CN111053789A

CN111053789A - Methods and compositions for modulating the immune system

Info

Publication number: CN111053789A
Application number: CN201911176703.9A
Authority: CN
Inventors: 肖宏; 陈禧莹; 杨涛; 陈筠
Original assignee: Hunan Nutrition Tree Biotechnology Co ltd
Current assignee: Hunan Nutrition Tree Biotechnology Co ltd
Priority date: 2019-11-26
Filing date: 2019-11-26
Publication date: 2020-04-24

Abstract

The invention discloses a composition for regulating an immune system, which is prepared by the following steps of S1: culturing a microorganism in a culture medium; wherein the microorganism is Lactobacillus, Bacillus bifidus, Streptococcus, Bacillus, enterococcus, and Klebsiella; s2: exposing the microorganism to a biological, chemical or physical stress causing the microorganism to release a stress product into the culture medium; s3: removing microorganisms from the culture medium and isolating the stress release product; s4: filtering and separating the stress release product by using a filter, and removing the stress release product with the molecular weight of more than 10KDa after separation; a stress release product of less than 10kDa is obtained. A method of modulating the immune system comprises administering to an animal an effective amount of a 0.5-10 kDa stress release product.

Description

Methods and compositions for modulating the immune system

Technical Field

The present invention relates to methods and compositions for modulating an immune response in an animal or human; more particularly, the present invention relates to the modulation of immune responses in animals or humans by administration of Stress Response Factors (SRFs) produced by lactic acid bacteria to activate and regulate circulating macrophages.

Background

Researches show that the lactobacillus can cause stress reaction to release polymer nucleic acid, protein and peptide polysaccharide and hydrolysate thereof when the external environment changes; the stress products can improve the activity and survival rate of the microorganisms under the condition of environmental change; therefore, the stress products are added into the storage environment of the lactic acid bacteria, so that the commercial application value of improving the survival rate is realized, and the stress products can improve the immune system of the host of the lactic acid bacteria when being released from the places where the lactic acid bacteria exist, such as the mouth, the nose, the oral cavity, the intestinal tract, even the vagina, and the like of animals or human beings; induced stress product release is common to commensal bacteria, such as overcrowding, nutrient deprivation, and exposure to antibiotics; researches find that when the molecular weight of the stress oligomer is less than 10kDa, the stress oligomer is easy to absorb particularly between 500 and 3000Da, has no toxicity and has the activity of regulating an immune system; macrophages can be activated to release cytokines, the levels of which are believed to help fight infection and down regulate the levels of cytotoxins produced by over-activation of the immune system following infection, preventing vascular perforation.

When microorganisms attempt to invade sterile tissues (e.g., from the outer ear to the inner ear, nose to sinus, vagina to uterus), nutritional deficiencies are encountered which release stress oligomers to alert the host to penetration of potential pathogenic bacteria into sterile areas or tissues; as sentinel cells, macrophages circulate in the blood and lymph and reside in specific endothelial cells, tissues and organs; they are the first line of defense of the host, releasing interleukin signals and destroying microorganisms and diseased cells present in the host. Twenty different interleukins can be released, modified, amplified, confined and suppressed by recipient cells when the immune system is stimulated. Therefore, macrophage signaling is very critical for initiating and carrying out an appropriate immune response.

During infection, bacterial endotoxins (lipopolysaccharide, LPS), which bind to macrophage CD-14 surface receptors, upregulate and induce the release of higher levels of IL-1, IL-6 and TNF; these signs can induce "septic shock" such as fever, fatigue, cardiovascular hypotension, renal failure, etc. There are over a dozen different species of lactic acid bacteria associated with animals that are found to release stress response factors in stress; however, the distribution and oligomer or monomer content of the sfps are different in different test species. If the polymer with the molecular weight of more than 10kDa is injected into a mouse body, toxic reactions such as diarrhea and the like can be generated; and the test of the oligomers with the molecular weight of less than 0.5kDa by human peripheral blood macrophages does not induce the release of external interleukins; however, the fraction of the intermediate oligomers with molecular weights between 0.5 and 10kDa, in addition to being non-toxic, can activate and regulate macrophages, thereby protecting experimental mice from being harmed after the injection of a lethal dose of endotoxin; thus, not all bacterial strains, the oligomers released are effective in protecting animals from bacteria.

