CN112458157A - In vitro metabolic activity detection system based on intestinal microorganisms - Google Patents
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- 230000000968 intestinal effect Effects 0.000 title claims abstract description 66
- 230000002503 metabolic effect Effects 0.000 title claims abstract description 57
- 244000005700 microbiome Species 0.000 title claims abstract description 42
- 238000001514 detection method Methods 0.000 title claims abstract description 41
- 238000000338 in vitro Methods 0.000 title claims abstract description 36
- 230000000694 effects Effects 0.000 claims abstract description 33
- 235000013406 prebiotics Nutrition 0.000 claims abstract description 31
- 206010012601 diabetes mellitus Diseases 0.000 claims abstract description 24
- 238000000855 fermentation Methods 0.000 claims abstract description 24
- 230000004151 fermentation Effects 0.000 claims abstract description 24
- 238000012163 sequencing technique Methods 0.000 claims abstract description 20
- 238000004519 manufacturing process Methods 0.000 claims abstract description 17
- 230000002550 fecal effect Effects 0.000 claims abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
- 239000006041 probiotic Substances 0.000 claims abstract description 13
- 235000018291 probiotics Nutrition 0.000 claims abstract description 13
- 241000894006 Bacteria Species 0.000 claims abstract description 11
- 230000015556 catabolic process Effects 0.000 claims abstract description 11
- 238000006731 degradation reaction Methods 0.000 claims abstract description 11
- 230000004060 metabolic process Effects 0.000 claims abstract description 10
- 238000012216 screening Methods 0.000 claims abstract description 10
- 239000002253 acid Substances 0.000 claims abstract description 6
- 239000000090 biomarker Substances 0.000 claims abstract description 6
- 230000003902 lesion Effects 0.000 claims abstract description 4
- 239000008280 blood Substances 0.000 claims description 30
- 210000004369 blood Anatomy 0.000 claims description 30
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 25
- 239000008103 glucose Substances 0.000 claims description 25
- 230000002159 abnormal effect Effects 0.000 claims description 12
- 201000009104 prediabetes syndrome Diseases 0.000 claims description 11
- 208000002705 Glucose Intolerance Diseases 0.000 claims description 6
- 230000001771 impaired effect Effects 0.000 claims description 6
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 5
- 230000037396 body weight Effects 0.000 claims description 5
- 150000004666 short chain fatty acids Chemical class 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 3
- 230000002757 inflammatory effect Effects 0.000 claims description 3
- 230000000529 probiotic effect Effects 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 6
- 206010018429 Glucose tolerance impaired Diseases 0.000 description 5
- 208000001280 Prediabetic State Diseases 0.000 description 5
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 241000186000 Bifidobacterium Species 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 244000005709 gut microbiome Species 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 206010022489 Insulin Resistance Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
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Abstract
The invention provides an in vitro metabolic activity detection system based on intestinal microorganisms, which comprises the following steps: acquiring a fecal sample, and screening the fecal sample through a fecal detector; reserving a stool sample without organic lesions according to the screening result; performing activity detection on the organic lesion-free excrement sample; according to the activity detection result, the live intestinal bacteria metabolism value; comparing the intestinal bacteria metabolism value with prestored information, and performing gene sequencing on the large difference of the comparison values; determining the structural characteristics of the intestinal flora according to the gene sequencing result; obtaining the biomarker of the early stage of diabetes according to the structural characteristics of the intestinal flora. The intestinal microorganism in-vitro metabolic activity detection system is utilized to carry out in-vitro fermentation on the excrement of different subtypes and normal people, detect the metabolic response values corresponding to different multiple prebiotics, including gas production, acid production, the degradation degree of the prebiotics and the promotion effect on the probiotics, and comprehensively reflect the whole metabolic level of the intestinal microorganism.
Description
Technical Field
The invention relates to the technical field of intestinal microorganism detection, in particular to an in-vitro metabolic activity detection system based on intestinal microorganisms.
