CN113925875A - Application of zinc gluconate in preparation of bacteroid regulator - Google Patents

Application of zinc gluconate in preparation of bacteroid regulator Download PDF

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CN113925875A
CN113925875A CN202111290382.2A CN202111290382A CN113925875A CN 113925875 A CN113925875 A CN 113925875A CN 202111290382 A CN202111290382 A CN 202111290382A CN 113925875 A CN113925875 A CN 113925875A
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bacteroides
zinc gluconate
treating
regulator
vitro
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刘庆军
沈鹤霄
张帆
李国龙
熊云
刘慧敏
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Maintain Biomedical Wuhan Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/30—Zinc; Compounds thereof
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D13/00—Finished or partly finished bakery products
    • A21D13/06—Products with modified nutritive value, e.g. with modified starch content
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D2/00—Treatment of flour or dough by adding materials thereto before or during baking
    • A21D2/08—Treatment of flour or dough by adding materials thereto before or during baking by adding organic substances
    • A21D2/14—Organic oxygen compounds
    • A21D2/145—Acids, anhydrides or salts thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A23C9/1522—Inorganic additives, e.g. minerals, trace elements; Chlorination or fluoridation of milk; Organic salts or complexes of metals other than natrium or kalium; Calcium enrichment of milk
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/16—Agglomerating or granulating milk powder; Making instant milk powder; Products obtained thereby
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/16—Inorganic salts, minerals or trace elements
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/191—Carboxylic acids, e.g. valproic acid having two or more hydroxy groups, e.g. gluconic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Abstract

The invention discloses an application of zinc gluconate in preparing a bacteroides regulator. According to the invention, through a zinc gluconate intervention experiment, the zinc gluconate is found and verified to have the effect of regulating the probiotic Bacteroides for the first time, and the abundance of the Bacteroides can be remarkably increased, so that the zinc gluconate can be prepared into a corresponding regulating medicament for preventing and/or treating intestinal flora disorder and assisting in treating diseases such as inflammatory bowel disease, colon cancer and autism related to Bacteroides, and has an important significance for maintaining the stability of the intestinal flora and assisting in treating related diseases caused by the flora disorder. Meanwhile, zinc gluconate is utilized to promote the Bacteroides to proliferate in a large quantity in vitro, so that the Bacteroides can be cultured in vitro in a large scale to prepare the microbial inoculum.

