A composition for solid preservation of Enterococcus faecium and a preservation method thereof

Abstract

The present invention belongs to the field of microorganisms, and specifically relates to a composition for solid preservation of Enterococcus faecium and a preservation method. The carrier used in the method is a medical stone-chitosan composite adsorbent, a protective agent is sodium glutamate and lactose, the moisture content of the bacterial powder is less than 1%, the bacterial powder prepared by the method has a high bacterial content, and the survival rate reaches more than 80% after being stored at room temperature for 1 year, which solves the problem that the fermentation liquid of Enterococcus faecium is difficult to preserve. Compared with other technologies, it has the characteristics of low cost, easy operation and outstanding preservation effect.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to a composition for solid preservation of Enterococcus faecium and a preservation method. Background Art

[0002] In the traditional breeding process, antibiotics are used in large quantities. Although the economic value of farmed animals is increased, it brings certain harms, such as the increase of bacterial resistance and the decline of animal immunity. Human consumption of large amounts of "antibiotic foods" seriously affects human health, inducing the increase of human resistance. Once a disease occurs in the future, it will be untreatable. Beneficial intestinal microorganisms, as a substitute for antibiotics, can have a good improvement effect. As a beneficial factor, it can maintain the balance of intestinal bacteria and increase animal immunity.

[0003] Enterococcus faecium is a type of lactic acid bacteria, an intestinal symbiotic bacterium, sensitive to most antibiotics, and a safe strain. Its mechanism of action is as follows: (1) It secretes antibacterial peptides to prevent and control diseases. It can grow and reproduce on the intestinal mucosa of animals. It inhibits the adsorption and proliferation of harmful bacteria by secreting bacteriocins, organic acids and other substances, and is used to regulate the microecological balance of the intestinal environment; (2) It secretes digestive enzymes to promote feed conversion and increase the decomposition of proteins into amides and amino acids, thereby improving the absorption and conversion rate of feed; (3) It enhances cellular immunity, enhances the activity of macrophages and monocytes, promotes antibody production, improves immunity, and reduces diarrhea rate and mortality; as a microecological preparation, it can be used in combination with other probiotics or alone. It has a wide range of uses and development prospects in livestock and poultry farming and aquaculture.

[0004] Although this type of strain has good application prospects, it is difficult to store. Solid storage can greatly extend the storage time, but most manufacturers' products are still mainly low-temperature storage, and cannot be stored at room temperature or high temperature. At the same time, the preparation process is complicated, the equipment investment cost is high, and the storage effect is unstable. Therefore, it is urgent to find a low-cost and good storage method. Summary of the invention

[0005] In view of the shortcomings of the existing room temperature preservation technology of Enterococcus faecium, the present invention provides a composition for solid preservation of Enterococcus faecium and a preservation method. The carrier used in the method is a medical stone-chitosan composite adsorbent, the protective agent is sodium glutamate and lactose, the moisture content of the bacterial powder is less than 1%, the bacterial powder prepared by the method has a high bacterial content, and the survival rate reaches more than 80% after being stored at room temperature for 1 year, which solves the problem that the fermentation liquid of Enterococcus faecium is difficult to preserve. Compared with other technologies, it has the characteristics of low cost, easy operation and outstanding preservation effect.

[0006] The technical solution of the present invention is:

[0007] A composition for solid preservation of Enterococcus faecium, comprising a medical stone-chitosan composite adsorbent and a protective agent, wherein the protective agent is preferably a combination of sodium glutamate and lactose.

[0008] The preparation method of the medical stone-chitosan composite adsorbent is as follows: remove organic matter and other impurities from the medical stone, wash it to neutrality, dry it, and crush it to above 80 meshes; dissolve chitosan with a deacetylation degree of 85% to 95% in an acetic acid solution; mix the crushed medical stone with the chitosan solution, soak it, and dry it to obtain the medical stone-chitosan composite adsorbent.

