A composition, method and application for improving the stress resistance of Enterococcus faecium

Abstract

The present invention belongs to the technical field of feed additives, and specifically provides a composition for improving the stress resistance of Enterococcus faecium. By mass percentage, it includes: 1% succinic acid, 0.05% sodium citrate, 0 - 1.2% thickener, 12% carrier, and the balance is water. All raw materials used in the composition for improving the stress resistance of Enterococcus faecium provided by the present invention are raw materials permitted to be added in the feed additive industry, ensuring the compliance and safety of the product. The present invention also provides a method for post-treating Enterococcus faecium with this composition to prepare heat-resistant and acid-resistant Enterococcus faecium. This method is simple and easy to implement, and through the synergistic effect of each component and the control of pH during the preparation process, the heat-resistant retention rate of the obtained Enterococcus faecium preparation at 75°C can be increased to 53.8%, and at the same time, the gastric acid tolerance at pH 2.5 can be increased to 60.5%, significantly improving the stress resistance of Enterococcus faecium and enabling it to better adapt to industrial production applications.

Description

Technical Field

[0001] The present invention belongs to the technical field of feed additives, and particularly relates to a composition, a method and an application for improving the stress resistance of Enterococcus faecium. Background Art

[0002] With the development of biotechnology, the production of feed microecological preparations has increased rapidly, and the quality has also been significantly improved. Microecological preparations can improve feed utilization rate, promote animal growth, improve the ecological environment and prevent and control animal diseases, avoiding the negative impacts caused by adding substances such as antibiotics and hormones, and having obvious economic benefits and positive environmental protection significance.

[0003] Enterococcus faecium is a probiotic, which plays an important role in maintaining the ecological balance of the animal intestinal flora, especially showing outstanding performance in the intestinal health care, epidemic prevention and treatment of diseases of young animals. Many enterprises have made it into a microecological feed additive, but Enterococcus faecium has poor stress resistance and cannot effectively maintain its activity in production and processing, which limits the large-scale application of Enterococcus faecium. Summary of the Invention

[0004] The purpose of the present invention is to overcome the problems that Enterococcus faecium has poor stress resistance and cannot effectively maintain its activity in production and processing.

[0005] To this end, the present invention provides a composition for improving the stress resistance of Enterococcus faecium, which includes, by mass percentage: 1% of succinic acid, 0.05% of sodium citrate, 0 - 1.2% of a thickener, 12% of a carrier, and the balance is water.

[0006] Specifically, the above-mentioned thickener includes locust bean gum.

[0007] Specifically, the above-mentioned carrier includes starch.

[0008] The present invention also provides a temperature- and acid-resistant Enterococcus faecium preparation, which includes: Enterococcus faecium and the above-mentioned composition for improving the stress resistance of Enterococcus faecium.

[0009] The present invention also provides a preparation method for the above-mentioned temperature- and acid-resistant Enterococcus faecium preparation, which includes the following steps:

[0010] (1) Prepare a composition for improving the stress resistance of Enterococcus faecium;

[0011] (2) Mix the composition with the Enterococcus faecium fermentation broth to obtain a composite fermentation broth;

[0012] (3) Adjust the pH of the composite fermentation broth, and dry it while stirring to obtain a temperature- and acid-resistant Enterococcus faecium preparation.

[0013] Specifically, in the above step (2), the volume ratio of the composition to the Enterococcus faecium fermentation broth is 1:1.

[0014] Specifically, in the above step (3), the pH of the composite fermentation broth is adjusted to 4.0 - 5.0.

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

[0016] All raw materials used in the composition for improving the stress resistance of Enterococcus faecium provided by the present invention are raw materials permitted to be added in the feed additive industry, ensuring the compliance and safety of the product. The present invention also provides a method for post-treating Enterococcus faecium with this composition to prepare heat-resistant and acid-resistant Enterococcus faecium. This method is simple and easy to implement. Through the synergistic effect of each component and the control of pH during the preparation process, the heat-resistant retention rate of the obtained Enterococcus faecium preparation can be increased to 53.8% at 75°C, and at the same time, the gastric acid tolerance at pH 2.5 can be increased to 60.5%, significantly improving the stress resistance of Enterococcus faecium and enabling it to better adapt to industrial production applications. 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 composition for improving the stress resistance of Enterococcus faecium, which includes, by mass percentage: 1% succinic acid, 0.05% sodium citrate, 0 - 1.2% thickener, 12% carrier, and the balance is water. Among them, the thickener includes locust bean gum; the carrier includes starch.

[0019] The present invention also provides a heat-resistant and acid-resistant Enterococcus faecium preparation, which includes: Enterococcus faecium and the above composition for improving the stress resistance of Enterococcus faecium. This Enterococcus faecium preparation is prepared through the following steps:

[0020] (1) Prepare the composition for improving the stress resistance of Enterococcus faecium;

[0021] (2) Mix the composition with the Enterococcus faecium fermentation broth in a volume ratio of 1:1 to obtain a composite fermentation broth; the Enterococcus faecium fermentation broth can be prepared by conventional Enterococcus faecium fermentation techniques to meet the requirements, and no limitation is imposed on this;

[0022] (3) Adjust the pH of the composite fermentation broth to 4.0 - 5.0, and dry while stirring to obtain the heat-resistant and acid-resistant Enterococcus faecium preparation.

[0023] The following is a study on the effects of the composition, method and application for improving the stress resistance of Enterococcus faecium through specific embodiments.

