CN108524925B

CN108524925B - Inactivated yeast injection

Info

Publication number: CN108524925B
Application number: CN201810359624.0A
Authority: CN
Inventors: 张勇; 石有斐
Original assignee: Shandong Agricultural University; Weifang Huaying Biotechnology Co Ltd
Current assignee: Shandong Agricultural University; Weifang Huaying Biotechnology Co Ltd
Priority date: 2018-04-03
Filing date: 2018-04-20
Publication date: 2020-12-01
Anticipated expiration: 2038-04-20
Also published as: CN108524925A

Abstract

The invention relates to an injection for enhancing immune function, resisting bacteria and viruses, belonging to the field of biological medicines. The main component of the medicine is inactivated yeast, and the medicine is prepared by inactivating live yeast. The injection administration of the inactivated yeast can enhance the immune function of the organism, has the functions of antibiosis and antivirus, can be used for preventing, treating or assisting in treating various bacterial diseases and viral diseases of human beings or animals, and various diseases such as low immune function of the organism caused by various reasons, and has wide application prospect.

Description

Inactivated yeast injection

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to an injection for enhancing immune function, resisting bacteria and viruses.

Background

At present, researches show that the yeast has good probiotic characteristics of regulating intestinal balance, promoting feed conversion, improving the immune function of organisms and the like, and is mostly used as a feed additive for livestock and poultry breeding. Yeast has been studied as a feed additive for enhancing the immune function of swine (patent publication No. CN 106387398A). In addition, yeast extracts or their active ingredients (such as zymosan, yeast manno-oligosaccharide, yeast beta-glucan) are used in many studies to improve the immune function of the body (reference [1 ]: Molist F, Eerden E V, parameier H K, Vuorenmaa J. influence of hydrolyzed yeast extracts on the immune function and growth performance of weaned piglets. feed exposition, 2014, 12: 32. reference [2 ]: Tyroc, Homophor, Liuyongqiang, Songzhe, Van Yong Shi, Happy. influence of yeast manno-oligosaccharide on the production performance and immune function of swine and chicken. 2017, 5: 24-26. reference [3 ]: cinnanmin, Zhang Haibo, Xiyanghong, Zhang Yangzhi, Zhang Xiao. In addition, an injection of sulfated or carboxymethyl yeast glucan obtained by chemically modifying yeast glucan for enhancing the immune function of animals has been proposed (patent publication Nos. CN102028704A and CN 102048686A).

In the past, we found that inactivated lactic acid bacteria have excellent immunopotentiating, antibacterial and antiviral effects when administered intravenously to animals such as mice, cattle, pigs and dogs, and reported multiple international and national inventions (patent application numbers: PCT/CN2016/09844, 201610809584.6, 201610811028.2, 201610810757.6, 201610810758.0, 201610809345.0, 201610809582.7, 201610809583.1, 201610809676.4, 201610811285.6 and 201610811770.3). Based on the above researches, we speculate that the injection prepared by inactivating the saccharomycetes has an immune enhancement effect on animal injection, and the examination and the discovery of documents and patent databases show that the direct inactivation of the saccharomycetes as the injection is not reported at present, and the research and the discovery that the inactivation of the saccharomycetes as the injection has good immune enhancement, antibacterial and antiviral effects on animal injection. The medicine has the advantages of low cost, no medicine residue and the like for animal medication, and particularly has wide application prospect for the prevention and control of epidemic diseases of livestock.

Disclosure of Invention

The injection has the main component of inactivated saccharomycete, and the inactivated saccharomycete is gram stained and observed under oil microscope to maintain the shape of the inactivated saccharomycete as the contour and shape of the live bacteria before inactivation.

The injection contains 10 inactivated yeast per ml⁵—10¹²And (4) respectively.

The yeast is selected from Saccharomyces, Torulopsis, Candida, Willemm, Pichia, Saccharomyces, Torulopsis, Schleromyces, Rhodotorula, Schizosaccharomyces pombe, and Babylonia.

Further, the inactivation method of the yeast is any one selected from high temperature inactivation, high temperature and high pressure inactivation, ultraviolet inactivation, chemical agent inactivation and radiation inactivation.

Still further, the injection also comprises a pharmaceutically acceptable adjuvant, wherein the adjuvant contains enough salt or monosaccharide to ensure that the injection suspension is the same as or similar to the osmotic pressure of blood.