Background
At present, the incidence of pre-diabetes is very high, but the degree of importance and the adherence rate of treatment are very low. The unfavorable control to the early stage of diabetes leads to the obvious increase of the incidence and the death rate of diabetes and cardiovascular diseases in China and brings catastrophic attack to the fragile sanitary economy in China.
A great deal of research shows that the intestinal microorganisms play an important role in the occurrence and development of diabetes. Prediabetes-a major cause of insulin resistance, may have different pathogenesis in the light and overweight prediabetes, and intestinal flora and especially its metabolites are important causes; thus, a system capable of detecting intestinal microorganisms is lacking
Disclosure of Invention
The invention provides an in-vitro metabolic activity detection system based on intestinal microorganisms, which is used for judging the incidence of diabetes by the intestinal microorganisms according to the detected intestinal microorganisms and carrying out statistical analysis on the detected intestinal microorganisms.
The invention provides an in vitro metabolic activity detection system based on intestinal microorganisms, which is characterized by comprising the following steps of:
acquiring a fecal sample, and screening the fecal sample through a fecal detector;
reserving a stool sample without organic lesions according to the screening result;
performing activity detection on the organic lesion-free excrement sample;
according to the activity detection result, the live intestinal bacteria metabolism value;
comparing the intestinal bacteria metabolism value with prestored information, and performing gene sequencing on the large difference of the comparison values;
determining the structural characteristics of the intestinal flora according to the gene sequencing result;
obtaining the biomarker of the early stage of diabetes according to the structural characteristics of the intestinal flora.
Preferably, the activity detection comprises the steps of:
inoculating and culturing the fecal sample;
fermenting the inoculated and cultured excrement sample to obtain fermentation liquor;
and analyzing and detecting the fermentation liquor to obtain the activity detection information of the intestinal bacteria.
Preferably, the fermentation time for fermenting the inoculated and cultured excrement sample is 18-30 hours;
the activity detection information includes: the gas production value of the fermentation liquor, the short-chain fatty acid value of the fermentation liquor, the degradation rate value and the probiotic value.
Preferably, the gene sequencing is 16s rRNA gene sequencing.
Preferably, the pre-stored information includes normal values, blood glucose values and body weight values of the activity detection information;
the blood glucose values include a normal blood glucose type and an abnormal blood glucose type, and the body weight values include: obese and non-obese.
Preferably, the activity detection information further includes metabolic response values of inflammatory factors and prebiotics.
Preferably, the metabolic response values comprise: the gas production and acid production of the prebiotics, the degradation degree of the prebiotics and the promotion effect on the probiotics.
Preferably, the abnormal blood glucose types include: impaired obese fasting glucose regulation, impaired non-obese fasting glucose regulation, obese impaired glucose tolerance, and non-obese impaired glucose tolerance;
the normal blood glucose types include: obese normal and non-obese normal;
and comparing the metabolic response value with the abnormal blood sugar type and the normal blood sugar type to obtain metabolic response values corresponding to the abnormal blood sugar type and the normal blood sugar type.
The working principle and the beneficial effects of the invention are as follows:
the in-vitro metabolic activity detection system based on the intestinal microorganisms provided by the invention utilizes the in-vitro metabolic activity detection system of the intestinal microorganisms to carry out in-vitro fermentation on the excrement of different subtypes and normal people, detects the metabolic response values corresponding to different multiple prebiotics, including gas production, acid production, the degradation degree of the prebiotics and the promotion effect on the probiotics, and more comprehensively reflects the whole metabolic level of the intestinal microorganisms. Both too high and too low metabolic capacity may be a manifestation of imbalanced gut microflora structure. By comparing the metabolic response values of intestinal microorganisms of different individuals to specific prebiotics, characteristic values of different subtypes are found to reflect the difference in individual flora structures.
On the basis of analyzing the metabolic characteristic value, aiming at the metabolic response value with obvious difference among subtypes, deeply sequencing the flora structure after in vitro fermentation, analyzing the flora structure of excrement, and determining the main characteristics of the intestinal flora at the early stage of diabetes by combining the difference of the metabolic response values of in vitro fermentation of prebiotics. Furthermore, the biomarkers in the early stage of diabetes are determined by combining each metabolic response value and the structural characteristics of the flora of in vitro fermentation.
