CN116076717A - Composition for regulating intestinal microbial environment and preparation method and application thereof - Google Patents
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract
The invention discloses a composition for regulating intestinal microbial environment, a preparation method and application thereof. The composition comprises fructus Toosendan, herba et Gemma Agrimoniae, mori fructus, withania somnifera-lactone and selenium-enriched yeast. The composition of the invention provides food for intestinal probiotics by adopting specific components, promotes the growth and propagation of beneficial bacteria, simultaneously inhibits the growth and activity of conditional pathogenic bacteria and maintains the steady balance of intestinal microorganisms. The composition of the invention can obviously improve the ratio of the lactobacillus helveticus to the conditional pathogenic bacteria, promote the proliferation of the lactobacillus helveticus, further improve anxiety and cognitive problems caused by beneficial bacteria such as the lactobacillus helveticus and the like, and reduce the occurrence risk of metabolic diseases.
Description
Technical Field
The invention relates to the technical field of medical health-care foods, in particular to a composition for regulating intestinal microbial environment, a preparation method and application thereof.
Background
The large number of microorganisms exist in human intestinal tracts, and the group of microorganisms depend on the intestinal life of the human body, and simultaneously help the human body to complete various physiological and biochemical functions. Intestinal microorganisms not only play an important role in bridging between diet and host, but also regulate human health as well as metabolic products, so that the intestinal microorganisms are closely related to human health. The intestinal microorganisms reach microecological balance through dynamic physiological action with a host, and bacteria and endotoxin translocation in intestinal tracts are effectively prevented. When the normal microflora is influenced by the host and the external environment, the original balance is destroyed, the intestinal flora is dysregulated, and the variety, the quantity and the proportion of the normal intestinal flora are abnormally changed and are converted into a pathological combined state, so that the host is pathogenic.
When the immune function of the organism is reduced or the state of the flora is changed, the intestinal dominant flora is changed to cause liver pus, lung, endometritis, intra-abdominal infection and the like, and even bacteremia occurs. An increased proportion of conditional pathogenic bacteria such as Eggerthella lenta leads to a further deregulation of the intestinal microbial environment. Many beneficial bacteria in the human intestinal tract can ameliorate these problems, for example, lactobacillus helveticus and lactobacillus reuteri can relieve stress and anxiety, improve cognition; lactobacillus acidophilus has the functions of regulating intestinal microbial flora, inhibiting pathogenic bacteria, enhancing immunity, reducing blood lipid and the like; lactobacillus salivarius and Bifidobacterium adolescentis have antiinflammatory and antiaging effects. Therefore, it is very necessary to maintain the balance of intestinal beneficial bacteria and conditionally pathogenic bacteria.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a composition for regulating intestinal microbial environment, a preparation method and application thereof, so as to solve the problems.
The invention is realized in the following way:
the invention provides a composition for regulating intestinal microbial environment, which comprises the following components of szechwan chinaberry fruit, hairyvein agrimonia herb and bud, mulberry, withania somnifera-lactone and selenium-enriched yeast;
the composition comprises, by weight, 25-50 parts of szechwan chinaberry fruit, 15-35 parts of hairyvein agrimony, 5-30 parts of mulberry, 5-21 parts of withania somnifera-lactone and 1-5 parts of selenium-enriched yeast.
In some embodiments, the composition comprises, by weight, 35-45 parts of szechwan chinaberry fruit, 23-28 parts of hairyvein agrimony, 10-25 parts of mulberry, 9-17 parts of withania somnifera-lactone and 1-5 parts of selenium-enriched yeast.
In some embodiments, the intestinal microbial environment includes beneficial bacteria; the beneficial bacteria include at least one of Lactobacillus helveticus, lactobacillus acidophilus, lactobacillus reuteri, lactobacillus salivarius, bifidobacterium animalis and Bifidobacterium adolescentis;
preferably, the beneficial bacteria are lactobacillus helveticus.
In some embodiments, the intestinal microbial environment further comprises a conditionally pathogenic bacteria; the conditional pathogenic bacteria include at least one of Eggerthella lenta and Escherichia coli.
The invention also provides a preparation method of the composition, which comprises the step of uniformly mixing the components according to the proportion to obtain the composition.
The invention also provides application of the composition in preparing foods or medicines for promoting intestinal beneficial bacteria.
In some embodiments, the intestinal beneficial bacteria include at least one of lactobacillus helveticus, lactobacillus acidophilus, lactobacillus reuteri, lactobacillus salivarius, bifidobacterium animalis, and bifidobacterium adolescentis;
preferably, the beneficial bacteria are lactobacillus helveticus and lactobacillus acidophilus.
