CN106434905A - Method for evaluating intestinal beneficial function of dietary polyphenols - Google Patents
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract
The invention discloses a method for evaluating an intestinal beneficial function of dietary polyphenols. A polyphenol sample is added to a nitrogen-containing culture medium which contains different carbon sources or is free from the carbon sources, and the polyphenol sample is cultivated together with fecal microorganisms in an anaerobic condition at 37 DEG C for 24h. Differences between situations before and after conducting cultivation as well as differences in pH values, floras and short-chain fatty acid compositions of culture systems added with and not added with the polyphenol sample are compared, and metabolites of the polyphenols are detected. With the application of the method provided by the invention, the intestinal beneficial function of the dietary polyphenols can be systematically and scientifically evaluated.
Description
Technical field
The invention belongs to Food Science and Engineering field is and in particular to a kind of side of the prebiotic function of evaluation meals polyphenol intestinal
Method.
Background technology
In human body intestinal canal particularly large intestine, large number of microorganism in perch, and wherein bacterial number can reach 1014Individual, it
Can form an extremely complex ecosystem in intestinal, have close contacting with body health.Enteric microorganism species
Reach more than 500 kinds, mainly by Firmicutes (Firmicutes), Bacteroidetes (Bacteroidetes), Proteobacteria
(Proteobacteria), actinomycetes door (Actinobacteria), Fusobacterium door (Fusobacteria) and wart micrococcus luteus door
(Verrucomicrobia) 6 big class composition, wherein with Firmicutes and Bacteroidetes as dominant microflora, can account for total
More than the 90% of microorganism species.The metabolism of multiple nutritional components, the change of its flora in enteric microorganism wide participation meals
Closely related with the health level of body.As the microecosystem that human body is the hugest, the most complicated, enteric microorganism itself and generation
Thank to product and can not only adjust health, serve important function served as bridge more between meals and host.More and more grind
Study carefully and also confirm that intestinal microbial population composition and its produced metabolite can change because of the change of meals composition, and then to body
Nutrition and Metabolism and health level produce material impact.Dietary frequency can adjust dysbacteriosiss, alleviates metabolic syndrome.
Meals polyphenol is the secondary metabolite being widely present in the foods such as veterinary antibiotics, corn and Folium Camelliae sinensis, due to tool
Have the chemical characteristic of multiple active hydroxyl groups so as to free radical scavenging, chelated metal ions and with specific cells protein binding
Play a significant role etc. in biological process, there are antioxidation, antibacterial, obesity, anti-inflammatory, antitumor, protection cardiovascular and cerebrovascular vessel
With functions such as nervous system.Meals polyphenol is most of or enters colon with intrinsic form, and with enteric microorganism, phase occurs
Interaction.Meals polyphenol enters colon and by microbial metabolism therein, and can pass through intestinal microflora in its metabolic process
The metabolite of change and different structure produces material impact to body.Meals polyphenol has very big shadow to microorganism in colon
Ring, there is prebiotic effect.Polyphenol runs into microorganism after entering colon, polyphenol occurs two-way phase interaction with faecal flora group
With：On the one hand, polyphenol can affect microbiologic population's composition, is likely to be of the effect of prebioticses；On the other hand, microorganism can turn
Change polyphenol and produce metabolite.Metabolism in enteric microorganism for the plant polyphenol and bioconversion in research meals, analyze its generation
Thanking product, intestinal microflora change and the mutual relation between them has become the important of clear and definite meals polyphenol mechanism of action
Direction.
Content of the invention
It is an object of the present invention to provide a kind of method evaluating the prebiotic function of meals polyphenol intestinal.
For achieving the above object, the technical scheme is that：Evaluate the prebiotic function of meals polyphenol intestinal it is characterised in that
Comprise the following steps：
(1) take 3 healthy volunteers (do not suffer from diarrhoea in three months, not using antibiotic and probiotic products, there is no Digestive
System disease) feces, after mixed in equal amounts, with half Guang ammonia of autoclaved 0.85% sodium chloride solution or the 0.5g/L of filtration sterilization
Acid solution dilutes 10 times, is stirred until homogeneous suspension, 500 revs/min of centrifugations, takes supernatant, obtains 10% feces bacteria suspension；
(2) (formula is to prepare nitrogenous culture medium：Peptone 1.6g/L, yeast extract 1.6g/L, soybean protein isolate
1.0g/L, sodium chloride 0.1g/L, potassium dihydrogen phosphate 0.08g/L, magnesium sulfate 0.01g/L, calcium chloride 0.01g/L, sodium bicarbonate
2.0g/L, chlorhematin 0.02g/L, cysteine 0.5g/L, bile salt 0.5g/L, "diazoresorcinol" 1.0mg/L, tween
802.0mL/L, vitamin K 10 μ L/L, pH hydrochloric acid is adjusted to 7.0), and prepare the (10.0g/ containing different carbon source on this basis
L, available carbon source includes but is not limited to glucose, Fructose, sucrose, Lactose, oligofructose, starch, pectin, inulin etc.)