According to the invention, through an in-vitro rapid detection system for the activity of intestinal microorganisms, different sub-diabetes early-stage intestinal microorganism metabolism databases are established, and the clinical functions of intestinal flora and the correlation with the early-stage diabetes onset are systematically evaluated and verified; further, an in-vitro evaluation method for improving the glucose tolerance by prebiotics is explored, and an individualized optimization intervention scheme suitable for different patients with sub-diabetes at the early stage, such as prebiotics, probiotics and the like, is established; then, the guiding effect of the prediction models of different subtypes on treatment is verified through random contrast research, and finally, a standard intervention treatment scheme is formed.
The system for detecting the metabolic activity of the intestinal microorganisms in vitro is characterized in that the intestinal microorganisms are cultured in vitro, and the metabolic activity difference of the intestinal microorganisms of different individuals is detected by detecting the metabolic response value of the intestinal microorganisms under a specific carbon source, so that the metabolic products are covered comprehensively, the degradation rate of the gas, the short-chain fatty acid and the carbon source is included, and the content of probiotics can be detected. Not only makes up the defect that the metabolic activity of intestinal microorganisms cannot be detected by 16S rRNA sequencing, but also is quicker and cheaper than metagenome sequencing, and is suitable for disease screening.
On the other hand, as the prebiotics are selected as the carbon source for in vitro culture, the problem of intervention in the early stage of diabetes is solved while detection is carried out, and the prebiotics with the highest butyric acid production or the prebiotics with the strongest growth promotion of bifidobacteria can be selected from the detected culture medium as the food for intervening special medical application. Prebiotics are currently available in the market, are simple and cheap, can be developed into special medical food, and are more easily accepted by patients with pre-diabetes. And the excrement of the patient is a non-invasive sample, so that the patient can accept the excrement more easily. Further realizes the formation of different treatment specifications of diabetes mellitus at the early stage, and is applied to the diabetes mellitus patients.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and drawings.
The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a block flow diagram of the present invention.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
According to fig. 1, the invention provides an in vitro metabolic activity detection system based on intestinal microorganisms, which is characterized by comprising the following steps:
acquiring a fecal sample, and screening the fecal sample through a fecal detector;
reserving a stool sample without organic lesions according to the screening result;
performing activity detection on the organic lesion-free excrement sample;
according to the activity detection result, the live intestinal bacteria metabolism value;
comparing the intestinal bacteria metabolism value with prestored information, and performing gene sequencing on the large difference of the comparison values;
determining the structural characteristics of the intestinal flora according to the gene sequencing result;
obtaining the biomarker of the early stage of diabetes according to the structural characteristics of the intestinal flora.
The activity detection comprises the following steps:
inoculating and culturing the fecal sample;
fermenting the inoculated and cultured excrement sample to obtain fermentation liquor;
and analyzing and detecting the fermentation liquor to obtain the activity detection information of the intestinal bacteria.
The in-vitro metabolic activity detection system based on the intestinal microorganisms provided by the invention utilizes the in-vitro metabolic activity detection system of the intestinal microorganisms to carry out in-vitro fermentation on the excrement of different subtypes and normal people, detects the metabolic response values corresponding to different multiple prebiotics, including gas production, acid production, the degradation degree of the prebiotics and the promotion effect on the probiotics, and more comprehensively reflects the whole metabolic level of the intestinal microorganisms. Both too high and too low metabolic capacity may be a manifestation of imbalanced gut microflora structure. By comparing the metabolic response values of intestinal microorganisms of different individuals to specific prebiotics, characteristic values of different subtypes are found to reflect the difference in individual flora structures.
On the basis of analyzing the metabolic characteristic value, aiming at the metabolic response value with obvious difference among subtypes, deeply sequencing the flora structure after in vitro fermentation, analyzing the flora structure of excrement, and determining the main characteristics of the intestinal flora at the early stage of diabetes by combining the difference of the metabolic response values of in vitro fermentation of prebiotics. Furthermore, the biomarkers in the early stage of diabetes are determined by combining each metabolic response value and the structural characteristics of the flora of in vitro fermentation.
