A proliferation medium for improving the heat resistance of Enterococcus faecium and its application

Abstract

The present invention belongs to the technical field of microorganisms, and specifically provides a proliferation medium for improving the heat resistance of Enterococcus faecium, comprising: 1-3% glucose, 1-3% peptone, 0.5-1.5% yeast extract powder, and 0.2-1.5% γ-aminobutyric acid, with the balance being water. The present invention also provides a method for improving the heat resistance of Enterococcus faecium using the above medium and a method for spray-drying to prepare solid Enterococcus faecium powder. The proliferation medium for improving the heat resistance of Enterococcus faecium provided by the present invention can effectively improve the heat resistance of Enterococcus faecium cells themselves, thereby increasing the viable cell retention rate of Enterococcus faecium during high-temperature drying. Under the same production process conditions, the viable cell yield of the product is increased, and the production cost of Enterococcus faecium is greatly reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a proliferation culture medium capable of improving the heat resistance of Enterococcus faecium and an application thereof. Background Art

[0002] Probiotics are beneficial microbial products containing live bacteria. Feeding probiotics can improve the intestinal microbial flora of animals, increase feed conversion rate, enhance animal immunity, and achieve the purpose of preventing and curing diseases and improving production performance. Enterococcus faecium belongs to the genus Lactobacillus. It is an intestinal probiotic isolated from the intestines of animals with good biosafety and probiotic properties. During metabolism, it secretes L-lactic acid and non-specific immunomodulatory factors, and produces a variety of nutrients that are beneficial to animal growth. Therefore, it is often used as a microbial feed additive to enhance animal intestinal health.

[0003] After the proliferation of Enterococcus faecium through liquid culture, its heat resistance is poor, so the vacuum freeze-drying method is used in production to obtain solid bacterial powder. However, the disadvantages of vacuum freeze-drying are high equipment requirements, high energy consumption, and time-consuming. In addition, the product also needs to be stored at low temperatures, which requires good cold chain technology, which is very unfavorable for commodity production. In recent years, the spray drying method for the production of microecological preparations has gradually emerged. The advantages are large production capacity and the product can be stored at room temperature. However, the inlet air temperature of spray drying usually reaches above 150°C, which has high requirements for the heat resistance of the dried material. If the material is not heat-resistant, the high temperature after spray drying will kill most of the live bacteria, resulting in a low yield of live bacteria. Therefore, improving the heat resistance of Enterococcus faecium cells allows spray drying to be used in industrial production, greatly reducing production costs. Summary of the invention

[0004] The purpose of the present invention is to overcome the problems in the prior art that after Enterococcus faecium is proliferated through liquid culture, the heat resistance is poor and the drying cost is high.

[0005] To this end, the invention provides a proliferation culture medium for improving the heat resistance of Enterococcus faecium, comprising: 1-3% glucose, 1-3% peptone, 0.5-1.5% yeast extract powder and 0.2-1.5% gamma-aminobutyric acid, and the balance is water.

[0006] The present invention also provides a method for improving the heat resistance of Enterococcus faecium, comprising the following steps:

[0007] (1) inoculating Enterococcus faecium into an activation medium for activation to obtain activated Enterococcus faecium;

[0008] (2) The activated Enterococcus faecium is inoculated into the above-mentioned proliferation medium for proliferation culture to obtain Enterococcus faecium with improved heat resistance.

[0009] Specifically, in the above step (2), the proliferation culture conditions are 37°C, 150 rpm, and culture for 15 - 20 h.

[0010] Specifically, in the above step (1), the activation medium includes 1 - 3% glucose, 1 - 3% peptone, 0.5 - 1.5% yeast extract powder, and the balance is water.

[0011] Specifically, in the above step (1), the activation conditions are 37°C, 150 rpm, and culture for 8 - 10 h.

[0012] The present invention also provides a method for preparing Enterococcus faecium solid bacterial powder, comprising the following steps: culturing Enterococcus faecium by using the above method for improving the heat resistance of Enterococcus faecium to obtain a culture solution of Enterococcus faecium with improved heat resistance, adding a carrier to the culture solution of Enterococcus faecium, and then performing spray drying to obtain Enterococcus faecium solid bacterial powder.

[0013] Specifically, based on the mass of the culture solution of Enterococcus faecium, the addition amount of the carrier is 15 - 30%.

[0014] Specifically, spray drying is carried out under the conditions of an inlet air temperature of 160 - 200°C and an outlet air temperature of 75 - 90°C.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0016] The proliferation medium for improving the heat resistance of Enterococcus faecium provided by the present invention can effectively improve the heat resistance of Enterococcus faecium cells themselves, thereby increasing the viable bacteria retention rate of Enterococcus faecium during the high-temperature drying process. Under the same production process conditions, the viable bacteria yield of the product is increased, and the production cost of Enterococcus faecium is greatly reduced. Specific Embodiments

[0017] The technical solutions in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Although the representative embodiments of the present invention have been described in detail, those of ordinary skill in the technical field to which the present invention belongs will understand that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0018] The present invention provides a proliferation medium for improving the heat resistance of Enterococcus faecium, comprising: 1 - 3% glucose, 1 - 3% peptone, 0.5 - 1.5% yeast extract powder, and 0.2 - 1.5% γ-aminobutyric acid, and the balance is water.