Culture medium；
(3) step (1) is obtained 10% feces bacteria suspension according to not containing that 1/9 ratio is separately added into that step (2) prepares
In carbon source and the culture medium containing different carbon source, every kind of cultivating system prepares 6 parts, and wherein 3 parts add polyphenol sample to be assessed
(crude extract rich in polyphenol or purified polyphenol sample), in addition 3 parts are not added with polyphenol sample as comparison；
(4) cultivating system of configuration in step (3) is placed in anaerobism glove box or aerobic sealing tank or anaerobic jar etc.
In oxygen-free environment, cultivate 24 hours for 37 DEG C；
(5) respectively before starting culture and culture 24 as a child, sampling and measuring pH in the cultivating system from step (4),
Measure specific Microflora using quantitative polyase chain reaction (qPCR) or fluorescence in situ hybridization (FISH) or denatured gradient coagulates
The method detection group composition of gel electrophoresis (DGGE) or high-flux sequence, using chromatographic process (liquid chromatograph or gas chromatogram) point
Analyse composition and the content of each cultivating system Short-Chain Fatty Acids (SCFAs), using the fall of liquid chromatography-mass spectrometry polyphenol
Solution product.
Product of the present invention has advantages below：1. simple experiment, by the way of in-vitro simulated fermentation, experimental period
Short, sample analysis are convenient；2. evaluation methodology science, using several kinds of carbon source, assesses the prebiotic work(of polyphenol under the conditions of different culture media
Can, reduce carbon source to the impact measuring polyphenol activity.
Brief description
Fig. 1 is the flow chart of the prebiotic function evaluation methods of meals polyphenol intestinal
Specific embodiment
The term being used in the present invention, unless otherwise stated, typically has those of ordinary skill in the art and generally manages
The implication of solution.
With reference to specific embodiment and with reference to data, the present invention is described in further detail.It should be understood that these embodiments are only
It is in order to demonstrate the invention, rather than limit the scope of the present invention by any way.
In the examples below, the various processes not described in detail and method are conventional methods as known in the art.
Embodiment 1：
(1) take 3 healthy volunteers (do not suffer from diarrhoea in three months, not using antibiotic and probiotic products, there is no Digestive
System disease) feces, after mixed in equal amounts, with half Guang ammonia of autoclaved 0.85% sodium chloride solution or the 0.5g/L of filtration sterilization
Acid solution dilutes 10 times, is stirred until homogeneous suspension, 500 revs/min of centrifugations, takes supernatant, obtains 10% feces bacteria suspension；
(2) (formula is to prepare nitrogenous culture medium：Peptone 1.6g/L, yeast extract 1.6g/L, soybean protein isolate
1.0g/L, sodium chloride 0.1g/L, potassium dihydrogen phosphate 0.08g/L, magnesium sulfate 0.01g/L, calcium chloride 0.01g/L, sodium bicarbonate
2.0g/L, chlorhematin 0.02g/L, cysteine 0.5g/L, bile salt 0.5g/L, "diazoresorcinol" 1.0mg/L, tween
802.0mL/L, vitamin K 10 μ L/L, pH hydrochloric acid is adjusted to 7.0), and prepare on this basis glucose containing 10.0g/L,
Sucrose, oligofructose and inulin and each 6 bottles of the culture medium without any carbohydrate；
(3) step (1) is obtained 10% feces bacteria suspension and be separately added into, according to 1/9 ratio, the culture that step (2) is prepared
In base, wherein 3 parts of every kind of cultivating system add EGCG (EGCG, 5.0g/ to be assessed
L), in addition 3 parts be not added with polyphenol sample as comparison；
(4) cultivating system of configuration in step (3) is placed in anaerobism glove box, cultivates 24 hours for 37 DEG C；
(5) respectively before starting culture and culture 24 as a child, sampling and measuring pH in the cultivating system from step (4),
Measure Lactobacillus-Enterococcus spp, Bifidobacterium spp, Bacteroides- using FISH
Prevotella, Clostridium histolyticum and Eubacterium-Clostridium number change, using gas phase
The composition of SCFAs in each cultivating system of chromatography, using the catabolite of liquid chromatography-mass spectrometry polyphenol.
