CN111440841A - Method for detecting influence of dietary fiber on activity of human intestinal bacteria - Google Patents
Method for detecting influence of dietary fiber on activity of human intestinal bacteria Download PDFInfo
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- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
Abstract
The scheme discloses a method for detecting the influence of dietary fibers on the activity of human intestinal bacteria in the field of bacterial activity detection, which comprises the following steps of detecting bacteroides: firstly, preparing a bacteroid culture medium; secondly, culturing the bacteroides till the bacteroides reaches the logarithmic growth phase and collecting the bacteroides, washing the bacteroides by using a bacteroides culture medium, and then diluting the bacteroides liquid concentration to OD by using the bacteroides culture medium600The value is 0.2-0.23, thirdly, 100 mu L diluted bacteroid bacterial liquid and 100 mu L dietary fiber solution are respectively added into each hole of a 96-hole plate, the final bacterial concentration in each hole is 0.09-0.11, then the mixture is cultured in a constant-temperature anaerobic incubator at 37 ℃ for 72 hours, and the OD of each 12 hours is recorded600A value; fourthly, recording the OD of the bacteroides according to each time point600Values, growth curves of bacteroides under the action of dietary fibers were plotted. According to the scheme, the growth state of the bacteroides in the culture medium is more attached to the growth state of the bacteroides in the human intestinal tract by optimizing the bacteroides culture medium, so that the accuracy of a detection result is improved.
Description
Technical Field
The invention belongs to the field of bacterial activity detection, and particularly relates to a method for detecting the influence of dietary fibers on the activity of human intestinal bacteria.
Background
The intestinal flora refers to microorganisms in the intestinal tract, and about 10 trillion microorganisms exist in the human intestinal tract, and the microorganisms have very important functions in aspects of regulating and controlling nutrient absorption, maintaining epithelial cell development, regulating innate immunity and the like. Firmicutes and bacteroidetes occupy the largest number.
Dietary fiber is an edible carbohydrate polymer containing three or more monomeric units and is resistant to endogenous digestive enzymes and therefore is neither hydrolyzed nor absorbed in the small intestine. Because the dietary fiber can not be digested and degraded by the human body, and the intestinal flora planted in the intestinal lumen of the human body contains rich polysaccharide hydrolase, the dietary fiber is mostly metabolized and degraded by the intestinal flora and has certain influence on the intestinal flora, thereby influencing the health of a host. Most of the healthy dietary fibers are called prebiotics, and the corresponding beneficial bacteria are acquired microorganisms which are beneficial to the health of a host after being taken for a certain amount, the most common probiotics are mainly lactobacillus and bifidobacterium, and the common prebiotics which can be selectively decomposed and utilized are fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS), inulin and the like.
In the preparation process of foods, beverages and the like, dietary fibers are often required to be added, so that the intestinal tracts of human bodies are improved through the dietary fibers, and the quality of the foods or the beverages is further improved. Currently, the activity of human intestinal bacteria is mainly detected by culturing the intestinal bacteria by using a culture medium and then adding dietary fibers into the culture medium to detect the effect of the dietary fibers on the activity of the human intestinal bacteria. The culture medium in the prior art has unreasonable components, so that the difference between the growth state of the cultured microorganisms and the growth state of the microorganisms in the intestinal tract is larger, and the error of the dietary fiber on the detection result of the activity of human intestinal bacteria is larger.
Disclosure of Invention
The invention aims to provide a method for detecting the influence of dietary fibers on the activity of human intestinal bacteria, and aims to solve the problem that the detection result is influenced by culturing microorganisms to be detected by adopting a culture medium with unreasonable components in the prior art.
The method for detecting the influence of dietary fibers on the activity of human intestinal bacteria in the scheme comprises the following steps of detecting bacteroides:
step one, preparation of a bacteroid culture medium: 100mM KH2PO4(pH 7.2)，15mM NaCl，8.5mM(NH4)2SO44mM L-cysteine, 1.9. mu.M hemin, 200. mu.M L-histidine, 100nM MgCl2，1.4nM FeSO4·7H2O，50μM CaCl21 μ g/m L vitamin K35ng/m L vitamin B12；
Step two, culturing the bacteroides till the bacteroides reaches the logarithmic growth phase and collecting the bacteroides, washing the collected bacteroides by using a bacteroides culture medium, and diluting the bacteroides liquid concentration to OD by using the bacteroides culture medium600The value is 0.2 to 0.23;
step three, respectively adding 100 mu L diluted bacteroid bacteria liquid and 100 mu L dietary fiber solution into each hole of a 96-hole plate to ensure that the final thallus concentration in each hole is OD6000.09 to 0.11; then cultured in a 37 ℃ constant temperature anaerobic incubator for 72 hours, and the OD was recorded every 12 hours600A value;
step four, recording the OD of the bacteroides according to each time point600Values, growth curves of bacteroides under the action of dietary fibers were plotted.