The invention also provides application of the composition in preparing food or medicine for inhibiting enteropathogenic bacteria.
In some embodiments, the enteropathogenic bacteria include at least one of Eggerthella lenta and Escherichia coli.
The invention also provides a food which comprises the composition for regulating the intestinal microbial environment.
The invention has the following beneficial effects:
the composition of the invention provides food for intestinal probiotics by adopting specific components, promotes the growth and propagation of beneficial bacteria, simultaneously inhibits the growth and activity of conditional pathogenic bacteria and maintains the steady balance of intestinal microorganisms. The composition of the invention can obviously improve the ratio of the lactobacillus helveticus to the conditional pathogenic bacteria, promote the proliferation of the lactobacillus helveticus, further improve anxiety and cognitive problems caused by beneficial bacteria such as the lactobacillus helveticus and the like, and reduce the occurrence risk of metabolic diseases.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Since imbalance of beneficial bacteria and conditional pathogenic bacteria in intestinal tracts can lead to disease generation, researches show that lactobacillus helveticus and lactobacillus reuteri can relieve stress and anxiety and improve cognition; lactobacillus acidophilus has the functions of regulating intestinal microbial flora, inhibiting pathogenic bacteria, enhancing immunity, reducing blood lipid and the like; lactobacillus salivarius and Bifidobacterium adolescentis have antiinflammatory and antiaging effects. In order to increase the proportion of beneficial bacteria including lactobacillus helveticus, the invention provides a composition for regulating the intestinal microbial environment, which comprises the following components of szechwan chinaberry fruit, hairyvein agrimonia herb and bud, mulberry, withania somnifera-lactone and selenium-enriched yeast;
the composition comprises, by weight, 25-50 parts of szechwan chinaberry fruit, 15-35 parts of hairyvein agrimony, 5-30 parts of mulberry, 5-21 parts of withania somnifera-lactone and 1-5 parts of selenium-enriched yeast.
In order to obtain better regulation effect, the inventor further optimizes the composition, and the optimized composition comprises, by weight, 35-45 parts of szechwan chinaberry fruit, 23-28 parts of hairyvein agrimony, 10-25 parts of mulberry, 9-17 parts of withania somnifera-lactone and 1-5 parts of selenium-enriched yeast.
According to the experimental results of the inventors, beneficial bacteria which can be regulated by the composition of the present invention include at least one of lactobacillus helveticus, lactobacillus acidophilus, lactobacillus reuteri, lactobacillus salivarius, bifidobacterium animalis and bifidobacterium adolescentis; more preferably, the beneficial bacterium that can be modulated is lactobacillus helveticus.
According to the experimental results of the inventors, the conditional pathogenic bacteria that the composition of the present invention can inhibit include at least one of Eggerthella lenta and Escherichia coli.
In the invention, the preparation method of the composition comprises the step of uniformly mixing the components according to the proportion to obtain the composition.
In addition, the composition of the present invention can be prepared as a food having the function of improving intestinal microbiota, in particular, the food can increase the ratio of beneficial bacteria to conditional pathogenic bacteria and inhibit the growth of conditional pathogenic bacteria.
The composition of the invention can also be prepared into a medicine which has the function of improving intestinal microbiota, in particular to the medicine which can increase the ratio of beneficial bacteria to conditional pathogenic bacteria, promote the growth of the beneficial bacteria and inhibit the growth of the conditional pathogenic bacteria.
The beneficial bacteria include at least one of Lactobacillus helveticus, lactobacillus acidophilus, lactobacillus reuteri, lactobacillus salivarius, bifidobacterium animalis and Bifidobacterium adolescentis; more preferably, the beneficial bacterium that can be modulated is lactobacillus helveticus. The conditional pathogenic bacteria include at least one of Eggerthella lenta and Escherichia coli.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The present example provides a composition for regulating the microbial environment of the intestinal tract, comprising the following components:
380g of szechwan chinaberry fruit, 280g of hairyvein agrimony, 150g of mulberry, 110g of withania somnifera-lactone and 80g of selenium-enriched yeast.
Example 2
The present example provides a composition for regulating the microbial environment of the intestinal tract, comprising the following components:
400g of szechwan chinaberry fruit, 250g of hairyvein agrimony, 100g of mulberry, 170g of withania somnifera-lactone and 80g of selenium-enriched yeast.