The preparation formulation for preparing the inactivated yeast injection used in the present invention comprises: powder injection, suspension injection, etc. The powder injection is prepared by spray drying or freeze drying, and is prepared into suspension when in use.

The preparation is an injection.

Preferably, the inactivated yeast is a single bacterium, gram staining is carried out on the inactivated yeast, the inactivated yeast keeps the complete thallus shape under the observation of a microscope, the outline and the shape of the inactivated yeast are consistent with those of live bacteria before inactivation, and the administration mode of the medicine is injection administration.

Further, the inactivated yeast is a mixture of more than two kinds of inactivated yeast, gram staining is carried out on the inactivated yeast, observation is carried out under a microscope, the inactivated yeast mainly keeps a complete thallus form, and the drug administration mode is injection drug administration. The inactivated yeast is obtained by any one inactivation method of high-temperature high-pressure inactivation, ultraviolet inactivation, chemical reagent inactivation or radiation inactivation. The yeast is selected from Saccharomyces, Torulopsis, Candida, Willemm, Pichia, Saccharomyces, Torulopsis, Schleromyces, Rhodotorula, Schizosaccharomyces pombe, and Babylonia.

The adjuvant of the injection is selected from octylphenoxy polyethoxyethanol, tyloxapol, sorbitan polyethylene glycol monooleate, polyoxyethylene monostearate, polyoxyethylene derivative, tween-80, sodium polyalkylbenzenesulfonate, sodium lauryl sulfate, polysaccharide sulfate alkali metal salt, dextran sulfate sodium, dioctyl sulfosuccinate, acacia gum, arabic latex, polyvinylpyrrolidone, ethyl polysilicate, ethanol, glycerol, sorbitol, honey, agar, starch, dextrose, fructose, malt extract, cocoa powder, tartaric acid, citric acid, sodium citrate, carrageenan, alginic acid, sodium alginate, tannic acid, cyclamic acid, mineral oil, eucarlin, saccharin sodium, ghatti gum, karaya gum, tragacanth gum, pectin, carageenan, gelatin, carboxymethyl cellulose, cellulose sulfate, methyl cellulose, sodium sulfonate, sodium alginate, sodium cyclamate, sodium oleate, sodium saccharin, ghatti gum, karaya gum, tragacanth gum, pectin, carrageenan, sodium gluconate, Sodium carboxymethylcellulose, sodium cellulose acetate sulfate, sodium hydroxyethylcellulose, methylpolysiloxane, potassium sorbate, kaolin, diatomaceous earth, bentonite, aluminum silicate, aluminum hydroxide, colloidal aluminum hydroxide, magnesium aluminum silicate, magnesium montmorillonite, magnesium trisilicate, sodium aluminum magnesium silicate, sodium bicarbonate, sodium carbonate, methyl paraben, propyl paraben, ethyl vanillin, lemon oil, orange peel oil, vanillin, casein, and the like.

Preferably, the yeast of the present invention is selected from: saccharomyces cerevisiae (Latin name: Saccharomyces cerevisiae, available from China center for Industrial culture Collection of microorganisms; accession number: CICC1012), and Torulopsis delbrueckii (Latin name: Torulaspora delbrueckii, available from China center for Industrial culture Collection of microorganisms; accession number: CICC 1004). The 2 yeasts are inactivated by a conventional method respectively, and then are administered to the veins of the mice, and the intravenous administration of the 2 inactivated yeasts is found to enhance the immune function of the mice and have antibacterial and antiviral effects.

The inactivated yeast injection can be used for preventing, treating or assisting in treating various bacterial diseases, viral diseases, fungal diseases, parasitic diseases, cancers, low body immune function and other diseases of human beings or animals caused by various reasons.

No one attempts to use the inactivated yeast for injection administration to improve the immune function of the organism and study on antibiosis and antivirus, and the safety problem of the inactivated yeast injection administration is probably considered mainly. However, currently, there are some drugs that are administered by injecting harmful bacteria, such as bacillus calmette-guerin, which is a viable tuberculosis vaccine, and can be used for the adjuvant treatment of preventing tuberculosis and tumor by oral administration, subcutaneous injection, intraperitoneal injection or intratumoral injection. The corynebacterium parvum preparation is a dead bacteria suspension of the corynebacterium parvum and is used for adjuvant therapy of tumors by subcutaneous, intramuscular, intratumoral or intravenous drip and the like. In addition, there are group A streptococci preparations, Pseudomonas aeruginosa preparations, Mycobacterium phlei preparations, and the like. In addition, whole cells of bacteria belonging to the genera Rhodococcus, Gordonia, Nocardia, Dietzia, Tsukamurella and Nocardioides have been administered by injection as immune modifiers (patent publication No. CN 1735431A).