The beneficial effect of this scheme: according to the scheme, the growth state of the bacteroides in the culture medium is more attached to the growth state of the bacteroides in the human intestinal tract by optimizing the bacteroides culture medium, so that the accuracy of a detection result is improved.
Further, the step two is to culture the bacteroides specifically as follows: using a TSB culture medium containing 5% fetal calf serum to recover bacteroides which is frozen at-80 ℃, and culturing for 24h in an anaerobic incubator under the following conditions: 10% CO2，10％H2，80％N2. Anaerobic culture by thawing iceThe bacteroides is cultured in a culturing mode, so that other variable factors can be reduced, and the subsequent detection accuracy can be improved.
Further, the bacteroides includes at least one of bacteroides thetaiotaomicron, bacteroides ovatus, bacteroides fragilis, and bacteroides cellulolyticus. Bacteroides Thetaiotaomicron (BT), Bacteroides Ovatus (BO), Bacteroides Fragilis (BF) and Bacteroides Cellulolyticus (BC) are important Bacteroides, and at least one of the four Bacteroides is detected, so that the cognition of people on dietary fibers influencing the growth of Bacteroides is improved, and the method is applied to links such as food processing.
Further, the method for detecting the influence of the dietary fiber on the activity of the human intestinal bacteria also comprises the detection of the probiotics, and comprises the following specific steps:
step one, preparing a probiotic culture medium, namely 6.5 g/L of bactopeptone, 5.0 g/L of soybean peptone, 2.5 g/L of tryptone and 3.0 g/L2.0.0 g/L of yeast extract30.2g/L，NaCl 4.5g/L，MgSO4·7H2O0.5g/L，CaCl2·2H2O 0.45g/L，MnSO4·H2O 0.2g/L，FeSO4·7H2O 0.005g/L，ZnSO4·7H2O0.005 g/L-cysteine 0.4 g/L, hemin 0.005 g/L, vitamin K30.005g/L；
Step two, culturing the probiotics until the probiotics reach logarithmic phase and collecting the probiotics, washing the collected probiotics by using a probiotic culture medium, and then diluting the concentration of the probiotic liquid to OD (origin-destination-plus-destination) by using the probiotic culture medium600The value is 0.2 to 0.23;
step three, respectively adding 100 mu L diluted probiotic bacteria liquid and 100 mu L dietary fiber solution into each hole of a 96-hole plate to ensure that the final thallus concentration in each hole is 0.09-0.11 of OD600, then culturing for 72h in a constant-temperature anaerobic incubator at 37 ℃, and recording the OD of each 12h600A value;
step four, recording the OD of the bacteroides according to each time point600Value, plotting the growth of Bacteroides under the action of dietary fiberLong curve.
Because the probiotics belong to microorganisms beneficial to human bodies in intestinal tracts, the subsequent optimization of food and the like can be guided by detecting the influence of dietary fibers on the activity of the probiotics in the intestinal tracts.
Further, the step two of culturing probiotics comprises the following specific steps: the probiotic bacteria stored at-80 ℃ were thawed using MRS medium until their growth reached logarithmic growth phase. The MRS culture medium is used for recovering the probiotics, so that the probiotics can grow normally, other variable factors can be reduced, and the subsequent detection accuracy can be improved.
The probiotics which are important to Bifidobacterium longum (Bifidobacterium longum, B L), lactobacillus rhamnosus, (L actinobacillus rhamnosus, L GG) and lactobacillus reuteri (L actinobacillus reuteri, L R) are detected, so that the cognition of people on the influence of dietary fibers on the growth of the probiotics is improved, and the probiotics is applied to links of food processing and the like.