Description

Application of zinc gluconate in preparation of bacteroid regulator
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an application of zinc gluconate in preparation of a bacteroides regulator.
Background
The intestinal microorganisms refer to a large number of microorganisms existing in the intestinal tract of a human body, and the microorganisms depend on the intestinal life of the human body and help the human body to complete various physiological and biochemical functions. As the most huge and complex microecosystem of human body, the intestinal microorganisms not only play an important bridge role between diet and host, but also can regulate human health by themselves and metabolites. The intestinal flora is closely related to human health, and achieves microecological balance through dynamic physiological action with a host, thereby effectively preventing translocation of bacteria and endotoxin in the intestinal tract. Based on this, it is possible to help the treatment of certain diseases by changing the composition of the intestinal microbial flora, and the intestinal microbes are considered to be one of the possible therapeutic targets of various diseases at present.
Bacteroides is a common intestinal probiotic, and has been reported to be associated with intestinal diseases, such as Takahashi, Kenichiro et al (2016), Reduced Absolute of butyl-Producing Bacteria in the Fecal microbiological Community in Crohn's disease, diagnostic, 93(1),59-65, Bacteroides is reported to have the effect of modulating inflammatory bowel disease, Crohn, etc., and the research of Sarkowski Mazmania in the California institute of microbiology has found that Bacteroides fragilis (Bacteroides fragilis) in Bacteroides has an important role in improving the symptoms of autism and reducing the degree of anxiety.
Zinc gluconate is used as a zinc supplement medicine and is mainly used for treating various problems caused by zinc deficiency, such as malnutrition, growth and development retardation of children, anorexia, oral ulcer, acne and the like. However, no relevant report is found about the regulation effect of zinc gluconate on intestinal probiotic bacteroides.
Disclosure of Invention
Aiming at the blank of the prior art, the invention provides the application of zinc gluconate in the preparation of Bacteroides regulators, and the invention verifies that the zinc gluconate has the effects of regulating probiotic Bacteroides and promoting the proliferation of the Bacteroides for the first time through a zinc gluconate intervention experiment, so that the zinc gluconate can be prepared into corresponding probiotic regulation medicines for preventing and/or treating intestinal flora disorder and assisting in treating diseases such as inflammatory bowel diseases, colon cancer, autism and the like related to the Bacteroides; meanwhile, the growth promoter can be prepared to be used for promoting the proliferation of Bacteroides in vitro and realizing the large-scale culture of the Bacteroides in vitro.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides application of zinc gluconate in preparing Bacteroides regulators or regulating medicaments.
The invention provides application of a composition containing zinc gluconate in preparing Bacteroides modulators or drugs for modulation.
Further, the zinc gluconate promotes the proliferation of Bacteroides.
Further, the zinc gluconate achieves the effect of improving inflammatory bowel diseases, colon cancers and/or autism by increasing the abundance of Bacteroides.
Furthermore, the medicine also comprises auxiliary materials.
Further, the medicine is tablets, capsules, powder, pills, granules or solutions.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, by collecting the intestinal flora and simulating the in vivo environment in vitro to perform a zinc gluconate intervention experiment, the effect of regulating the probiotic Bacteroides of zinc gluconate is found and verified for the first time, and the abundance of the Bacteroides can be remarkably increased, so that the Bacteroides can be prepared into a corresponding regulating medicament for preventing and/or treating intestinal flora disorder and assisting in treating diseases such as inflammatory bowel disease, colon cancer and autism related to Bacteroides, and the method has an important significance for maintaining the stability of the intestinal flora and assisting in treating related diseases caused by the flora disorder. Meanwhile, zinc gluconate is utilized to promote the Bacteroides to proliferate in a large quantity in vitro, so that the Bacteroides can be cultured in vitro in a large scale to prepare the microbial inoculum.
Drawings
FIG. 1 is the result of measuring the abundance of Bacteroides Bacteroides in the test group and the control group in example 1 of the present invention;
FIG. 2 is a graph showing the growth of Bacteroides against zinc gluconate in example 1 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Because the intestinal flora from the human body can not be completely colonized in a mouse body, the human excrement is taken as an intestinal flora sample, an in-vivo simulation experiment is carried out in vitro, and the regulation effect on bacteroides after the intervention treatment of zinc gluconate is observed, and the specific operation process is as follows:
1. fecal sample handling
(1) Preparation of PBS buffer solution
Taking 1000ml as an example, the following drugs are weighed and recorded separately by an electronic analysis day and poured into a 1000ml beaker, wherein the drugs comprise: KH (Perkin Elmer)2PO4 0.24g，Na2HPO4·12H2O 2.90g，NaCl 8.00g，KCl 0.20g。
800ml of ultrapure water is measured by a measuring cylinder, poured into a beaker, put into a stirring rotor, put on a magnetic heating stirrer and stirred until the ultrapure water is completely dissolved, then the pH of the solution is measured by a calibrated pH meter, and the pH is adjusted to 7.4 +/-0.05 by 0.1M HCl or NaOH.
And (3) draining the completely dissolved and pH-adjusted solution into a 1000ml volumetric flask by using a glass rod, washing the beaker, pouring the beaker into the volumetric flask, fixing the volume to a scale mark, plugging a cover, and turning over for 10 times to ensure that the solution is fully mixed.
Pouring the solution with constant volume into a clean yellow glass bottle with a cover, and putting 500ml of the solution in each 1000ml bottle to prevent the solution from being sprayed out during autoclaving. The cap of the yellow glass bottle is unscrewed and then placed into an autoclave for 15 minutes at the temperature of 121 ℃. The cap is quickly screwed down after removal. After cooling to room temperature, the solution is stored in a refrigerator at 4 ℃ for 6 months.
(2) Fecal sample handling
In the case of 10g, the final concentration is 5%.
A one hundredth balance is used to weigh 10g of the fecal sample, an automatic pipette is used to take a proper amount of PBS buffer solution to add into a centrifuge tube, and the mixture is fully mixed on a shaker. And then, evenly distributing the completely mixed sample into new 50ml centrifuge tubes on average, and adding a proper amount of PBS buffer solution into each tube.
