CN113925854B - Application of chlorogenic acid in preparation of Anaerosticpes growth promoter - Google Patents

Application of chlorogenic acid in preparation of Anaerosticpes growth promoter Download PDF

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CN113925854B
CN113925854B CN202111296910.5A CN202111296910A CN113925854B CN 113925854 B CN113925854 B CN 113925854B CN 202111296910 A CN202111296910 A CN 202111296910A CN 113925854 B CN113925854 B CN 113925854B
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chlorogenic acid
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anaerosticpes
intestinal flora
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刘庆军
沈鹤霄
张帆
李国龙
刘慧敏
熊云
彭文聪
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Maintain Biomedical Wuhan Co ltd
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Abstract

The invention discloses an application of chlorogenic acid in preparing an Anaerosticpes growth promoter, which is verified for the first time that chlorogenic acid has the effect of regulating intestinal flora and promoting proliferation of probiotics vibrio butyricum by performing an intervention experiment on intestinal flora by chlorogenic acid derived from coffee bean extract, so that the chlorogenic acid can be prepared into a corresponding growth promoter for in vitro promotion of proliferation fermentation of Anaerosticpes, and the industrialized efficient preparation of the probiotics is realized; can also be made into corresponding medicines or health foods, which can be directly acted on the body to improve intestinal flora disorder or assist in treating Anaerosticides related diseases including: the preparation method has the advantages that various diseases such as fatty liver, colon cancer, constipation and depression are treated by the preparation method, and the preparation method is used for preparing corresponding auxiliary treatment medicines, functional foods or health care foods, so that the preparation method has extremely high application value.

