler intestinal microorganisms constructed by the present invention can restore the dominant flora of the original flora in the body to a certain extent.

[0050] Embodiment 2:

[0051] Ten healthy AA broilers of similar weight at 28d and 42d were selected, and the in vitro simulation systems of intestinal microorganisms at 28 and 42 days of age were constructed according to the steps of Example 1, and the cecal contents cultured for 16h were inoculated into an anaerobic fermentation tank for fermentation. The fermentation system was operated after 4h, and probiotics were added to the fermentation broth as the experimental group, and the fermentation broth without probiotics was used as the control group. The fermentation broth was collected regularly during the fermentation period, and the microorganisms in the fermentation broth were determined to study the effect of exogenous probiotics on intestinal microorganisms.

[0052] Probiotics include: Clostridium butyricum 4×10 8 CFU / g, Bacillus subtilis 8×10 8 CFU / g, Bacillus coagulans 8×10 8 CFU / g, glucose oxidase 1000U / g; based on the volume of fermentation liquid, the addition amount is 0.4g / L.

[0053] The dynamic monitoring results of the cecal flora growth curve are as follows: Figure 4 As shown in the figure, the growth curves of the experimental group and the control group were basically synchronized. In the early stage of in vitro culture, the bacterial density in the probiotic preparation group was higher; in the later stage of in vitro culture, the bacterial density in the probiotic preparation group was relatively lower.

[0054] Depend on Figure 5 It can be seen that after adding probiotics to the in vitro fermentation system of cecal microorganisms of two groups of different ages, the number of Escherichia coli in the experimental group was significantly reduced, and there were differences in the effects on Lactobacillus. This shows that the addition of probiotics can reduce the number of harmful bacteria in the in vitro fermentation system and increase the number of beneficial bacteria in the fermentation system to varying degrees.

[0055] The diversity analysis of intestinal microorganisms showed that Figure 6 As shown in the figure, "28d" and "42d" are the original flora of the cecum of broiler chickens at different ages. Although the diversity of microorganisms in the in vitro fermentation system is reduced, its specific microbial composition still needs to be analyzed. From the "phylum level", regardless of whether the microecological preparations are added, the bacteria of the three phyla, Firmicutes, Bacteroidetes and Proteobacteria, are dominant in the in vitro fermentation system. After the addition of microecological preparations, the relative abundance level of Bacteroidetes in the in vitro fermentation system at 28 days old increased ( Figure 6 A), while in the 42-day-old in vitro fermentation system, the relative abundance of Firmicutes was upregulated ( Figure 6 C).

[0056] From the "genus" level analysis, in the 28-day in vitro fermentation system ( Figure 6 B): The original cecal microbial community of broiler chickens mainly includes: Oscillospiraceae, Bacteroides, Barnesella, and Ruminococcaceae. In the in vitro fermentation system, it is mainly composed of unclassified_f__Oscillospiraceae, Escherichia coli, Bacteroides, and Ruminococcus. Compared with the original cecal microbial community in the in vitro fermentation system at 28 days old, some genera are different, but they are generally consistent, which is consistent with the results in the early establishment of the fermentation system. In the 42-day-old fermentation system, the original cecal microbial community mainly includes: Bacteroides, Ruminococcus, Barnesella, unclassified_o__Clostridia_UCG-014; in the in vitro fermentation system, it is mainly composed of Bacteroides, Negativibacillus, Acinetobacter, and Oscillospiraceae.

[0057] A type of Acinetobacter appeared in the in vitro fermentation system. This type of bacteria belongs to the Proteobacteria and is a type of pathogenic bacteria. Acinetobacter baumannii (Ab), which belongs to the same type, is a non-fermenting Gram-negative bacterium and a conditional pathogen. From the results, it can be seen that the exogenous addition of microecological preparations can reduce the abundance of this genus.

[0058] The above examples are merely illustrative of the present invention and do not constitute a limitation on the protection scope of the present invention. All designs that are the same or similar to the present invention fall within the protection scope of the present invention.

Claims

1. A method for constructing an in vitro simulation system of intestinal microorganisms, characterized in that: The following steps are involved: The cecal contents are taken, mixed evenly and then inoculated into an anaerobic fermentation tank for fermentation, and the rate of adding the culture medium is controlled; during the fermentation period, the pH is controlled at 6.0-7.0, and the fermentation is carried out for 1-7 days to obtain a fermentation liquid containing intestinal microorganisms.

2. The method for constructing an in vitro simulation system of intestinal microorganisms according to claim 1, characterized in that: The culture medium comprises: 6-10 g / L tryptone, 5-9 g / L yeast extract powder, 4-8 g / L sodium chloride, 1.0-3.0 g / L glucose, 2.4-4.4 g / L beef extract powder, and 0.3-0.8 g / L L-cysteine ​​hydrochloride. ， Soluble starch 6-12g / L, agar powder 6-10g / L.

3. The method for constructing an in vitro simulation system of intestinal microorganisms according to claim 1, characterized in that: The addition rate of the culture medium is 3 to 10 mL / L / h.

4. The method for constructing an in vitro simulation system of intestinal microorganisms according to claim 1, characterized in that: The inoculation amount of the cecal contents is 1-2 g / L based on the volume of the culture medium in the fermenter.

5. The method for constructing an in vitro simulation system of intestinal microorganisms according to claim 1, characterized in that: The pH was adjusted with sodium hydroxide.

6. Use of the method as described in any one of claims 1 to 5 in studying the effects of exogenously added probiotics on intestinal microorganisms.

7. A method for evaluating the effect of an exogenous additive, characterized in that: The following steps are involved: (1) constructing an in vitro intestinal microbial simulation system using the method described in any one of claims 1 to 5; (2) Dividing the in vitro intestinal microbial simulation system into an experimental group and a control group, and adding the exogenous additive to be tested to the experimental group; (3) The control group and the experimental group continued to ferment under the same conditions, and the growth of the bacterial flora in the fermentation liquid was dynamically monitored. Based on the differences in the bacterial flora between the experimental group and the control group, the effect of the exogenous additive was evaluated.

8. The method for evaluating the effect of an exogenous additive according to claim 7, characterized in that: The amount of exogenous additive added is 0.1-0.4 g / L based on the volume of the fermentation liquid containing intestinal microorganisms.

Images