The research of the invention discovers that the inactivated saccharomycete intravenous injection has the functions of improving the immunity and preventing and treating bacterial diseases and virus diseases. The yeast as a probiotic bacterium has safer characteristic of inactivation injection administration. The injection of the inactivated yeast can improve the immune function of the organism, and the mechanism that the injection of the inactivated yeast can improve the immune function of the organism is probably that after the inactivated yeast is injected and administrated, the organism takes thalli of the inactivated yeast as foreign substances for recognition and aims to remove the foreign substances so as to enhance the immune function of the organism; it is also possible that the inactivated yeast with intact thallus form is contacted with immune cells to act on Toll-like receptors on the immune cells, thereby activating the immune system. The antibacterial and antiviral effects of the inactivated yeast injection may be the result of enhancing the immune function of the body, and may also be the effect of substances produced by the body after catabolism of the inactivated yeast after injection.

Drawings

FIG. 1 is a gram stain oil-lens observation photograph of live Saccharomyces cerevisiae

FIG. 2 is an oil-lens observation photograph of gram stain of inactivated Saccharomyces cerevisiae

Detailed Description

Example 1

The method comprises the steps of inoculating Saccharomyces cerevisiae (purchased from China center for preservation and management of industrial microbial strains; Latin name: Saccharomyces cerevisiae, preservation number: CICC1012) to a culture medium (prepared by weighing 20g of peptone, 20g of glucose and 10g of yeast powder, adding water to 1000mL, sterilizing at 121 ℃ for 20 min), culturing in a constant-temperature oscillator at 37 ℃ for 24 hours at the rotating speed of 160r/min, centrifuging for 5 minutes at 3000 rpm, removing the upper-layer culture solution, retaining the precipitate, adding sterile physiological saline to wash the precipitate, centrifuging for 5 minutes, repeatedly washing for 3 times, adding sterile physiological saline to mix with the precipitate, and preparing a suspension. Taking a certain amount of Saccharomyces cerevisiae normal saline suspension, and counting bacteria by using a THOMA bacteria counting plate to make each 1ml of suspension contain 10⁹And (3) saccharomyces cerevisiae thalli. Inactivating the prepared saccharomyces cerevisiae normal saline suspension for 15min at the temperature of 121 ℃ and under the pressure of 0.12MPa to obtain the inactivated saccharomyces cerevisiae injection. Respectively gram-staining live saccharomyces cerevisiae and inactivated saccharomyces cerevisiae, observing thallus forms under an oil lens (see attached figure 1 and attached figure 2), comparing to find that the inactivated bacteria and the viable bacteria keep consistent thallus profiles and forms, and counting bacteria finds that the quantity of the thallus is not obviously changed before and after inactivation. And centrifuging the inactivated saccharomyces cerevisiae normal saline suspension, discarding the supernatant, retaining the precipitate, extracting DNA, amplifying 16S rDNA by adopting PCR, and identifying the type of the saccharomyces cerevisiae by sequencing.

Example 2

Inoculating torulospora delbrueckii (torulospora delbrueckii, purchased from China center for culture Collection of industrial microorganisms; latin name: Torulaspora delbrueckii, preservation number: CICC1004) to a culture medium (prepared by weighing 20g of peptone, 20g of glucose and 10g of yeast powder, adding water to 1000mL, sterilizing at 121 ℃ for 20 min), culturing in a constant-temperature oscillator at 37 ℃ for 24 hours at a rotating speed of 160r/min, centrifuging for 5 minutes at 3000 r, removing the upper culture solution, retaining the precipitate, adding sterile physiological saline to wash the precipitate, centrifuging for 5 minutes, repeatedly washing for 3 times, adding sterile physiological saline, and uniformly mixing with the precipitate to prepare a suspension. Taking a certain amount of the physiological saline suspension of the Torulopsis delensiana, and passing through THOMA bacteriaThe plates were counted for bacterial count to 10 per ml suspension⁹Torulopsis sp. Inactivating the prepared physiological saline suspension of the Torulaspora delbrueckii for 15min at the temperature of 121 ℃ and the pressure of 0.12MPa to obtain the inactivated Torulaspora delbrueckii injection.