Description

Application of chlorogenic acid in preparation of Anaerosticpes growth promoter
Technical Field
The invention belongs to the technical field of probiotic adjustment, and particularly relates to application of chlorogenic acid in preparation of an Anaerosticpes growth promoter.
Background
Chlorogenic acid (Chlorogenic acid) is depsipeptide generated by Caffeic acid (Caffeic acid) and quinic acid (quinic acid, 1-hydroxyhexahydrogallic acid), and is a phenylpropanoid compound generated by plants in the way of shikimic acid during aerobic respiration, and is derived from seeds of Rubiaceae plants such as small fruit coffee, medium fruit coffee and large fruit coffee, namely, extract of green coffee beans. Researches show that the composition has wide antibacterial and antiviral effects, oxidation resistance and aging resistance, and blood pressure reducing and cardiovascular protection effects. However, the regulation effect of intestinal probiotics such as Anaerosticpes is not reported in the related research at present.
Intestinal flora is a large number of microflora which colonize the intestinal tract of a human body, has complex structure and various bacterial types, including symbiotic bacteria, conditional pathogenic bacteria, pathogenic bacteria and the like, is not only used for digesting and absorbing food, but also has been shown to be related to various diseases by a plurality of researches. Many scientists believe that the intestinal flora can be considered as another organ of the human body and can be used as one of the targets for disease treatment by regulating the colonial structures of intestinal microorganisms or additionally supplementing probiotics to improve the disease.
A variety of intestinal probiotics associated with constipation and intestinal disease are reported in the literature "Zhuang, min et al Abundance of Probiotics and Butyrate-Production Microbiome Manages Constipation via Short-Chain Fatty Acids Production and Hormones secret. Molecular Nutrition & Food Research,2019", "Francoco Strati, et al interleaved gut microbiota in Rett syndrome. Microbiol, 2016" and "Louise Brunkwall et al. Self-reported bowel symptoms are associated with differences in overall gut microbiota composition and enrichment of Blautia in a population-based cow. Journal of Gastroenterology and hepatology 2020": anaerostics, blauthia and Faecalibacterium. Furthermore, in the document "Ren Shimeng, intestinal flora modification in patients with non-alcoholic fatty liver disease" is disclosed that intestinal flora in patients with non-alcoholic fatty liver disease (NAFLD) is abundantly modified compared to healthy persons, wherein partial bacteria such as Anaerostipes are associated with NAFLD and related inflammatory reactions. Document "Kobel et al effective junction of Inpatient Psychotherapy for Patients With and Without Migratory Background: do They Benefit Equally? Intestinal microorganisms Anaerosticpes and Blauthia associated with depression are disclosed in Frontiers in Psychiatry,2020 ".
Disclosure of Invention
The invention aims to provide the application of chlorogenic acid in preparing an Anaerosticpes growth promoter, and through an intervention experiment on intestinal flora by chlorogenic acid derived from coffee bean extract, the chlorogenic acid has the effects of regulating the intestinal flora and promoting the growth and proliferation of the Anaerosticpes, so that the chlorogenic acid can be prepared into a growth promoter or a medicine or a health food, can be used for in vitro promoting the proliferation and fermentation of probiotics or improving the disorder of the intestinal flora in vivo, and can be used for assisting in treating diseases related to the Anaerosticpes, such as: fatty liver, colon cancer, constipation, depression, etc.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides application of chlorogenic acid in preparing an Anaerosticpes growth promoter.
The invention also provides a method of promoting proliferation of Anaerosticpes, the method comprising: adding chlorogenic acid into a GAM culture medium according to the mass concentration of 5% to prepare a chlorogenic acid-GAM culture medium, and culturing probiotics by using the chlorogenic acid-GAM culture medium.
Further, the chlorogenic acid is derived from coffee bean extract.
The invention also provides application of chlorogenic acid or a composition containing chlorogenic acid in preparing medicines, functional foods and/or health-care foods for improving intestinal flora disorder.
Further, the chlorogenic acid can improve fatty liver, colon cancer, constipation and/or depression by promoting proliferation of vibrio butyricum Anaerostickers.
Further, the medicine also comprises pharmaceutically acceptable auxiliary materials.
Further, the dosage form of the medicine is granule, tablet, capsule, powder, pill or solution.
Further, the functional food or health food comprises: solid beverage, liquid beverage, biscuit, cake food, can, dairy product and oral liquid.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, through simulating the intestinal environment in vitro and taking intestinal flora collected in healthy human body excrement as a sample to carry out chlorogenic acid intervention experiments, the chlorogenic acid derived from coffee bean extract is found and verified for the first time to have the effect of regulating the intestinal flora and promoting the proliferation of probiotics vibrio anaerosis, so that the chlorogenic acid can be prepared into a corresponding growth promoter for promoting the proliferation and fermentation of probiotics in vitro, and the industrial efficient preparation of the probiotics is realized; can also be made into corresponding medicines or health foods, which can be directly acted on the body to improve intestinal flora disorder or assist in treating Anaerosticides related diseases including: the preparation method has the advantages that various diseases such as fatty liver, colon cancer, constipation and depression are treated by the preparation method, and the preparation method is used for preparing corresponding auxiliary treatment medicines, functional foods or health care foods, so that the preparation method has extremely high application value.
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FIG. 1 shows the results of abundance determinations and significance analyses of the flora in the test and control groups of example 1 of the present invention;
FIG. 2 is a graph showing the growth of Anaerosticpes in the test and control groups of example 1 of the present invention.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention.
Example 1
Considering that intestinal flora derived from a human body cannot be completely colonized in a mouse body, human body excrement is directly taken as an intestinal flora sample after being treated, the intestinal environment in the human body is simulated in vitro, chlorogenic acid extracted from coffee beans is used for intervention treatment, and the intestinal flora change after the chlorogenic acid intervention treatment is observed, wherein the specific operation process is as follows:
1. fecal sample treatment
(1) PBS buffer solution preparation
Taking 1000ml as an example, the following drugs were weighed separately by electronic analysis and poured into a 1000ml beaker, which included: KH (KH) 2 PO 4 0.24g，Na 2 HPO 4 ·12H 2 O 2.90g，NaCl 8.00g，KCl 0.20g。
800ml of ultrapure water was weighed into a beaker, stirred until completely dissolved, then the pH of the above solution was measured with a pH meter, and the pH of the solution was adjusted to 7.4.+ -. 0.05 with HCl or NaOH.