Further, OD of Bacteroides and/or probiotics was recorded using Micro plants Reader600The value is obtained. Recording of OD of Bacteroides and/or Probiotics Using a Microplates Reader600The method can improve the recording accuracy and reduce errors caused by manual operation.
Further, collecting bacteroides and/or probiotics in logarithmic growth phase by adopting a low-speed centrifugation mode. The bacillus and/or the probiotics can be collected by low-speed centrifugation, so that the harm to the bacillus and/or the probiotics can be reduced, and the incidental impurities are less.
Further, the low speed centrifugation is specifically 3000rpm 5 min. The bacteroides and/or probiotics are collected in a 3000 rpm-5 min mode, so that impurities are reduced, and harm to the bacteroides and/or probiotics is reduced.
Drawings
FIG. 1A is a plot of the growth of BT, BO, BF, and BC using fructooligosaccharides;
FIG. 1B is a graph of growth curves of B L, L GG and L R using fructooligosaccharides;
FIG. 2A is a plot of BT, BO, BF, and BC growth with isomaltooligosaccharides;
FIG. 2B is a graph of the growth curves of B L, L GG and L R using isomaltooligosaccharides;
FIG. 3A is a graph of BT, BO, BF, and BC growth curves using pectin;
FIG. 3B is a graph of growth curves of B L, L GG and L R using pectin;
FIG. 4A is a graph of the growth of BT, BO, BF, and BC using xylo-oligosaccharides;
FIG. 4B is a graph of the growth of B L, L GG and L R using xylo-oligosaccharides;
FIG. 5A is a graph showing the growth curves of BT, BO, BF, and BC using sweet potato fibers;
FIG. 5B is a graph showing growth curves of B L, L GG and L R using sweet potato fibers.
Detailed Description
In order that the objects and advantages of the invention will be more clearly understood, the following description is given in conjunction with the accompanying examples. It is to be understood that the following text is merely illustrative of one or more specific embodiments of the invention and does not strictly limit the scope of the invention as specifically claimed.
The dietary fiber in the examples is derived from the product fiber of Ailikang (Suzhou) Biotechnology GmbH; it comprises fructo-oligosaccharide, isomalto-oligosaccharide, pectin, xylo-oligosaccharide, sweet potato fiber, soybean fiber, corn fiber, guar gum, konjaku flour, resistant dextrin, etc.
A method for detecting the influence of dietary fiber on the activity of human intestinal bacteria comprises the following steps:
step one, preparation of a bacteroid culture medium: 100mM KH2PO4(pH 7.2)，15mM NaCl，8.5mM(NH4)2SO44mM L-cysteine, 1.9. mu.M hemin, 200. mu.M L-histidine, 100nM MgCl2，1.4nM FeSO4·7H2O，50μM CaCl21 μ g/m L vitamin K35ng/m L vitamin B12；
The method for culturing and detecting the bacteroides comprises the following steps: using a composition containing 5% fetal bovine serumThe TSB medium was thawed in an anaerobic incubator (10% CO) in four Bacteroides Thetaiotaomicron (BT), Bacteroides Ovatus (BO), Bacteroides Fragilis (BF) and Bacteroides Cellulolyticus (BC)) at-80 deg.C2，10％H2，80％N2) After 24h of culture, the cells were collected by low speed centrifugation (3000rpm 5min) when they reached the logarithmic phase. Washing the bacteroid thallus once by using the bacteroid culture medium, and diluting the bacteroid thallus concentration to OD by using the bacteroid culture medium600The value was 0.2. finally, 100. mu. L diluted bacterial solution and 100. mu. L constant concentration dietary fiber raw material solution were added to each well of a 96-well plate, respectively, so that the final bacterial cell concentration in each well was OD6000.1. Subsequently cultured in a 37 ℃ constant temperature anaerobic incubator for 72 hours, and the OD was recorded every 12 hours using a Micro platesReder600The value is obtained.