Example 3
The present example provides a composition for regulating the microbial environment of the intestinal tract, comprising the following components:
450g of szechwan chinaberry fruit, 230g of hairyvein agrimony, 180g of mulberry, 90g of withania somnifera-lactone and 50g of selenium-enriched yeast.
Example 4
The present example provides a composition for regulating the microbial environment of the intestinal tract, comprising the following components:
350g of szechwan chinaberry fruit, 250g of hairyvein agrimony, 250g of mulberry, 120g of withania somnifera-lactone and 30g of selenium-enriched yeast.
Comparative example 1
The comparative example provides a composition for regulating the microbial environment of intestinal tracts, which comprises the following components:
300g of szechwan chinaberry fruit, 300g of hairyvein agrimony, 200g of mulberry, 120g of withania somnifera-lactone and 80g of selenium-enriched yeast.
Comparative example 2
The comparative example provides a composition for regulating the microbial environment of intestinal tracts, which comprises the following components:
500g of szechwan chinaberry fruit, 150g of hairyvein agrimony, 150g of mulberry, 150g of withania somnifera-lactone and 50g of selenium-enriched yeast.
Comparative example 3
The comparative example provides a composition for regulating the microbial environment of intestinal tracts, which comprises the following components:
380g of chinaberry bark, 280g of hairyvein agrimony, 150g of mulberry, 110g of withania somnifera-lactone and 80g of selenium-enriched yeast.
Experimental example 1
This experimental example is an in vitro experiment performed on the compositions of examples 1-4 and comparative examples 1-3, and the specific procedure is as follows:
1. fecal sample treatment
(1) PBS buffer solution preparation
Taking 1000ml as an example, the following medicines were weighed separately by electronic analysis and poured into a 1000ml beaker, comprising: KH (KH) 2 PO 4 0.24g，Na 2 HPO 4 ·12H 2 O2.90g,NaCl 8.00g,KCl 0.20g. The PH of the above solution was measured with a calibrated PH meter and ph=7.4±0.05 was adjusted with 0.1M HCl or NaOH. Then put into an autoclave at 121 ℃ for 15 minutes. After cooling to room temperature, the solution was stored in a refrigerator at 4℃for 6 months.
(2) Fecal sample treatment
Taking 10g as an example, the final concentration is 10%.
10g of fecal sample was weighed out with a one percent balance, an appropriate amount of the above PBS buffer was added to the centrifuge tube with an automatic pipette, and thoroughly mixed on a shaker. The completely mixed samples were then split equally into new 50ml centrifuge tubes, and an appropriate amount of PBS buffer was added to each tube.
Filtering in a biosafety cabinet after uniformly mixing, and sequentially passing diluted samples through filter screens of 20 meshes, 50 meshes, 100 meshes and 200 meshes. Collecting filtrate into a centrifuge tube, placing the centrifuge tube into a centrifuge, balancing, centrifuging at 6000G and 4 ℃ for 15min, and discarding the supernatant. After weighing the precipitate, the volume was fixed with PBS at a final concentration of 5% to obtain an intestinal microorganism sample.
2. Preparation of basic culture medium
Preparing a basal medium for culturing intestinal microorganisms: GAM medium, for example 1000ml. 60g of the modified GAM broth drug was weighed out with an electronic analytical balance and poured into a 1000ml beaker. 800ml of ultrapure water was measured with a measuring cylinder, poured into a beaker, put into a stirring rotor, put on a magnetic heating stirrer, stirred until completely dissolved, and fixed to 1000ml.121 ℃,15 minutes. After sterilization, the bottle cap is immediately screwed up and cooled to room temperature.
3. Preparation of the Mixed solution
The compositions of examples 1-4 and comparative examples 1-3 were dissolved in PBS buffer to a 5% mixture by mass.
4. Grouping and intervention
On a sterile bench, the above GAM medium was dispensed into glass tubes with a 1ml pipette, 2ml per tube. Next, the mixture of the compositions of examples 1 to 4 and comparative examples 1 to 3 was dispensed by a 1ml pipette, and the mixture was placed in the above-mentioned glass tube containing GAM medium, 2ml per tube, to be used as an experimental group. Meanwhile, a comparison group is also arranged, and the grouping is specifically as follows:
experimental group: 2mL of GAM culture medium+1 mL of mixed solution+1 mL of intestinal microbial sample+2 mL of PBS buffer;
control group: 2mL GAM medium+1 mL intestinal microbial sample+3 mL PBS buffer.