[0009] In the above preparation method, the method for removing impurities such as organic matter in the medical stone is preferably to remove them by soaking and filtering with a hydrochloric acid solution, and washing the medical stone with deionized water until it is neutral.

[0010] In the above preparation method, the acetic acid solution used is preferably 3% to 5% acetic acid solution, the mass fraction of the chitosan solution is preferably 0.5% to 1%, and the mass ratio of the crushed medical stone to the chitosan solution is preferably 1:1 to 2.

[0011] The crushed medical stone is mixed with the chitosan solution, soaked, dried and preferably further crushed to a size of 150 mesh or more.

[0012] When the protective agent of the solid storage composition for Enterococcus faecium is a combination of sodium glutamate and lactose, the ratio of the two is 1:3 to 3:1, preferably 1:1.

[0013] The method for solid preservation of Enterococcus faecium using the above composition comprises:

[0014] (1) adding 1% to 3% (m / v, g / ml) of medical stone-chitosan composite adsorbent to the fermentation broth of Enterococcus faecium, stirring and adsorbing for 10 min to 30 min, and centrifuging at 5000 rpm to obtain a centrifugal heavy liquid;

[0015] (2) Add 0.4% to 1.5% of protective agent to the centrifugal heavy liquid, mix well and evenly, let it stand for a period of time, dry it, and then store it at room temperature.

[0016] The drying method in the above step (2) can be medium temperature drying, and the specific drying temperature can be 40° C. to 55° C.; the water content after drying is less than 1%.

[0017] Medical stone is a medicinal mineral with a porous structure, physiological activity and good adsorption and ion exchange properties. Chitosan is the deacetylation product of chitin, and its macromolecular chain contains many amino groups and hydroxyls, which has good adsorption and flocculation effects on metal ions, organic matter, proteins, etc. Medical stone-chitosan composite adsorbent, that is, chitosan molecules enter the internal pores of medical stone, change the internal structure of medical stone, expand its interlayer spacing, and enhance the ability of medical stone and chitosan to adsorb Enterococcus faecium alone.

[0018] The inventors have found through research that -NH2, α-COOH and γ-COOH in sodium glutamate play a protective role on microorganisms, and lactose has a strong binding force on the phospholipid membrane of Enterococcus faecium. During the drying and storage process, the medical stone-chitosan composite adsorbent, as an excipient, provides a large amount of porous amorphous structure for the dry powder. Sodium glutamate and lactose enhance and synergistically replace water molecules to be adsorbed on the surface of phospholipids, proteins and amorphous structures, interact with the phospholipid bilayer, promote the formation of the glassy state of Enterococcus faecium dry powder, and improve the recovery rate of Enterococcus faecium dry powder. In addition, sodium glutamate also has an antioxidant effect, inhibits the oxidation of triglycerides and the formation of free radicals to prevent irreversible damage to the cell membrane, and improves the survival rate of Enterococcus faecium dry powder.

[0019] In addition, the research experiment also found that when the medical stone-chitosan composite adsorbent and the protective agent are a combination of sodium glutamate and lactose, the preservation effect is better, and this effect cannot be achieved by any other combination of adsorbent and protective agent.

[0020] The solid preservation method for improving the survival rate of Enterococcus faecium is characterized in that the drying method is medium temperature drying, the drying temperature is 40°C to 55°C, and the final moisture content is less than 1%. The lower moisture content can maintain the dormant state of Enterococcus faecium and prolong the preservation time.

[0021] The beneficial effects of the present invention are:

[0022] The solid-preserved combined species carrier used in the present invention is a medical stone-chitosan composite adsorbent. Both medical stone and chitosan are natural environmentally friendly materials. The internal structure combination of medical stone and chitosan enhances their ability to adsorb Enterococcus faecium alone, greatly improving the adsorption efficiency.