Embodiment 2：
(1) with the step (1) of example 1；
(2) with the step (2) of example 1, the carbon source of wherein selection is changed to glucose, Lactose, oligofructose, pectin and chrysanthemum
Sugar；
(3) with the step (3) of example 1, sample wherein to be assessed is changed to grape seed polyphenols (2.5g/L)；
(4) cultivating system of configuration in step (3) is placed in aerobic sealing tank, cultivates 24 hours for 37 DEG C；
(5) respectively before starting culture and culture 24 as a child, sampling and measuring pH in the cultivating system from step (4),
Method using high-flux sequence measures the Bacterial community of each culture systems, using in each cultivating system of gas chromatographic analysiss
The composition of SCFAs, using the catabolite of liquid chromatography-mass spectrometry polyphenol.
Embodiment 3：
(1) with the step (1) of example 1；
(2) with the step (2) of example 1, the carbon source of wherein selection is changed to sucrose, Lactose, starch, pectin and inulin；
(3) with the step (3) of example 1, sample wherein to be assessed is changed to black tea extract (10.0g/L)；
(4) cultivating system of configuration in step (3) is placed in anaerobic jar, cultivates 24 hours for 37 DEG C；
(5) respectively before starting culture and culture 24 as a child, sampling and measuring pH in the cultivating system from step (4),
Using the method for qPCR measure total bacterium in each cultivating system, Firmicutes, Actinobacteria, Bacteroidetes,
The quantity of Proteobacteria, Bifidobacterium, Lactobacillus, Eubacterium and Clostridium,
Using the composition of SCFAs in each cultivating system of liquid-phase chromatographic analysis, the degraded using liquid chromatography-mass spectrometry polyphenol is produced
Thing.
Be only the present invention in sum preferably applies example, is not used for limiting the practical range of the present invention.I.e. all according to
The equivalence changes that the content of scope of the present invention patent is done all should be the technology category of the present invention with modifying.
Claims (7)
1. a kind of evaluate the prebiotic function of meals polyphenol intestinal method, methods described comprise external Anaerobic culturel, Bacterial community and
Methanogenesis, comprise the following steps：
(1) take 3 healthy volunteers (do not suffer from diarrhoea in three months, not using antibiotic and probiotic products, there is no digestive system disease
Disease) feces, after mixed in equal amounts, with the half Guang ammonia of the 0.5g/L through 0.85% sodium chloride solution of moist heat sterilization or filtration sterilization
Acid solution dilutes 10 times, is stirred until homogeneous suspension, 500 revs/min of centrifugations, takes supernatant, obtains 10% feces bacteria suspension；
(2) prepare nitrogenous culture medium, and prepare the culture medium containing different carbon source on this basis；
(3) step (1) is obtained 10% feces bacteria suspension and be separately added into, according to 1/9 ratio, the not carbonaceous sources that step (2) is prepared
In the culture medium containing different carbon source, every kind of cultivating system prepares 6 parts, and wherein 3 parts add polyphenol sample to be assessed, in addition
3 parts are not added with polyphenol sample as comparison；
(4) cultivating system of configuration in step (3) is placed in oxygen-free environment, cultivates 24 hours for 37 DEG C；
(5) respectively start culture before and cultivate 24 hours after, sample analysis pH, flora in the cultivating system from step (4)
Composition and metabolite.
2. the method evaluating the prebiotic function of meals polyphenol intestinal as claimed in claim 1 is it is characterised in that step (2) is described nitrogenous
Culture medium prescription is：Peptone 1.6g/L, yeast extract 1.6g/L, soybean protein isolate 1.0g/L, sodium chloride 0.1g/L, phosphorus
Acid dihydride potassium 0.08g/L, magnesium sulfate 0.01g/L, calcium chloride 0.01g/L, sodium bicarbonate 2.0g/L, chlorhematin 0.02g/L,
Cysteine 0.5g/L, bile salt 0.5g/L, "diazoresorcinol" 1.0mg/L, Tween 80 2.0mL/L, vitamin K 10 μ L/L, pH
Adjusted to 7.0 with hydrochloric acid.