Therefore, the research suggests that the SRFs, the oligomeric stress response factors of which the molecular weight is between 0.5 and 10kDa, are natural immunomodulators and can be safely used for protecting animals and human beings from infection and over-stimulating the immune systems of the animals and the human beings. In addition, stress response factors contain compounds that can be used to modulate the expression of surface receptors on macrophages, re-tuning the dysfunctional immune system.

Immune activation and release of regulatory factors can be used to broadly improve the immune response of humans or animals by dietary or pharmaceutical preparations; fermented products (e.g. milk, cheese, yoghurt) contain live bacteria, which release stress response factors SRFs when transferred to a nutritional deficient mouth environment. If formulated in a form that extends residence time in the mouth (e.g., a buccal tablet), more SRFs are released to activate and modulate the local immune response.

Technical detection and analysis show that the oligomeric SRFs exist and can be effectively activated and improve the immune system efficiency of animals when lactic acid bacteria are transferred to saliva or the growth of probiotics with unfavorable nutrient substances in the environment; through this discovery, there are biologically effective requirements for developing a probiotic formulation that is sterile, stable, does not require refrigeration, and provides a known dosage.

Disclosure of Invention

The object of the present invention is to provide oral medication, food, topical preparations and even injectable drugs for human health to help prevent infections. It is another object of the present invention to provide local immunostimulation in food products for oral or parenteral use, using SRFs as starting materials and protective agents against other bacteria.

In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:

a composition for regulating immune system is prepared by the following steps,

s1: culturing a microorganism in a culture medium; wherein the microorganism is Lactobacillus, Bacillus bifidus, Streptococcus, Bacillus, enterococcus, and Klebsiella;

s2: exposing the microorganism to a biological, chemical or physical stress causing the microorganism to release a stress product into the culture medium;

s3: removing microorganisms from the culture medium and isolating the stress release product;

s4: filtering and separating the stress release product by using a filter, and removing the stress release product with the molecular weight of more than 10KDa after separation; a stress release product of less than 10kDa is obtained.

Further, the stress applying step in S2 may be one or more of the following methods,

a. separating the microorganisms from the culture medium by centrifugation, and reintroducing the microorganisms into the non-nutritive isotonic solution;

b. adding antibiotics to prepare sensitive microorganisms;

c. adding additional microorganisms to the culture medium;

d. reducing the volume of the culture medium;

e. removing nutrients from the culture medium;

f. the pH of the medium was changed.

Further defined, the non-nutritive isotonic solution includes 0.9% sodium chloride.

Further defined, the non-nutritive isotonic solution is 0.1M phosphate buffer at ph 7.5.

Further defined, the concentration of the microbial cells of said microorganism is 3 x 10⁹～5*10⁹CFU per ml.

Further limited, the microorganism is a field microorganism.

Further defined, the pH in the medium is 7.5.

Further defined, the culture temperature in the medium is 40 ± 1 ℃.

A method for modulating the immune system, comprising administering to an animal an effective amount of a 0.5-10 kDa stress release product; modulating an immune response in an animal includes:

a. stimulating an immune response by activating macrophages, releasing immunostimulatory interleukins IL-1, IL-6 and TNF, e.g. to prevent or fight infection;

b. down-regulation of macrophage CD-14 receptor prevents endotoxin overstimulation leading to overproduction of IL-1, IL-6 and TNF and systemic related inflammation, cardiovascular dysfunction, shock and death;

c. downregulation of the macrophage's CD-16 receptor prevents IL-10 overstimulation from resulting in the conversion of the terminating macrophage to a cytotoxic phenotype and has the potential to excessively destroy host cells, such as endothelial cells and T cells lining the lining the lining blood vessels.