Filtering in a biological safety cabinet after uniformly mixing, and sequentially passing the diluted samples through 20-mesh, 50-mesh, 100-mesh and 200-mesh filter screens. Collecting filtrate in a centrifuge tube, placing the centrifuge tube in a centrifuge, balancing, centrifuging at 6000G and 4 deg.C for 15min, and discarding supernatant. After weighing the precipitate, the volume was determined with a PBS solution at a final concentration of 5% to obtain an intestinal flora sample.
2. Intervention test of Zinc gluconate
(1) Preparation of basic culture medium
A basic culture medium GAM for culturing intestinal flora was prepared, taking 1000ml as an example. 60g of the modified GAM broth was weighed on an electronic analytical balance and poured into a 1000ml beaker. 800ml of ultrapure water is weighed by a measuring cylinder, poured into a beaker, put into a stirring rotor and put on a magnetic heating stirrer to be stirred until the ultrapure water is completely dissolved. The above completely dissolved solution was drained to a 1000ml volumetric flask with a glass rod, and the beaker was rinsed with a small amount of ultrapure water and poured into the volumetric flask, and repeated 3 times. The liquid in the volumetric flask is fixed to the scale mark by ultrapure water, and the flask is plugged with a cover and then turned upside down for 10 times to ensure that the solution is fully mixed.
Pouring the solution with constant volume into a clean yellow glass bottle with a cover, and putting 500ml of culture medium in each 1000ml bottle to prevent the solution from being sprayed out during autoclaving. The glass bottle with the yellow cap containing the culture medium was unscrewed and placed in a pulse autoclave using the liquid program at 121 ℃ for 15 minutes. After sterilization, the bottle cap is immediately screwed down and cooled to room temperature.
(2) Preparation of zinc gluconate-GAM culture medium
On a sterile operating table, the GAM medium was dispensed into glass tubes with 2ml per tube using a 1ml pipette. The black cap was placed over the bottle mouth and masked slightly. Then, a 1ml pipette was used to take a zinc gluconate solution (purchased from Ci-Anoectosai, 10% by mass) and add it into the glass tube containing GAM medium, 2ml per tube, to prepare a test group. Meanwhile, a blank control group is also arranged, each group has 3 repetitions, and the grouping is as follows:
test groups: 2mL of GAM medium +2mL of zinc gluconate solution +1mL of PBS buffer;
control group: 2mL of GAM medium +3mL of PBS buffer;
after confirming that each glass tube cover is screwed, the glass tube cover is transferred to an anaerobic box transfer box and needs to be disinfected by 84 disinfectant before being placed. After placing in an anaerobic tank, unscrewing the bottle cap and replacing O2Replacement was carried out for 12 hours.
The intestinal flora samples obtained after the treatment are respectively inoculated into a test group (zinc gluconate-GAM culture medium) and a blank control group (GAM culture medium-PBS buffer), and each tube contains 1 ml.
After culturing for 72 hours in an anaerobic box, observing the growth condition of the flora, photographing and keeping.
And centrifuging the test group sample and the control group sample at 10000rpm for 3min, discarding the supernatant, treating the precipitate with liquid nitrogen, sending the precipitate to Wuhan Aikanjian biotechnology limited, and respectively measuring the abundance of the Bacteroides in the test group and the control group, namely the percentage of the Bacteroides in all detected strains, wherein the measurement result of the abundance of the Bacteroides is shown in Table 1.
And the change of the abundance before and after the intervention of the zinc gluconate is significantly analyzed, and the analysis result is shown in figure 1, wherein P is less than 0.01, namely the change of the abundance has extremely significant difference; p < 0.05, i.e. there was a significant difference in abundance change.
TABLE 1 variation of Bacteroides abundance for different treatment groups
Figure BDA0003334501960000051
The results show that the abundance of Bacteroides in the intestinal flora is remarkably improved compared with the control group after the zinc gluconate is used for prognosis. The results show that the zinc gluconate has the efficacy of regulating Bacteroides, can effectively prevent or treat diseases such as Inflammatory Bowel Diseases (IBD), colon cancer and autism related to the Bacteroides when acting in vivo, and can be used for large-scale multiplication culture of the Bacteroides when acting in vitro.
3. Drawing of growth curves
The change of the growth curve of Bacteroides before and after the treatment with zinc gluconate is observed, and the determination method is as follows:
a100. mu.l sterile tip was prepared and sterilized in an autoclave at 115 ℃ for 20 mins. The liquid transfer gun and the sterile 96-well plate are sterilized by alcohol in advance and then put into a biological safety cabinet for ultraviolet sterilization for 30 minutes. Then transferred into an anaerobic box for sampling and determining OD600The value is obtained.
The Bacteroides Bacteroides is cultured by adopting zinc gluconate as a test group, and a culture solution without adding the zinc gluconate is used as a control group, and the test group is subjected to timing sampling and determination. The culture solution is fully shaken up and then sampled, the amount of 100 mul of sample is taken for three times from each culture bottle, the samples are respectively and longitudinally added, three to five groups of continuous gradients are transversely diluted, and each culture bottle uses a sterile gun head. By analogy, all the culture solutions were assayed.
The system used, Tecan i-control,2.0.10.0, set the system values as follows:
Figure BDA0003334501960000061
the data obtained from the system are shown in table 2.
TABLE 2 Effect of Zinc gluconate on Bacteroides
Figure BDA0003334501960000062
Figure BDA0003334501960000071
According to the data, the measuring point time is taken as an X axis, the percentage of the measured OD value is taken as a Y value, corresponding growth curve graphs of the test group and the control group are drawn, the measuring result is shown in figure 2, the results show that Bacteroides in the test group and the control group start to grow rapidly after two hours, the growth speed is high, 6-12h is a logarithmic growth phase, and the growth phase enters a plateau phase after 12 h. The decay phase was removed after 28 h. The growth curves of the test group and the control group are compared, and the Bacteroides grows more vigorously under the culture of the zinc gluconate, which shows that the zinc gluconate has a promoting effect on the growth of the Bacteroides.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Claims (6)