[0019] The present invention also provides a method for improving the heat resistance of Enterococcus faecium, comprising the following steps:

[0020] (1) Inoculate Enterococcus faecium into the activation medium, and incubate it at 37°C with a rotation speed of 150 rpm for 8 - 10 h to obtain activated Enterococcus faecium;

[0021] Among them, the activation medium includes 1 - 3% glucose, 1 - 3% peptone, 0.5 - 1.5% yeast extract powder, and the balance is water;

[0022] (2) Inoculate the activated Enterococcus faecium into the above - mentioned proliferation medium, and incubate it at 37°C with a rotation speed of 150 rpm for 15 - 20 h to obtain Enterococcus faecium with improved heat resistance.

[0023] The present invention also provides a method for preparing solid Enterococcus faecium powder, which includes the following steps: Cultivate Enterococcus faecium by using the method for improving the heat resistance of Enterococcus faecium to obtain a culture solution of Enterococcus faecium with improved heat resistance. After adding a carrier to the culture solution of Enterococcus faecium, perform spray drying under the conditions of an inlet air temperature of 170°C and an outlet air temperature of 80°C to obtain solid Enterococcus faecium powder.

[0024] The carrier is preferably corn starch, and the addition amount of the carrier is 15 - 30 based on the mass of the culture solution of Enterococcus faecium.

[0025] Next, the proliferation medium for improving the heat resistance of Enterococcus faecium and the effects of its application in the present invention are studied through specific examples.

[0026] Example 1:

[0027] This example provides a proliferation medium for improving the heat resistance of Enterococcus faecium, which includes: 2% glucose, 2% peptone, 1% yeast extract powder, and 0.25% γ - aminobutyric acid, and the balance is water. Sterilize the above - mentioned medium at 115°C for 30 min and then reserve it for use.

[0028] This example also provides a method for improving the heat resistance of Enterococcus faecium, which includes the following steps:

[0029] 1. Activation

[0030] Inoculate the cryopreserved Enterococcus faecium strain into the activation medium, and incubate it at 37°C with a rotation speed of 150 rpm for 8 h to obtain activated Enterococcus faecium;

[0031] Among them, the activation medium includes 2% glucose, 2% peptone, 1% yeast extract powder, and the balance is water.

[0032] 2. Proliferation

[0033] Inoculate the activated Enterococcus faecium into the above - mentioned proliferation medium, and incubate it at 37°C with a rotation speed of 150 rpm for 15 h to obtain a fermentation broth of Enterococcus faecium with improved heat resistance.

[0034] The Enterococcus faecium after the proliferation culture was diluted to 10 with physiological saline. -6 Then, it was treated at 60 °C for 5 min, and the viable count of Enterococcus faecium was counted by the dilution plate coating method. Taking the viable count in the Enterococcus faecium culture solution without heat treatment as the control, the viable count retention rate after heat treatment was calculated. For Enterococcus faecium treated at 60 °C for 5 min, the viable count before high-temperature treatment was 3.60×10 8 CFU / mL, and the viable count after high-temperature treatment was 1.15×10 8 CFU / mL, and the viable count retention rate reached 31.9%.

[0035] 20% corn starch was added as a carrier to the Enterococcus faecium culture solution after proliferation culture, and spray drying was carried out under the conditions of an inlet air temperature of 170 °C and an outlet air temperature of 80 °C. The same method was used to detect the viable count after spray drying. The viable count after drying was 9.82×10 8 CFU / g, and the drying yield was 68.06%.

[0036] Comparative Example 1:

[0037] This comparative example studied the effect of the proliferation medium on the heat resistance of Enterococcus faecium, and the specific steps were as follows.

[0038] 1. Activation

[0039] The cryopreserved Enterococcus faecium strain was inoculated into the activation medium, and activated at 37 °C and 150 rpm for 8 h to obtain the activated Enterococcus faecium.

[0040] Among them, the activation medium included 2% glucose, 2% peptone, 1% yeast extract powder, and the balance was water.

[0041] 2. Proliferation

[0042] The activated Enterococcus faecium was inoculated into the proliferation medium, and proliferated at 37 °C and 150 rpm for 15 h to obtain the proliferated Enterococcus faecium fermentation broth. The difference between the proliferation medium in this comparative example and that in Example 1 was that it did not contain γ-aminobutyric acid.

[0043] The Enterococcus faecium after the proliferation culture was diluted to 10 with physiological saline. -6 Then, it was treated at 60 °C for 5 min, and the viable count of Enterococcus faecium was counted by the dilution plate coating method. Taking the viable count in the Enterococcus faecium culture solution without heat treatment as the control, the viable count retention rate after heat treatment was calculated. For Enterococcus faecium treated at 60 °C for 5 min, the viable count before high-temperature treatment was 4.50×10 8 CFU / mL, and the viable count after high-temperature treatment was 4.85×10 7 CFU / mL, and the viable count retention rate was 10.8%, which was only 34% of the retention rate in Example 1.