The invention has the beneficial effects that:

the present invention teaches the selection of microorganisms and the conditions used to bring them to generate the maximum of oligomeric SRFs (optimal between 0.5 and 3 kDa); the present invention teaches two natural and improved conditions: the fresh milk contains about 10 per ml of fresh milk which is instantly sterilized at high temperature⁴The microorganism of CFU, fermented milk product (milk, yogurt, cheese) contains about 10 per ml⁶～10⁸CFU microorganisms harmless to human flora; transferring these microorganisms to a less nutritious oral environment by feeding, releases certain amounts of SRFs, which, if increased residence time in the mouth can be achieved by gelling or thickening agents, can increase the level of immune stimulation; it is thought that this explains the benefits of regular consumption of fermented foods for immune stimulation, fresh vegetables containing high levels of harmless bacteria, stimulate oral local macrophages by releasing SRFs during feeding.

Detailed Description

In order that those skilled in the art can better understand the present invention, the following embodiments are provided to further illustrate the present invention.

Under normal and naturally occurring stress, lactic acid bacteria can release products known as Stress Response Factors (SRFs). The product with the molecular weight less than 10kDa is nontoxic, and the 0.5-10 kDa oligomer can activate and regulate macrophage.

The compositions of the invention may be used to stimulate the immune system orally or parenterally or locally, to elicit an immune response that prevents or reduces infection by activating macrophages to release cytokines, particularly IL-1, IL-6 and TNF; over-stimulation of local inflammatory responses, such as rheumatoid arthritis and other autoimmune diseases, by counteracting the potentially pathological effects of macrophages; or systemic inflammatory responses such as septic shock.

A composition for regulating immune system is prepared by the following steps,

s2: exposing the microorganism to a biological, chemical or physical stress that causes the microorganism to release a stress product into the culture medium;

s3: removing the microorganisms from the culture medium and isolating the stress release product;

s4: filtering and separating the stress release product by using a filter, and removing the stress release product with the molecular weight of more than 10KDa after separation; obtaining a stress release product of 0.5-10 kDa.

Preferably, when the microorganism is propagated to a certain thallus concentration, stress response factors SRFs are prepared by one or more of the following methods, a, the microorganism is separated from the culture medium by centrifugation, and then the microorganism is placed into the non-nutritive isotonic solution again; b. adding antibiotics to prepare sensitive microorganisms; c. adding additional microorganisms to the culture medium; d. reducing the volume of the culture medium; e. removing nutrients from the culture medium; f. changing the pH value of the culture medium; to influence the bioavailability of the nutrient composition in the culture medium by the microorganisms.

The best method for applying stress stimulation to the microorganisms is as follows: in mid-log phase of growth (about 10 per ml)⁹CFU) the microorganisms were removed from the medium and resuspended in 5-fold concentration of non-nutritive phosphate buffer (pH7.5) at 40 ℃ for 12 hours, and the concentration of stress response factors SRFs was monitored using UV254 nm; SRFs containing molecular weights greater than 10kDa are toxic, so all species greater than 10kDa are removed by filtration means. The supernatant may therefore be filtered so as to delete all the SRFs above 10kDa and to retain in solution the fraction with a molecular weight below 10 kDa. In addition, we can obtain more SRFs by continuous stress stimulation.

The amount of SRFs released depends on: (1) the concentration of the lactobacillus; the optimum level is 3 x 10⁹～5*10⁹CFU per ml; at higher concentrations, fewer SRFs are released per cell; (2) the severity of the stress on the cells, the greater the stress, the more SRFs are released; (3) under the same conditions, strains screened from the field released more SRF than laboratory strains; (4) pH in the medium; at a pH below 4.8, the release is about one-fourth of that at pH 7.5; (5) temperature in the culture medium; release can be observed at 40 ℃, with an optimal temperature of about 40 ℃; (6) time; immediate release following the stress stimulus, plateau is reached in about 9 to 12 hours unless the supernatant is continuously removed.