[0023] Sodium glutamate, lactose and medical stone-chitosan composite adsorbent synergistically improve the recovery rate and survival rate of Enterococcus faecium dry powder.

[0024] The fermentation medium, fermentation method and preservation method disclosed in the present invention can maintain a high bacterial amount and bacterial activity at room temperature, and the stability is very high; and medium-temperature drying can be used in the bacterial body drying process, the equipment process requirements are simple, and the production cost and equipment investment cost of low-temperature drying can be greatly saved. DETAILED DESCRIPTION

[0025] The following is a further detailed description of the above contents of the present invention through specific implementation methods in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention, and unless otherwise specified, the following embodiments are all completed using conventional existing technologies.

[0026] The Enterococcus faecium fermentation broth used in the following examples of the present invention is obtained by the following method:

[0027] (1) Preparation of seed solution: After taking out the bacterial strain from -20°C, perform primary activation in MRS medium to obtain primary seed solution, and perform secondary transfer at a ratio of 5% to obtain secondary seed solution.

[0028] (2) Fermentation in tanks: The fermentation medium formula is: 3% glucose, 3% sucrose, 2.3% corn flour, 5% yeast powder, 1% ammonium sulfate, 0.1% calcium carbonate, 0.8% sodium acetate, and 0.006% manganese sulfate.

[0029] The fermentation medium was placed in a fermentation tank for sterilization. When the temperature was cooled to 30°C, it was inoculated at 5% (v / v) and the initial pH was adjusted to 6.0. During the fermentation process, the pH was adjusted once every hour with ammonia water, and ventilation was performed for 5 minutes every 2 hours with a ventilation rate of 1:2. When the pH of the fermentation liquid was maintained at 6.0, the fermentation was stopped, which was regarded as the fermentation end point.

[0030] The amount of Enterococcus faecium fermentation broth obtained was about 9.3×10 9 cfu / mL.

[0031] Example 1 Preparation of medical stone-chitosan composite adsorbent

[0032] Soak medical stone in 1:3 (m / v, g / mL) 0.1mol / L HCl solution for 24h, filter to remove organic matter and other impurities, wash medical stone with deionized water until neutral, dry, and crush to 80 mesh. Use 5% (v / v) acetic acid solution to slowly dissolve chitosan with a deacetylation degree of 90% to prepare a 0.5% chitosan solution. Mix 80-mesh medical stone and chitosan solution in a mass ratio of 1:1.3 to make a paste, make it fully infiltrated, place the paste in an oven, heat and dry, grind it into powder, and pass it through a 150-mesh sieve to obtain medical stone-chitosan composite adsorbent.

[0033] Example 2 Preparation of medical stone-chitosan composite adsorbent

[0034] Soak medical stone in 1:3.7 (m / v, g / mL) 0.1mol / L HCl solution for 24h, filter to remove organic matter and other impurities, wash medical stone with deionized water until neutral, dry, and crush into 80 mesh. Use 3% (v / v) acetic acid solution to slowly dissolve chitosan with a deacetylation degree of 95% to prepare a 0.8% chitosan solution. Mix 80-mesh medical stone and chitosan solution in a mass ratio of 1:1 to make a paste, make it fully infiltrated, place the paste in an oven, heat and dry, grind it into powder, and pass it through a 150-mesh sieve to obtain medical stone-chitosan composite adsorbent.

[0035] Example 3 Preparation of medical stone-chitosan composite adsorbent

[0036] Soak medical stone in 1:4 (m / v, g / mL) 0.1mol / L HCl solution for 24h, filter to remove organic matter and other impurities, wash medical stone with deionized water until neutral, dry, and crush into 80 mesh. Use 3.3% (v / v) acetic acid solution to slowly dissolve chitosan with a deacetylation degree of 85% to prepare a 1% chitosan solution. Mix 80-mesh medical stone and chitosan solution in a mass ratio of 1:2 to make a paste, make it fully infiltrated, place the paste in an oven, heat and dry, grind it into powder, and pass it through a 150-mesh sieve to obtain medical stone-chitosan composite adsorbent.