Step two, preparing a probiotic culture medium, namely 6.5 g/L of bactopeptone, 5.0 g/L of soybean peptone, 2.5 g/L of tryptone and 3.0 g/L2.0.0 g/L of yeast extract30.2g/L，NaCl 4.5g/L，MgSO4·7H2O0.5g/L，CaCl2·2H2O 0.45g/L，MnSO4·H2O 0.2g/L，FeSO4·7H2O 0.005g/L，ZnSO4·7H2O0.005 g/L-cysteine 0.4 g/L, hemin 0.005 g/L, vitamin K30.005g/L；
The method for culturing and detecting probiotics comprises thawing three probiotics (Bifidobacterium longum (B L), Lactobacillus rhamnosus (L bacteria bacillus, L GG) and Lactobacillus reuteri (L bacteria bacillus, L R)) frozen at-80 deg.C with MRS culture medium, centrifuging to collect thallus (3000rpm 5min) when its growth reaches logarithmic growth phase, cleaning thallus with probiotic culture medium, and diluting probiotic liquid concentration to OD with probiotic culture medium600Finally, 100 mu L of diluted probiotic bacteria liquid and 100 mu L of dietary fiber raw material solution with certain concentration are respectively added into each hole of a 96-hole plate, so that the final concentration of the probiotic bacteria in each hole is OD600＝0.1。Subsequently, the cells were incubated for 72h in a thermostated anaerobic incubator at 37 ℃ and the OD was recorded every 12h using a Micro plates Reader600The value is obtained.
Step three, drawing a growth curve: according to OD recorded at each time point600And (4) drawing growth curves of the bacteroides and the probiotics under the dietary fiber raw materials, and referring to the attached figures 1-5.
And (3) analyzing the growth curve result:
as shown in FIG. 1A, fructooligosaccharide significantly promoted the growth of 4 Bacteroides, with the best BT and BC effects and the highest OD600The value can reach about 0.6.
As shown in FIG. 1B, fructooligosaccharide significantly promoted the growth of Bifidobacterium longum with the highest OD600The value can reach about 0.5.
As shown in FIG. 2A, 4 species of Bacteroides could be grown in isomaltose hypgather as the sole carbon source, and the activities were relatively uniform, and the maximum OD was as high as possible600The value is around 0.5.
As shown in FIG. 2B, isomaltooligosaccharide has good bioactivity on Bifidobacterium longum, with maximum OD600Only about 0.7.
As shown in FIG. 3A, pectin as a sole carbon source also promoted the growth of 4 Bacteroides to some extent, with a maximum OD600The value is between 0.3 and 0.4.
As shown in FIG. 3B, Bifidobacterium longum can also grow using pectin as the sole carbon source with the maximum OD600The value is about 0.5.
As can be seen from FIG. 4A, xylo-oligosaccharide has weak activity on Bacteroides sp, BT, BC and B L can slowly utilize xylo-oligosaccharide for propagation, and the highest OD is600The value is 0.3-0.4.
As shown in FIG. 4B, Bifidobacterium longum can also grow slowly using xylooligosaccharide with maximum OD600The value is about 0.3.
As can be seen from FIG. 5A, the sweet potato fibers had better activity against 4 Bacteroides, the maximum OD600The value is about 0.6.
As shown in FIG. 5B, the sweet potato fibers have significant bioactivity and maximum OD for Bifidobacterium longum600Value exceeding 0.7, L GG in cultureIt can also be used for reproduction in later stage, with maximum OD600The value was 0.6, whereas L R failed to reproduce using sweet potato fiber.
In summary, it can be seen from the examples that dietary fiber components in Fiberwill, including fructo-oligosaccharide, isomalto-oligosaccharide, pectin, xylo-oligosaccharide and sweet potato fiber, can promote the growth of BT, BO, BF, BC and B L to some extent, and these results provide a favorable evidence support for the beneficial effects of Fiberwill on intestinal health.
The present invention is not limited to the above embodiments, and those skilled in the art can make various equivalent changes and substitutions without departing from the principle of the present invention after learning the content of the present invention, and these equivalent changes and substitutions should be considered as belonging to the protection scope of the present invention.