Example 3

The influence of the inactivated saccharomyces cerevisiae injection prepared in example 1 and the inactivated torulopsis delbrueckii injection prepared in example 2 on the phagocytic function of normal mouse mononuclear-macrophages is respectively detected by a carbon particle clearance test method. A clean-grade Kunming-breed white mouse with the weight of 18-22g is divided into a normal control group, an inactivated saccharomyces cerevisiae injection group and an inactivated Torulopsis delbrueckii injection group. Each group had 10 mice, half male and half female. Normal control group mice tail vein injection sterile normal saline, inactivation Saccharomyces cerevisiae injection group mice tail vein injection inactivation Saccharomyces cerevisiae suspension, inactivation Delaware injection group mice tail vein injection inactivation Delaware suspension. The administration volume of each group is 0.1mL/10g, and the administration is performed for 5 days by continuous tail vein injection, 1 time per day. 2 hours after the last administration, mice were injected with 0.05mL/10g India ink via the tail vein, and 40. mu.L of blood was collected from orbital venous plexus at 1min and 10min, respectively, and added with 4mL of 0.1% Na₂CO₃Shaking the solution, comparing the color with a spectrophotometer at 680nm wavelength, and measuring optical density (OD is used below₁And OD₁₀To represent the optical density of the blood samples taken at 1min and 10 min), the carbon clearance index K value was calculated according to the following formula. Carbon clearance index K ═ lgOD₁-lgOD₁₀)/(t₁₀-t₁). The significance of the experimental data was checked using SPSS11.5 software and the results are shown in table 1. As can be seen from Table 1, compared with the normal control group, the carbon clearance index K value of the inactivated Saccharomyces cerevisiae injection group and the inactivated Delaware Torulaspora delbrueckii injection group are respectively and remarkably improved. As shown above, intravenous administration of inactivated Saccharomyces cerevisiae and Torulopsis delbrueckii can improve the phagocytic function of monocyte-macrophage in normal mice, i.e., improve the nonspecific immune function of mice.

TABLE 1 Effect of intravenous injection of inactivated yeasts on phagocytic function of monocyte-macrophages in Normal mice

Note: indicates a significant difference P < 0.01 from the normal control group, and indicates a significant difference P < 0.05 from the normal control group.

Example 4

Respectively adopts iodine [2]¹²⁵I]Tumor necrosis factor-alpha radioimmunoassay kit and iodine¹²⁵I]The interferon-r radioimmunoassay kit detects the influence of the inactivated saccharomyces cerevisiae injection prepared in example 1 on the contents of TNF-alpha and IFN-gamma in mouse serum. The significance of the experimental data was checked using SPSS11.5 software and the results are shown in table 2. As can be seen from Table 2, compared with the normal control group, the inactivated Saccharomyces cerevisiae injection group significantly increased the TNF-alpha and IFN-gamma content in the mouse serum.

TABLE 2 results of the effect of intravenous injection of inactivated yeast on TNF-alpha and IFN-gamma content in mouse serum

Example 5

The control effect of the inactivated saccharomyces cerevisiae injection prepared in example 1 and the inactivated torulopsis delbrueckii injection prepared in example 2 on salmonella-infected mice was examined. The clean-grade Kunming white mice with the weight of 18-22g are divided into 4 groups, namely a normal control group, a salmonella group, an inactivated saccharomyces cerevisiae injection group and an inactivated delbrueckia frutescens injection group, wherein each group comprises 30 mice and each half of the mice is male and female. The tail vein of mice of a normal control group and a salmonella group is injected with sterile normal saline, and the tail vein injection dosage of an inactivated saccharomyces cerevisiae injection group and an inactivated delbrueckia sporophora injection group is 0.1mL/10g of corresponding injection respectively. After the administration, mice in the salmonella group, the inactivated saccharomyces cerevisiae injection group and the inactivated delbrueckia frugiperda injection group are respectively injected with salmonella enteritidis in tail vein at intervals of 24 hours (purchased from China veterinary culture collection center for microbiological culture collection, code CVCC 3377). The observation was continued for 20 days and the mice were scored for daily deaths. The experimental data were checked chi-square using SPSS11.5 software and the results are shown in table 3. As can be seen from Table 3, the inactivated Saccharomyces cerevisiae injection group and the inactivated Torulopsis delbrueckii injection group both significantly reduced the number of deaths in mice compared to the Salmonella control group. The inactivated saccharomyces cerevisiae injection and the inactivated torulopsis delbrueckii injection have the effect of preventing and treating mice infected with salmonella after intravenous administration.