[0044] Add 20% corn starch as a carrier to the culture solution of Enterococcus faecium after proliferation culture, and perform spray drying under the conditions of an inlet air temperature of 170 °C and an outlet air temperature of 80 °C. The viable count after spray drying was detected by the same method. The viable count after drying was 5.82×10 8 CFU / g, and the drying yield was only 32.3%, only 47.5% of that in Example 1.

[0045] Example 2:

[0046] This example provides a method for preparing Enterococcus faecium solid bacterial powder, which includes the following steps:

[0047] 1. Activation

[0048] Inoculate the cryopreserved Enterococcus faecium strain into the activation medium, and perform activation culture at 37 °C and 150 rpm for 10 h to obtain activated Enterococcus faecium;

[0049] Among them, the activation medium includes 2% glucose, 2% peptone, 1% yeast extract powder, and the balance is water.

[0050] 2. Proliferation

[0051] Inoculate the activated Enterococcus faecium into the proliferation medium, and perform proliferation culture at 37 °C and 150 rpm for 20 h to obtain a fermentation broth of Enterococcus faecium with improved heat resistance.

[0052] The proliferation medium includes 2% glucose, 2% peptone, 1% yeast extract powder, and 0.5% γ-aminobutyric acid, and the balance is water. It is used after sterilization at 115 °C for 30 min.

[0053] 3. Heat resistance detection

[0054] Dilute the Enterococcus faecium after proliferation culture to 10 -6 , and treat it at 60 °C for 5 min. The viable count of Enterococcus faecium was counted by the dilution plate coating method. Taking the viable count in the Enterococcus faecium culture solution without heat treatment as a control, calculate the viable count retention rate after heat treatment. Enterococcus faecium was treated at 60 °C for 5 min. The viable count before high-temperature treatment was 5.25×10 8 CFU / mL, and the viable count after high-temperature treatment was 2.21×10 8 CFU / mL, and the viable count retention rate reached 42.10%. Compared with Comparative Example 1, the retention rate increased by 3.9 times.

[0055] 4. Drying of the culture solution

[0056] Add 20% corn starch as a carrier to the culture solution of Enterococcus faecium after proliferation culture, and perform spray drying under the conditions of an inlet air temperature of 170 °C and an outlet air temperature of 80 °C. Detect the viable count after spray drying by the same method. The viable count after drying is 1.82×10 9 CFU / g, and the drying yield is 86.67%. Compared with Comparative Example 1, the yield is increased by 2.68 times.

[0057] In summary, the proliferation medium for improving the heat resistance of Enterococcus faecium provided by the present invention can effectively improve the heat resistance of Enterococcus faecium cells themselves, thereby increasing the viable cell retention rate of Enterococcus faecium during high-temperature drying. Under the same production process conditions, the viable cell yield of the product is increased, and the production cost of Enterococcus faecium is greatly reduced.

[0058] The above examples are only illustrative of the present invention and do not constitute a limitation on the protection scope of the present invention. Any design identical or similar to the present invention falls within the protection scope of the present invention.

Claims

1. A method for improving the heat resistance of Enterococcus faecium, characterized in that, It includes the following steps: (1) Inoculate Enterococcus faecium into an activation medium and activate it for 8 - 10 h to obtain activated Enterococcus faecium; (2) Inoculate the activated Enterococcus faecium into a proliferation medium for proliferation culture to obtain Enterococcus faecium with improved heat resistance; the proliferation medium includes 1 - 3% glucose, 1 - 3% peptone, 0.5 - 1.5% yeast extract powder, and 0.2 - 1.5% γ-aminobutyric acid, with the balance being water.

2. The method for improving the heat resistance of Enterococcus faecium according to claim 1, characterized in that: In step (2), the proliferation culture conditions are 37°C, 150 rpm, and culture for 15 - 20 h.

3. The method for improving the heat resistance of Enterococcus faecium according to claim 1, characterized in that: The activation medium in step (1) includes 1 - 3% glucose, 1 - 3% peptone, 0.5 - 1.5% yeast extract powder, with the balance being water.

4. The method for improving the heat resistance of Enterococcus faecium according to claim 1, characterized in that: The activation conditions in step (1) are 37°C, 150 rpm, and culture for 8 - 10 h.

5. A method for preparing solid bacterium powder of Enterococcus faecium, characterized in that, It includes the following steps: Culture Enterococcus faecium by using the method for improving the heat resistance of Enterococcus faecium according to any one of claims 2 - 4 to obtain a culture solution of Enterococcus faecium with improved heat resistance, add a carrier to the culture solution of Enterococcus faecium, and then perform spray drying to obtain a solid bacterial powder of Enterococcus faecium.

6. The preparation method of the Enterococcus faecium solid bacterial powder according to claim 5, characterized in that: Based on the mass of the culture solution of Enterococcus faecium, the addition amount of the carrier is 10 - 30%.

7. The preparation method of the Enterococcus faecium solid bacterial powder according to claim 5, characterized in that: Perform spray drying under the conditions of an inlet air temperature of 160 - 200°C and an outlet air temperature of 75 - 90°C.