According to the invention, through an in-vitro rapid detection system for the activity of intestinal microorganisms, different sub-diabetes early-stage intestinal microorganism metabolism databases are established, and the clinical functions of intestinal flora and the correlation with the early-stage diabetes onset are systematically evaluated and verified; further, an in-vitro evaluation method for improving the glucose tolerance by prebiotics is explored, and an individualized optimization intervention scheme suitable for different patients with sub-diabetes at the early stage, such as prebiotics, probiotics and the like, is established; then, the guiding effect of the prediction models of different subtypes on treatment is verified through random contrast research, and finally, a standard intervention treatment scheme is formed.
In one embodiment, the fermentation time for fermenting the inoculated fecal sample is 18-30 hours;
the activity detection information includes: the gas production value of the fermentation liquor, the short-chain fatty acid value of the fermentation liquor, the degradation rate value and the probiotic value.
The gene sequencing is 16s rRNA gene sequencing.
The pre-stored information comprises a normal value, a blood sugar value and a weight value of the activity detection information;
the blood glucose values include a normal blood glucose type and an abnormal blood glucose type, and the body weight values include: obese and non-obese.
The activity detection information also comprises the metabolic response values of the inflammatory factors and the prebiotics.
The metabolic response values include: the gas production and acid production of the prebiotics, the degradation degree of the prebiotics and the promotion effect on the probiotics.
The abnormal blood glucose types include: impaired obese fasting glucose regulation, impaired non-obese fasting glucose regulation, obese impaired glucose tolerance, and non-obese impaired glucose tolerance;
the normal blood glucose types include: obese normal and non-obese normal;
and comparing the metabolic response value with the abnormal blood sugar type and the normal blood sugar type to obtain metabolic response values corresponding to the abnormal blood sugar type and the normal blood sugar type.
The system for detecting the metabolic activity of the intestinal microorganisms in vitro is characterized in that the intestinal microorganisms are cultured in vitro, and the metabolic activity difference of the intestinal microorganisms of different individuals is detected by detecting the metabolic response value of the intestinal microorganisms under a specific carbon source, so that the metabolic products are covered comprehensively, the degradation rate of the gas, the short-chain fatty acid and the carbon source is included, and the content of probiotics can be detected. Not only makes up the defect that the metabolic activity of intestinal microorganisms cannot be detected by 16S rRNA sequencing, but also is quicker and cheaper than metagenome sequencing, and is suitable for disease screening.
On the other hand, as the prebiotics are selected as the carbon source for in vitro culture, the problem of intervention in the early stage of diabetes is solved while detection is carried out, and the prebiotics with the highest butyric acid production or the prebiotics with the strongest growth promotion of bifidobacteria can be selected from the detected culture medium as the food for intervening special medical application. Prebiotics are currently available in the market, are simple and cheap, can be developed into special medical food, and are more easily accepted by patients with pre-diabetes. And the excrement of the patient is a non-invasive sample, so that the patient can accept the excrement more easily. Further realizes the formation of different treatment specifications of diabetes mellitus at the early stage, and is applied to the diabetes mellitus patients.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (8)
1. An in vitro metabolic activity detection system based on intestinal microorganisms, characterized by comprising the following steps:
acquiring a fecal sample, and screening the fecal sample through a fecal detector;
reserving a stool sample without organic lesions according to the screening result;
performing activity detection on the organic lesion-free excrement sample;
according to the activity detection result, the live intestinal bacteria metabolism value;
comparing the intestinal bacteria metabolism value with prestored information, and performing gene sequencing on the large difference of the comparison values;
determining the structural characteristics of the intestinal flora according to the gene sequencing result;
obtaining the biomarker of the early stage of diabetes according to the structural characteristics of the intestinal flora.