3. the method evaluating the prebiotic function of meals polyphenol intestinal as claimed in claim 1 is it is characterised in that contain described in step (2)
The compound method of the culture medium of different carbon source is：Add the Portugal of 10.0g/L respectively in nitrogenous culture medium described in claim 2
Grape sugar, Fructose, sucrose, Lactose, oligofructose, starch, pectin, inulin etc. obtain a series of carbonaceous sources culture medium.Once test
The middle culture medium needing using multiple different carbon source, but above carbon source does not need all to select in once testing, the choosing of carbon source
Select and be also not limited to above 8 kinds.
4. the method evaluating the prebiotic function of meals polyphenol intestinal as claimed in claim 1 is it is characterised in that the described polyphenol of step (3)
Sample can be the crude extract or purified polyphenol sample rich in polyphenol.
5. the method evaluating the prebiotic function of meals polyphenol intestinal as claimed in claim 1 is it is characterised in that the described anaerobic of step (4)
Environment can be realized using the means such as anaerobism glove box, aerobic sealing tank, anaerobic jar.
6. the method evaluating the prebiotic function of meals polyphenol intestinal as claimed in claim 1 is it is characterised in that the described flora of step (5)
Composition analysis include measuring specific Microflora using quantitative polyase chain reaction (qPCR) or fluorescence in situ hybridization (FISH)
And the method analysis group composition of denaturing gradient gel electrophoresises (DGGE) or high-flux sequence.
7. the method evaluating the prebiotic function of meals polyphenol intestinal as claimed in claim 1 is it is characterised in that the described metabolism of step (5)
Product analysis include analyzing each cultivating system Short-Chain Fatty Acids (SCFAs) using chromatographic process (liquid chromatograph or gas chromatogram)
Composition and content, using chromatograph-mass spectrometer coupling method (liquid chromatograph mass spectrography) analyze polyphenol catabolite.
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Cited By (5)
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| CN107655985A (en) * | 2017-08-25 | 2018-02-02 | 南京农业大学 | A kind of evaluation method based on proteinaceous nutrient inside LC MS MS technologies |
| CN109439722A (en) * | 2018-10-24 | 2019-03-08 | 浙江工商大学 | Measuring method of the lactic acid bacteria based on gut simulation model to enteron aisle prebiotic effect |
| CN110305781A (en) * | 2019-07-05 | 2019-10-08 | 山东大学 | Microbiologic population's co-culture device and evaluation method for the evaluation of probiotics external activity |
| CN111808910A (en) * | 2020-07-22 | 2020-10-23 | 南京农业大学 | Method for evaluating activity of dietary polysaccharide |
| CN114606289A (en) * | 2022-03-24 | 2022-06-10 | 江南大学 | Method for in-vitro dynamic digestion and probiotic evaluation of food fat component |
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| CN104107194A (en) * | 2014-04-09 | 2014-10-22 | 南昌大学 | Making method of human intestinal micro-ecological system |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107655985A (en) * | 2017-08-25 | 2018-02-02 | 南京农业大学 | A kind of evaluation method based on proteinaceous nutrient inside LC MS MS technologies |
| CN109439722A (en) * | 2018-10-24 | 2019-03-08 | 浙江工商大学 | Measuring method of the lactic acid bacteria based on gut simulation model to enteron aisle prebiotic effect |
| CN110305781A (en) * | 2019-07-05 | 2019-10-08 | 山东大学 | Microbiologic population's co-culture device and evaluation method for the evaluation of probiotics external activity |
| CN110305781B (en) * | 2019-07-05 | 2022-03-18 | 山东大学 | Microbial community co-culture device and evaluation method for evaluating in-vitro activity of microecological preparation |
| CN111808910A (en) * | 2020-07-22 | 2020-10-23 | 南京农业大学 | Method for evaluating activity of dietary polysaccharide |
| CN114606289A (en) * | 2022-03-24 | 2022-06-10 | 江南大学 | Method for in-vitro dynamic digestion and probiotic evaluation of food fat component |
| CN114606289B (en) * | 2022-03-24 | 2023-08-08 | 江南大学 | Method for in-vitro dynamic digestion and probiotics evaluation of food fat components |
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