The following examples are provided to illustrate, but not to limit, the invention.

Example 1:

by measuring the release of compounds with A254 absorbance values, we thus approximately observed the release of SRFs, 2X 10¹⁰CFU was incubated at 37 deg.C under 0.1M phosphate buffer (pH7.5) for 10 hours, and then the mean absorbance from three experiments was determined as. + -. 50% per ml in units (AU),

example 2:

caseii propagated in MRS medium, as described in example 1, producing fractions of the stress response factors SRFs of less than 10kDa, where A₂₅₄9.250 or 9250AU/ml after the first inoculation culture; the release of SRFs every 16 hours for the second and third serial cultures yielded 12000AU/ml and 3250AU/ml, respectively.

Example 3: caseii-derived < 10kDaSRFs were prepared as described in example 2 and tested for their ability to activate macrophages, release interleukins and downregulate CD-14 and CD-16 surface receptors by selective macrophage depletion.

Peripheral blood to which an anticoagulant has been added is collected from a human body of a volunteer, and leukocytes are separated by centrifugation. Collection of buffy coat (buffycoat) containing leukocytes for transfer to wells of a microtiter plate at a concentration of 10 per well⁵(ii) a Adding RPMI1640 culture medium, culturing at 37 deg.C for 4 hr in carbon dioxide-rich environment, adsorbing macrophages in the titer plate to separate, and obtaining macrophage concentration of 1-3 x 10⁶And/ml. Phosphate Buffered Saline (PBS) at pH7.5 or phosphate solution containing SRFs at pH7.5 at 0.1M (pH7.5) was added in portions corresponding to one tenth of the volume of the medium in RPM, and incubated together under the above-mentioned culture conditions. During the incubation period, an aliquot was taken and the activated macrophages were counted in a hemocytometer.

Interleukins IL-1 α -6 α and TNF α, measured using a commercial cytokine kit (R & DSystems, Minneapolis, Minn.) the level of surface receptors was measured by addition of fluorescent agent to measure fluorescence of macrophages CD-14 and CD-16 specific monoclonal antibodies in a FACScan flow cytometer and the data was analyzed by the Lysis-1 program.

Percentage of macrophages with highly expressed CD-14 and CD-16。

Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. A composition for modulating the immune system, comprising: the preparation method comprises the following steps of,

2. The immune system modulating composition of claim 1, wherein: the stress applying step in S2 may be one or a combination of more of the following ways,

b. adding antibiotics to prepare sensitive microorganisms;

c. adding additional microorganisms to the culture medium;

d. reducing the volume of the culture medium;

e. removing nutrients from the culture medium;

f. the pH of the medium was changed.

3. The immune system modulating composition of claim 2, wherein: the non-nutritive isotonic solution comprises 0.9% sodium chloride.

4. The immune system modulating composition of claim 2, wherein: the non-nutritive isotonic solution is 0.1M phosphate buffer at pH 7.5.

5. The immune system modulating composition of claim 1, wherein: the concentration of the microorganism is 3 x 10⁹～5x10⁹CFU per ml.

6. The immune system modulating composition of claim 1, wherein: the microorganism is field microorganism.

7. The immune system modulating composition of claim 1, wherein: the pH in the medium was 7.5.

8. The immune system modulating composition of claim 1, wherein: the culture temperature in the medium is 40 +/-1 ℃.

9. A method of modulating the immune system, comprising: applying an effective dose of a stress release product of less than 10KDa to an animal; modulating an immune response in an animal includes:

a. stimulating an immune response by activating macrophages, releasing immunostimulatory interleukins IL-1, IL-6 and TNF;

c. downregulation of the macrophage's CD-16 receptor prevents IL-10 overstimulation from resulting in the conversion of terminal macrophages to a cytotoxic phenotype, and has the potential to excessively destroy host cells.