2. An in vitro intestinal microorganism-based metabolic activity assay system according to claim 1, wherein said activity assay comprises the steps of:
inoculating and culturing the fecal sample;
fermenting the inoculated and cultured excrement sample to obtain fermentation liquor;
and analyzing and detecting the fermentation liquor to obtain the activity detection information of the intestinal bacteria.
3. An in vitro intestinal microorganism-based metabolic activity detection system according to claim 2,
the fermentation time for fermenting the inoculated and cultured excrement sample is 18-30 hours;
the activity detection information includes: the gas production value of the fermentation liquor, the short-chain fatty acid value of the fermentation liquor, the degradation rate value and the probiotic value.
4. The in vitro intestinal microorganism-based metabolic activity detection system according to claim 1, wherein the gene sequencing is 16s rRNA gene sequencing.
5. The system of claim 1, wherein the pre-stored information includes normal values, blood glucose values and body weight values of the activity detection information;
the blood glucose values include a normal blood glucose type and an abnormal blood glucose type, and the body weight values include: obese and non-obese.
6. The in vitro intestinal microorganism-based metabolic activity assay system of claim 2, wherein said activity assay information further comprises metabolic response values of inflammatory factors and prebiotics.
7. The system of claim 6, wherein the metabolic response value comprises: the gas production and acid production of the prebiotics, the degradation degree of the prebiotics and the promotion effect on the probiotics.
8. The system of claim 5, wherein the abnormal blood glucose type comprises: impaired obese fasting glucose regulation, impaired non-obese fasting glucose regulation, obese impaired glucose tolerance, and non-obese impaired glucose tolerance;
the normal blood glucose types include: obese normal and non-obese normal;
and comparing the metabolic response value with the abnormal blood sugar type and the normal blood sugar type to obtain metabolic response values corresponding to the abnormal blood sugar type and the normal blood sugar type.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113345586A (en) * | 2021-06-09 | 2021-09-03 | 中南大学湘雅医院 | Gestational disease evaluation system |
| CN116287335A (en) * | 2023-02-21 | 2023-06-23 | 浙江大学 | Method for evaluating intestinal microecological regulation effect of arabinoxylans and application thereof |
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| CN105296620A (en) * | 2015-10-26 | 2016-02-03 | 上海市内分泌代谢病研究所 | Intestinal metagenome feature as type-2 diabetes acarbose curative effect selection marker |
| CN108117991A (en) * | 2017-11-03 | 2018-06-05 | 杭州海路医疗科技有限公司 | A kind of in-vitro simulated cultural method of enteric microorganism |
| CN109706235A (en) * | 2019-01-29 | 2019-05-03 | 广州康昕瑞基因健康科技有限公司 | A kind of the detection and analysis method and its system of intestinal microflora |
| CN111575393A (en) * | 2020-05-25 | 2020-08-25 | 深圳市领治医学科技有限公司 | Novel method for screening microecological traditional Chinese medicine by intestinal flora polygenomics |
| US20210239696A1 (en) * | 2018-05-09 | 2021-08-05 | Carbiotix Ab | Method for measuring and improving gut health |
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| CN105296620A (en) * | 2015-10-26 | 2016-02-03 | 上海市内分泌代谢病研究所 | Intestinal metagenome feature as type-2 diabetes acarbose curative effect selection marker |
| CN108117991A (en) * | 2017-11-03 | 2018-06-05 | 杭州海路医疗科技有限公司 | A kind of in-vitro simulated cultural method of enteric microorganism |
| US20210239696A1 (en) * | 2018-05-09 | 2021-08-05 | Carbiotix Ab | Method for measuring and improving gut health |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113345586A (en) * | 2021-06-09 | 2021-09-03 | 中南大学湘雅医院 | Gestational disease evaluation system |
| CN116287335A (en) * | 2023-02-21 | 2023-06-23 | 浙江大学 | Method for evaluating intestinal microecological regulation effect of arabinoxylans and application thereof |
| CN116287335B (en) * | 2023-02-21 | 2024-01-30 | 浙江大学 | Method for evaluating intestinal microecological regulation effect of arabinoxylans and application thereof |
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