Then draining the solution into a 1000ml volumetric flask by using a glass rod, pouring the solution remained in the beaker after flushing the beaker with a small amount of ultrapure water into the volumetric flask, repeating for 3 times, finally fixing the volume to a scale mark by using the ultrapure water, and turning upside down after a cover is plugged to ensure that the solution is fully and uniformly mixed.
Pouring the solution with fixed volume into a clean yellow cover glass bottle, unscrewing the cover, placing into an autoclave, and sterilizing at 121 ℃ for 15 minutes. And after the cover is taken out, the cover is quickly screwed down, and after the temperature is reduced to the room temperature, the cover is stored in a refrigerator at 4 ℃ for standby.
(2) Preparation of intestinal flora samples
Taking 10g as an example, 10g of fecal sample was weighed by a balance, the PBS buffer described above was added to a centrifuge tube and thoroughly mixed on a shaker. The completely mixed samples were then evenly dispensed into new 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 pellet, the pellet is resuspended in the above PBS buffer and the final concentration is 5% constant to give a sample of intestinal flora.
2. Intervention experiment of chlorogenic acid in coffee Bean extract
(1) Preparation of basic culture medium
A basal medium-GAM medium for culturing intestinal flora was prepared, for example 1000 ml. 60g of the modified GAM broth drug was weighed and dissolved in ultrapure water and stirred until completely dissolved. If the flask was drained with a glass rod to a 1000ml flask, the beaker was rinsed with a small amount of ultrapure water and then poured into the flask to avoid residue, and repeated 3 times. The ultrapure water is fixed to the scale mark, and is turned upside down after the cover is plugged until the ultrapure water is fully and uniformly mixed.
Pouring the solution with constant volume into clean yellow cover glass bottle, placing 500ml culture medium in every 1000ml bottle to prevent spraying during high pressure sterilization, unscrewing the bottle cap, placing into pulse autoclave, and sterilizing at 121deg.C for 15 min. After sterilization, the bottle cap is immediately screwed up and cooled to room temperature for standby.
(2) Chlorogenic acid-GAM culture medium preparation
On a sterile bench, the above GAM medium was dispensed into glass tubes with a 1ml pipette, 2ml per tube. A black cap was placed over the finish and micro-masked. Then, chlorogenic acid solution (coffee bean extract-chlorogenic acid was purchased from Shaanxi Huidae, 50% chlorogenic acid was dissolved in PBS buffer to a mass percent concentration of 5%) was taken with a 1ml pipette, and added to the above glass tube containing GAM medium, 2ml per tube, as a test group. And a blank control group is also arranged, each group is repeated for 3 times, and the grouping is specifically as follows:
test group: 2mL of GAM medium+2 mL of chlorogenic acid solution+1 mL of PBS buffer;
control group: 2mL 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 being put into an anaerobic box, the bottle cap is unscrewed to replace O 2 Replacement was required for 12 hours.
Intestinal flora samples obtained after the treatment were inoculated into 1ml of each tube in a test group (chlorogenic acid-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 samples of the test and control groups were then centrifuged at 10000rpm for 3min, the supernatant was discarded, the pellet was treated with liquid nitrogen and sent to the wuhan ai kang biotechnology Co., ltd for detection, and the abundance of the bacterial groups such as Anaerostictus and Parasutterella in the test and control groups, i.e. the percentage of the different bacterial groups in all species detected, were measured, respectively, and the abundance measurement results are shown in Table 1.
And carrying out significance analysis on the abundance change before and after chlorogenic acid intervention, wherein the analysis result is shown in figure 1, and the x represents that P is less than 0.01, namely the abundance change has extremely significant difference.
TABLE 1 flora abundance before and after chlorogenic acid intervention
Figure BDA0003334502190000061
The results show that after chlorogenic acid dry prognosis, the abundance of the intestinal probiotics vibrio anostictus is greatly increased compared with the control group, and part of intestinal microorganisms such as: the Parasutterella genus was not significantly different from the control group. The results show that chlorogenic acid has the effect of promoting the proliferation of the Anaerosticpes, so that the Anaerosticpes can be prepared into an Anaerosticpes growth promoter, can be used for culturing a large amount of probiotics in vitro to prepare probiotics, can also be prepared into medicines or health foods, and can directly act on probiotics in vivo to improve intestinal flora disorder and assist in treating fatty liver, colon cancer, constipation, depression and other diseases.
3. Growth curve determination
The change of the growth curve of Anaerosticpes before and after chlorogenic acid treatment is observed, and the measurement method is specifically as follows:
a100. Mu.l sterile gun head was prepared and sterilized in an autoclave at 115℃for 20 min. The pipette and the sterile 96-well plate are sterilized by alcohol in advance and then put into a biosafety cabinet for ultraviolet sterilization for 30 minutes. Then transferring the sample to an anaerobic box for sampling and measuring OD 600 Values.
The Anaerosticles is cultured by chlorogenic acid as a test group, and a culture solution without chlorogenic acid is used as a control group for timing sampling and measurement. The culture medium was thoroughly shaken and sampled, and 100. Mu.l of sample was taken three times per flask, added longitudinally, and diluted laterally to three to five consecutive gradients, each flask using a sterile gun head. Similarly, all the culture solutions were measured.
The system was used, tecan i-control,2.0.10.0, and the system values were set as follows:
Figure BDA0003334502190000071
the data derived from the system is shown in table 2.
TABLE 2 Effect of chlorogenic acid on Anaerosticpes
Figure BDA0003334502190000072
Based on the above data, the OD is measured by taking the measurement point time as the X axis 600 The percentage of the values is Y, the corresponding growth curves of the test group and the control group are drawn, the measurement results are shown in figure 2, and the results show that Anaerosticpes in the test group and the control group start to grow rapidly after two hours, 8-12h start to enter the logarithmic growth phase, 12-28h are the stationary phase, 28And after h, entering into the decay period. The growth curves of the control group and the test group can be obtained, and the growth of the Anaerosticpes is more vigorous under the culture of chlorogenic acid, which indicates that the chlorogenic acid has a promoting effect on the growth of the Anaerosticpes.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.

Claims (1)

1. Chlorogenic acid preparation in vitroAnaerostipesThe application of the growth promoter is characterized in that chlorogenic acid is added into a GAM culture medium according to the mass concentration of 5% to prepare a chlorogenic acid-GAM culture medium, and probiotics are cultured by the chlorogenic acid-GAM culture mediumAnaerostipes。
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