[0037] Embodiments 4 to 6

[0038] (1) adding the medical stone-chitosan composite adsorbent described in Example 1 to the fermentation liquid of Enterococcus faecium, stirring for adsorption, and centrifuging at 5000 rpm to obtain a centrifuged heavy liquid;

[0039] (2) Add a protective agent to the centrifugal heavy liquid, mix thoroughly and evenly, let it stand for a period of time, dry it at 40°C to 55°C until the water content is less than 1%, and then store it at room temperature.

[0040] Other different parameters are implemented according to Table 1:

[0041] Table 1 Parameters of the embodiments

[0042]

[0043] Experimental Example 1 Comparative test of adsorbents

[0044] The same amount of Enterococcus faecium fermentation broth was used to compare the medical stone-chitosan composite adsorbent prepared in Examples 1, 2, and 3 with a conventional diatomaceous earth adsorption carrier.

[0045] The specific processing methods are:

[0046] Treatment 1: Add 1% by mass volume ratio of the medical stone-chitosan composite adsorbent prepared in Example 1 to the fermentation broth, stir and adsorb at 5000rpm for 20min, add sodium glutamate powder to the centrifuged heavy liquid at a mass volume ratio of 0.5% after centrifugation, mix well, let stand for 1h, and then dry at 40°C, and the final moisture content is 0.8%.

[0047] Treatment 2: Add 3% of the medical stone-chitosan composite adsorbent prepared in Example 2 in a mass volume ratio to the fermentation broth, stir and adsorb at 5000rpm for 30min, add sodium glutamate powder to the centrifuged heavy liquid at a mass volume ratio of 0.5% after centrifugation, mix well, let stand for 1h, and then dry at 45°C, and the final moisture content is 0.6%.

[0048] Treatment 3: Add 2% of the medical stone-chitosan composite adsorbent prepared in Example 3 at a mass volume (g / mL) ratio to the fermentation broth, stir and adsorb at 5000rpm for 10 minutes, add sodium glutamate powder to the centrifuged heavy liquid at a mass volume ratio of 0.5% after centrifugation, mix well, let stand for 1 hour, and then dry at 55°C. The final moisture content is 0.7%.

[0049] Treatment 4: Add 3% diatomaceous earth by mass volume and stir at 5000rpm for 30 minutes. After centrifugation, add sodium glutamate powder to the centrifuged heavy liquid at a mass volume ratio of 0.5%. After fully mixing, let it stand for 1 hour and then dry it at 45°C. The final moisture content is 0.6%.

[0050] The prepared bacterial powder was placed at 54°C for an accelerated test to observe the effect of the adsorption carrier on the survival rate. The test results are shown in Table 2.

[0051] Table 2 Effects of different treatments on the survival rate of Enterococcus faecium under 54℃ accelerated conditions

[0052]

[0053]

[0054] The test showed that under the accelerated condition of 54℃, treatments 1, 2, and 3 had better stability as adsorption carriers, which was significantly higher than treatment 4. This indicated that the medical stone-chitosan composite adsorbent had stronger bacterial activity as an adsorption carrier of Enterococcus faecium, which was significantly better than the conventional adsorbent diatomaceous earth.

[0055] Experimental Example 2 Protective Agent Control Experiment

[0056] According to Table 3, adsorbents were added to 10 groups of fermentation broths for adsorption. After centrifugation, corresponding protective agents were added according to Table 3. After being fully mixed, the broths were dried at 45° C. to a water content of 1%.

[0057] Table 3 Different protective agent schemes

[0058]

[0059] The recovery rate and preservation effect are used as evaluation indicators: the recovered mass of solid bacterial powder is M, the bacterial amount per gram of bacterial powder is A1, the bacterial amount of fermentation liquid is A2, the volume of the fermentation liquid prepared by the bacterial powder is V, and the recovery rate = A1×M / (A2×V);

[0060] Colony count: The dilution plate method was used for counting, and the counting plate was an MRS plate with calcium carbonate.