1. Application of zinc gluconate in preparing Bacteroides regulator or regulating medicine is disclosed.
2. Use of a composition comprising zinc gluconate for the manufacture of a Bacteroides modulator or a modulating medicament.
3. The use according to claim 1 or 2, characterized in that said zinc gluconate promotes the proliferation of Bacteroides.
4. The use according to claim 3, wherein said zinc gluconate acts to ameliorate inflammatory bowel disease, colon cancer and/or autism by increasing the abundance of Bacteroides Bacteroides.
5. The use of claim 1 or 2, wherein the medicament further comprises an adjuvant.
6. Use according to claim 5, wherein the medicament is a tablet, capsule, powder, pill, granule or solution.
CN202111290382.2A 2021-07-27 2021-11-02 Application of zinc gluconate in preparation of bacteroid regulator Pending CN113925875A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114622026A (en) * 2022-04-02 2022-06-14 中南大学湘雅二医院 Bacteroides as objective index for predicting curative effect of autism

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
周长波: "双歧杆菌四联活菌片联合锌制剂治疗小儿腹泻的临床效果", 《实用临床医学》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114622026A (en) * 2022-04-02 2022-06-14 中南大学湘雅二医院 Bacteroides as objective index for predicting curative effect of autism

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Application publication date: 20220114