TABLE 3 results of the intravenous injection of inactivated yeast for the prevention and treatment of Salmonella lethal effects in mice

Note: indicates a significant difference P < 0.01 from the normal control group, and indicates a significant difference P < 0.05 from the normal control group; delta indicates a significant difference P < 0.01 compared with the salmonella group, and delta indicates a significant difference P < 0.05 compared with the salmonella group.

Example 6

The effect of the inactivated saccharomyces cerevisiae injection prepared in example 1 and the inactivated torulopsis delbrueckii yeast injection prepared in example 2 on the death count of mice infected with influenza PR8 strain was examined. The experiment selects 18-22g clean-grade Kunming white mice to be divided into a normal control group, a PR8 strain model group (namely an influenza virus PR8 strain infected mouse model group), an inactivated saccharomyces cerevisiae injection group and an inactivated Torulopsis delbrueckii injection group. 30 mice in each group, an inactivated saccharomyces cerevisiae injection group and an inactivated torulopsis delbrueckii injection group respectively carry out tail vein injection on corresponding injections with the volume of 0.1ml/10 g; sterile normal saline is injected into tail vein of normal control group and model group of PR8 strain, and the administration volume of each group is 0.1mL/10 g. Each group of mice was administered 1 time and at an interval of 24 hours, and except for the normal control group, each group of mice was inoculated with chick embryo allantoic fluid containing influenza virus PR8 strain in each 0.05mL of each nasal cavity under light ether anesthesia to infect influenza virus PR8 strain with the mice. The number of deaths in each group was recorded by observation over 10 days. The experimental data were checked chi-square using SPSS11.5 software and the results are shown in table 4. As can be seen from Table 4, the inactivated Saccharomyces cerevisiae injection group and the inactivated Torulopsis delbrueckii injection group have extremely significant and significant reduction in the number of mouse deaths compared with the PR8 strain model group, respectively. The above shows that the intravenous administration of the inactivated Saccharomyces cerevisiae and the inactivated Torulopsis delbrueckii can be used for preventing and treating viral diseases.

TABLE 4 Effect of intravenous injection of inactivated yeast on influenza PR8 strain-infected mice

Note: indicates a significant difference P < 0.01 from the normal control group, and indicates a significant difference P < 0.05 from the normal control group; Δ means a significant difference P < 0.01 from the model group of PR8 strain, and Δ means a significant difference P < 0.05 from the model group of PR8 strain.

Claims

1. An injection for improving immunity, resisting salmonella and influenza virus PR8 strain is prepared from the inactivated yeast through gram staining, observing under oil microscope, and adding 10 inactivated yeast to each ml of injection⁵—10¹²The injection is suspension, the administration mode is injection administration,

the yeast is saccharomyces cerevisiae, and the name of latin is as follows: saccharomyces cerevisiae, accession number: CICC 1012; or torulopsis delbrueckii, latin name: torula pora delbrueckii, accession number: the CICC 1004.

2. The injection according to claim 1, wherein the inactivation method is selected from any one of high temperature inactivation, high temperature and high pressure inactivation, ultraviolet inactivation, chemical agent inactivation and radiation inactivation.

3. The injectable formulation of claim 1, further comprising a pharmaceutically acceptable adjuvant comprising sufficient salts or monosaccharides to ensure that the injectable suspension is at or near osmolality.

4. The injection according to claim 1, wherein the injection is prepared into powder for injection by spray drying or freeze drying, and is prepared into suspension when in use.

5. The injection according to any one of claims 1 to 4, wherein the injection is administered intravenously, subcutaneously, intradermally, intramuscularly, intraperitoneally, intratumorally.

6. Use of the injection according to claims 1 to 5 for the preparation of a medicament for enhancing immune function, against salmonella and against influenza PR8 strain.