After each glass tube cover is confirmed to be screwed, the glass tube covers are transferred to an anaerobic box transfer box, and 84 disinfectant is needed for disinfection before the glass tube covers are placed. After the anaerobic tank is put into the anaerobic tank, the bottle cap is unscrewed to replace oxygen, and the replacement is needed for 12 hours.
Intestinal microbial samples obtained after the above treatment were inoculated into 1ml of each of a test group (composition-GAM medium) and a blank group (GAM medium-PBS buffer), respectively.
After culturing in an anaerobic box for 72 hours, the growth condition of the flora is observed, and the flora is photographed and retained.
The test and control samples were then centrifuged at 10000rpm for 3min, the supernatant was discarded, and the pellet was treated with liquid nitrogen and sent to wuhan ai health biotechnology limited to determine the abundance of intestinal flora in the test and control groups, respectively, wherein the abundance determination results of a portion of the intestinal flora are shown in table 1. And the abundance change before and after the intervention of part of intestinal flora is subjected to significance analysis. The results are analyzed as shown in Table 1:
TABLE 1 analysis of abundance changes before and after partial intestinal microbiota intervention
Note that: * P <0.05, #P <0.01.
The analysis results in table 1 show that: the composition can achieve the aim of regulation within the range of the composition, the abundance of the Lactobacillus helveticus (Lactobacillus helveticus) and the Lactobacillus acidophilus (Lactobacillus acidophilus) is obviously increased, the abundance of the Lactobacillus reuteri, the Lactobacillus salivarius, the bifidobacterium animalis and the bifidobacterium adolescentis is also obviously increased, and the significance of the Egget-slow bacteria (Eggerthella lenta) is reduced. While comparative examples 1-2 show that other schemes beyond the scope of the present invention do not have such an effect; comparing example 1 with comparative example 3, the composition of comparative example 3 showed an increase in the abundance of lactobacillus helveticus (Lactobacillus helveticus) and lactobacillus acidophilus (Lactobacillus acidophilus), but was not as significant as in example 1; eggerthella lenta (Eggerthella lenta), escherichia coli (Escherichia coli) was also reduced, but the effect was not as remarkable as in example 1; thus, it was confirmed that the composition ingredient szechwan chinaberry fruit employed in the present invention is not replaceable in the composition.
Experimental example 2
The experimental example verifies the inhibition effect of the composition on conditional pathogenic bacteria-escherichia coli, and the specific conditions are as follows:
1. preparation of basic culture medium
Preparing a basal medium for culturing intestinal microorganisms: GAM medium, for example 1000ml. 60g of the modified GAM broth drug was weighed out with an electronic analytical balance and poured into a 1000ml beaker. 800ml of ultrapure water was measured with a measuring cylinder, poured into a beaker, put into a stirring rotor, put on a magnetic heating stirrer, stirred until completely dissolved, and fixed to 1000ml.121 ℃,15 minutes. After sterilization, the bottle cap is immediately screwed up and cooled to room temperature.
2. Grouping and intervention
(1) Culture of Egget-slow bacteria
The purchased Egget-slow bacteria (Eggerthella lenta) were first inoculated into LB plates for activation and cultured anaerobically at 37℃for 24 hours. Then transferring to an anaerobic liquid culture medium for culturing for 24 hours, wherein the culture medium is as follows: 15g/L of peptone, 5g/L of yeast powder, 5g/L of soybean powder, 5g/L of beef powder, 5g/L of glucose, 5g/L of sodium chloride, 3g/L of soluble starch, 0.5g/L of cysteine, 2.5g/L of potassium dihydrogen phosphate, 0.005g/L of hemin, 0.001g/L of vitamin K and 0.2 of pH 7.3.
(2) Identification of Egget's disease
Extracting genome DNA of the original bacteria after pure amplification culture of the original bacteria. The 16S rRNA gene PCR amplification experiments were performed with forward primer 27F (5'-AGAGTTTGATCATGGCTCAG-3') and reverse primer 1492R (5'-TACGGCTACCTTGTACGACTT-3').
The reaction procedure was as follows, 96℃for 3min, 96℃for 30s, 58℃for 30s, 72℃for 1min, 35 cycles under this condition, 72℃for 10min, and after the PCR reaction was completed, 1% agarose was identified and the desired PCR product fragment was recovered using a gel recovery kit. The kit, the primer and the sequencing service are all completed by Aikangjian company. The sequence was aligned with the 16S rRNA gene of Egger lentus (Eggerthella lenta) in the GeneBank database.