[0061] Protective agent control test results:

[0062] (1) Drying difficulty of different protective agents:

[0063] Sodium glutamate, yeast peptone, maltodextrin and lactose are easier to dry, followed by sodium alginate, and sorbitol is the most difficult to dry.

[0064] (2) Effects of different protective agents on bacterial powder recovery rate:

[0065] Table 4 Effects of different protective agents on the final recovery rate of Enterococcus faecium at 45℃

[0066]

[0067] As shown in Table 4, the effects of different protective agents on the final recovery rate of Enterococcus faecalis under 45°C conditions: for single protective agent, treatment 1 (sodium glutamate) and treatment 6 (lactose) had high bacterial survival rates and high final recovery rates, while treatment 4 (maltodextrin) and treatment 5 (sodium alginate) had low bacterial survival rates and poor recovery effects; sodium glutamate and lactose were used simultaneously as protective agents in combination with medical stone-chitosan composite adsorbent, and the recovery effect was the best.

[0068] Experimental Example 3 Accelerated test of bacterial agent stability

[0069] The solid bacterial powder of each test group prepared in Experimental Example 2 was packaged in equal amounts and placed in a 54°C constant temperature incubator for constant temperature culture. The bacterial count was detected at 0d, 2d, 5d, 8d, 11d, and 14d. The results are shown in Table 5.

[0070] Table 5 Effects of different treatments on the survival rate of Enterococcus faecium under 54℃ accelerated conditions

[0071]

[0072] The survival rate of the bacterial powder prepared by using the medical stone-chitosan composite adsorbent in Example 6 as the adsorption carrier of Enterococcus faecium and sodium glutamate and lactose in a ratio of 1:1 as the protective agent was significantly higher than that of other control groups. The survival rate was 82.94% at 14 days, which is equivalent to the survival rate of the bacterial agent with a shelf life of 1 year under room temperature storage conditions, indicating that the technical solution of the present invention is significantly effective.

[0073] The above application examples are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A composition for solid preservation of Enterococcus faecium, characterized in that: The composition is composed of a medical stone-chitosan composite adsorbent and a protective agent; the protective agent is a combination of sodium glutamate and lactose, and the ratio of sodium glutamate to lactose is 1:1; The preparation method of medical stone-chitosan composite adsorbent is as follows: remove impurities from medical stone and wash to neutrality, dry and crush to above 80 mesh; dissolve chitosan with a deacetylation degree of 85% to 95% in acetic acid solution; mix the crushed medical stone with chitosan solution, soak, and dry to obtain medical stone-chitosan composite adsorbent; The acetic acid solution used is 3% to 5%, the mass fraction of the chitosan solution is 0.5% to 1%, and the mass ratio of the crushed medical stone to the chitosan solution is 1:1 to 2.

2. The composition for solid storage of Enterococcus faecium at room temperature according to claim 1, characterized in that: The crushed medical stone is mixed with the chitosan solution, soaked, dried and then further crushed to a size of more than 150 meshes.

3. A method for preserving Enterococcus faecium using the composition according to claim 1, characterized in that: This step includes: (1) Add 3% medical stone-chitosan composite adsorbent to the fermentation broth of Enterococcus faecium, stir and adsorb for 10 min to 30 min, and centrifuge to obtain a centrifugal heavy liquid; (2) Add 0.5% protective agent to the centrifugal heavy liquid, mix thoroughly and let stand for a period of time, then dry it and store it at room temperature.

4. The method for preserving Enterococcus faecium according to claim 3, characterized in that: The drying method in step (2) is medium temperature drying, and the specific drying temperature is 40°C to 55°C. The moisture content after drying is less than 1%.

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