Claims (9)
1. A method for detecting the influence of dietary fiber on the activity of human intestinal bacteria is characterized by comprising the following steps: comprises the detection of bacteroides, and comprises the following specific steps:
step one, preparation of a bacteroid culture medium: 100mM KH2PO4(pH 7.2)，15mM NaCl，8.5mM (NH4)2SO44mM L-cysteine, 1.9. mu.M hemin, 200. mu.M L-histidine, 100nM MgCl2，1.4nM FeSO4·7H2O，50μM CaCl21 μ g/m L vitamin K35ng/m L vitamin B12；
Step two, culturing the bacteroides till the bacteroides reaches the logarithmic growth phase and collecting the bacteroides, washing the collected bacteroides by using a bacteroides culture medium, and diluting the bacteroides liquid concentration to OD by using the bacteroides culture medium600The value is 0.2 to 0.23;
step three, respectively adding 100 mu L diluted bacteroid bacteria liquid and 100 mu L dietary fiber solution into each hole of a 96-hole plate to ensure that the final thallus concentration in each hole is 0.09-0.11, then culturing for 72h in a constant-temperature anaerobic incubator at 37 ℃, and recording the OD of each 12h600A value;
step four, recording the OD of the bacteroides according to each time point600Values, growth curves of bacteroides under the action of dietary fibers were plotted.
2. The method for detecting the influence of dietary fiber on the activity of human intestinal bacteria, according to claim 1, wherein the method comprises the following steps: the step two is to culture bacteroides, which comprises the following specific steps: using a TSB culture medium containing 5% fetal calf serum to recover bacteroides which is frozen at-80 ℃, and culturing for 24h in an anaerobic incubator under the following conditions: 10% CO2，10％H2，80％N2。
3. The method for detecting the influence of dietary fiber on the activity of human intestinal bacteria, according to claim 2, is characterized in that: the Bacteroides includes at least one of Bacteroides thetaiotaomicron, Bacteroides ovorans, Bacteroides fragilis and Bacteroides cellulolyticus.
4. The method for detecting the influence of dietary fiber on the activity of human intestinal bacteria according to any one of claims 1 to 3, wherein the method comprises the following steps: the method also comprises the detection of the probiotics, and comprises the following specific steps:
step one, preparing a probiotic culture medium, namely 6.5 g/L of bactopeptone, 5.0 g/L of soybean peptone, 2.5 g/L of tryptone and 3.0 g/L2.0.0 g/L of yeast extract30.2g/L，NaCl 4.5g/L，MgSO4·7H2O 0.5g/L，CaCl2·2H2O 0.45g/L，MnSO4·H2O 0.2g/L，FeSO4·7H2O 0.005g/L，ZnSO4·7H2O0.005 g/L-cysteine 0.4 g/L, hemin 0.005 g/L, vitamin K30.005g/L；
Step two, culturing the probiotics until the probiotics reach logarithmic phase and collecting the probiotics, washing the collected probiotics by using a probiotic culture medium, and then diluting the concentration of the probiotic liquid to OD (origin-destination-plus-destination) by using the probiotic culture medium600The value is 0.2 to 0.23;
step three, adding 100 mu L diluted probiotic bacteria liquid and 100 mu L dietary fiber into each 96-well plateMaintaining the solution so that the final concentration of the thallus in each hole is 0.09-0.11 at OD 600; then cultured in a 37 ℃ constant temperature anaerobic incubator for 72 hours, and the OD was recorded every 12 hours600A value;
step four, recording the OD of the bacteroides according to each time point600Values, growth curves of bacteroides under the action of dietary fibers were plotted.
5. The method for detecting the influence of dietary fiber on the activity of human intestinal bacteria, according to claim 4, wherein the method comprises the following steps: the step two is to culture probiotics by the specific steps of: the probiotic bacteria stored at-80 ℃ were thawed using MRS medium until their growth reached logarithmic growth phase.
6. The method for detecting the influence of dietary fiber on the activity of human intestinal bacteria, according to claim 5, wherein the method comprises the following steps: the probiotic bacteria comprise at least one of bifidobacterium longum, lactobacillus rhamnosus and lactobacillus reuteri.
7. The method for detecting the influence of dietary fiber on the activity of human intestinal bacteria, according to claim 6, wherein the method comprises the following steps: recording of OD of Bacteroides and/or Probiotics Using Micro plants Reader600The value is obtained.
8. The method for detecting the influence of dietary fiber on the activity of human intestinal bacteria, according to claim 7, wherein the method comprises the following steps: collecting bacteroides and/or probiotics in logarithmic growth phase by adopting a low-speed centrifugation mode.
9. The method for detecting the influence of dietary fiber on the activity of human intestinal bacteria, according to claim 8, wherein the method comprises the following steps: the low speed centrifugation is specifically 3000rpm 5 min.
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