(3) Operating procedure
On a sterile bench, the above GAM medium was dispensed into glass tubes with a 1ml pipette, 2ml per tube. Then, a 1ml pipette was used to take a mixture of the composition of example 1, and the mixture was added to the above glass tube containing GAM medium, 2ml each, and as an experimental group, a condition control group and a blank group were further provided, and three groups were arranged in parallel, and the following groups were specifically defined:
experimental group: 3ml of GAM medium+1 ml of composition mixture+1 ml of Eggerthella lenta+1 ml of PBS buffer;
condition control group: 3ml of GAM medium+1 ml of Eggerthella lenta+2 ml of PBS buffer;
blank group: 3ml GAM medium+3 ml PBS buffer
After each glass tube cover is confirmed to be screwed, the glass tube covers are transferred to an anaerobic box transfer box, and 84 disinfectant is needed for disinfection before the glass tube covers are placed. After the anaerobic tank is put into the anaerobic tank, the bottle cap is unscrewed to replace oxygen, and the replacement is needed for 12 hours.
The intestinal microbial samples obtained after the treatment are respectively inoculated into an experimental group, a conditional control group and a blank control group, wherein each tube is 1ml.
After culturing in an anaerobic box for 72 hours, the growth condition of the flora is observed, and the flora is photographed and retained.
The test and control samples were then centrifuged at 10000rpm for 3min, the supernatant was discarded, the pellet was treated with liquid nitrogen and sent to wuhan ai kang biosciences, ltd, to determine the abundance of intestinal flora in the test and control samples, respectively, i.e. the percentage of different intestinal microorganisms in all species detected, wherein the abundance determination of part of the intestinal flora is shown in table 2. And the change of abundance before and after the intervention of the slow Egget bacteria is subjected to significance analysis. The analysis results are shown in table 2:
TABLE 2 analysis of changes in abundance before and after Egget's disease prevention
Comparison blank group P <0.01, comparison condition control group #p <0.01.
The analysis results in Table 2 show that the composition of the invention has an inhibitory effect on Egget strain, as evidenced by a significant decrease in abundance after intervention by the composition.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A composition for regulating intestinal microbial environment is characterized by comprising the following components of szechwan chinaberry fruit, hairyvein agrimonia herb and bud, mulberry, withania somnifera-lactone and selenium-enriched yeast;
the composition comprises, by weight, 25-50 parts of szechwan chinaberry fruit, 15-35 parts of hairyvein agrimony, 5-30 parts of mulberry, 5-21 parts of withania somnifera-lactone and 1-5 parts of selenium-enriched yeast.
2. The composition according to claim 1, wherein the components of the composition comprise, in parts by weight, 35-45 parts of szechwan chinaberry fruit, 23-28 parts of hairyvein agrimony, 10-25 parts of mulberry, 9-17 parts of withania somnifera-lactone and 1-5 parts of selenium-enriched yeast.
3. The composition of claim 1, wherein the intestinal microbial environment comprises beneficial bacteria;
the beneficial bacteria comprise at least one of Lactobacillus helveticus, lactobacillus acidophilus, lactobacillus reuteri, lactobacillus salivarius, bifidobacterium animalis and Bifidobacterium adolescentis;
preferably, the beneficial bacteria are lactobacillus helveticus.
4. The composition of claim 1, wherein the intestinal microbial environment further comprises a conditionally pathogenic bacteria;
the conditional pathogenic bacteria include at least one of Eggerthella lenta and Escherichia coli.
5. The method for preparing the composition according to any one of claims 1 to 4, wherein the composition is obtained by uniformly mixing the components according to a ratio.
6. Use of a composition according to any one of claims 1 to 4 for the preparation of a food or pharmaceutical product for promoting intestinal beneficial bacteria.
7. The use according to claim 6, wherein the intestinal beneficial bacteria comprise at least one of lactobacillus helveticus, lactobacillus acidophilus, lactobacillus reuteri, lactobacillus salivarius, bifidobacterium animalis and bifidobacterium adolescentis;
preferably, the beneficial bacteria are lactobacillus helveticus and lactobacillus acidophilus.
8. Use of a composition according to any one of claims 1 to 4 for the preparation of a food or pharmaceutical product for inhibiting enteropathogenic bacteria.
9. The use according to claim 8, wherein the enteropathogenic bacteria comprise at least one of eglinium and escherichia coli.
10. A food product comprising a composition for regulating the intestinal microbial environment according to any one of claims 1 to 4.
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