[0024] Example 1:

[0025] This example provides a composition for improving the stress resistance of Enterococcus faecium, which, by mass percentage, includes: 1% succinic acid, 0.05% sodium citrate, 12% starch, 0.4% locust bean gum, and the balance is water.

[0026] Example 2:

[0027] This example provides a composition for improving the stress resistance of Enterococcus faecium, which, by mass percentage, includes: 1% succinic acid, 0.05% sodium citrate, 12% starch, 0.8% locust bean gum, and the balance is water.

[0028] Example 3:

[0029] This example provides a composition for improving the stress resistance of Enterococcus faecium, which, by mass percentage, includes: 1% succinic acid, 0.05% sodium citrate, 12% starch, 1.2% locust bean gum, and the balance is water.

[0030] Comparative Example 1:

[0031] This comparative example provides a composition, which, by mass percentage, includes: 1% succinic acid, 0.05% sodium citrate, 12% starch, and the balance is water.

[0032] Comparative Example 2:

[0033] This comparative example provides a composition, which, by mass percentage, includes: 12% starch, 1.2% locust bean gum, and the balance is water.

[0034] Comparative Example 3:

[0035] This comparative example provides a composition, which, by mass percentage, includes: 1% succinic acid, 0.05% sodium citrate, 1.2% locust bean gum, and the balance is water.

[0036] Example 4:

[0037] This example studied the effects of the compositions provided in Examples 1 - 3 and Comparative Examples 1 - 4 on improving the stress resistance of Enterococcus faecium. The specific steps are as follows.

[0038] 1. Fermentation broth of Enterococcus faecium

[0039] Inoculate the Enterococcus faecium seed solution at 8% into the fermentation medium (formulation of the medium: 52 g / L corn flour, 2.5 g / L urea, 6 g / L sodium acetate, 2.5 g / L dipotassium hydrogen phosphate, 0.04 g / L anhydrous magnesium sulfate, 0.03 g / L manganese sulfate, 3.5 g / L diammonium hydrogen citrate). The liquid filling volume is 60%, and ferment and culture at 37°C for 22 h.

[0040] 2. Acid-resistant and heat-resistant Enterococcus faecium preparation

[0041] Experimental group 1: Take 600 ml of the composition provided in Example 1 and mix it with 600 ml of Enterococcus faecium fermentation broth to obtain a composite fermentation broth; adjust the pH of the composite fermentation broth, and spray-dry while stirring to obtain an Enterococcus faecium preparation.

[0042] Experimental group 2: Take 600 ml of the composition provided in Example 2 and mix it with 600 ml of Enterococcus faecium fermentation broth to obtain a composite fermentation broth; adjust the pH of the composite fermentation broth, and spray-dry while stirring to obtain an Enterococcus faecium preparation.

[0043] Experimental group 3: Take 600 ml of the composition provided in Example 3 and mix it with 600 ml of Enterococcus faecium fermentation broth to obtain a composite fermentation broth; adjust the pH of the composite fermentation broth, and spray-dry while stirring to obtain an Enterococcus faecium preparation.

[0044] Control group 1: Take 600 ml of the composition provided in Comparative Example 1 and mix it with 600 ml of Enterococcus faecium fermentation broth to obtain a composite fermentation broth; adjust the pH of the composite fermentation broth, and spray-dry while stirring to obtain an Enterococcus faecium preparation.

[0045] Control group 2: Take 600 ml of the composition provided in Comparative Example 2 and mix it with 600 ml of Enterococcus faecium fermentation broth to obtain a composite fermentation broth; adjust the pH of the composite fermentation broth, and spray-dry while stirring to obtain an Enterococcus faecium preparation.

[0046] Control group 3: Take 600 ml of the composition provided in Comparative Example 3 and mix it with 600 ml of Enterococcus faecium fermentation broth to obtain a composite fermentation broth; adjust the pH of the composite fermentation broth, and spray-dry while stirring to obtain an Enterococcus faecium preparation.

[0047] Control group 4: Take 600 ml of water and mix it with 600 ml of Enterococcus faecium fermentation broth, adjust the pH of the fermentation broth, and spray-dry while stirring to obtain an Enterococcus faecium preparation.

[0048] 3. Evaluation of the stress resistance of Enterococcus faecium preparation

[0049] 3.1 Heat and humidity method for heat resistance

[0050] 3.1.1 Detection of the initial number of Enterococcus faecium

[0051] Detect the initial number of bacteria according to the following method, denoted as C0.

[0052] Sampling shall be carried out in accordance with the provisions of GB / T 14699.1. Accurately weigh two parallel samples of 0.3 - 3.0 g and add them to a sterilized and dried 250 mL Erlenmeyer flask (equipped with glass beads); the glass beads cover the bottom of the Erlenmeyer flask; accurately measure 97.0 - 99.7 mL of 0.85% sterilized normal saline (3.3) with a 100 mL sterilized graduated cylinder and pour it into the Erlenmeyer flask. Place the Erlenmeyer flask in a constant temperature vortex mixer and shake it at 170 rpm at room temperature for 30 min (products with ≥100 billion CFU / g shall be shaken for 60 min) to mix the bacterial powder and water evenly to prepare an initial bacterial suspension of 10-2; use a 1 mL pipette equipped with a sterile pipette tip (when the sample particles are large or the bacterial liquid is thick, cut the pipette tip obliquely within about 3 mm and aspirate 0.50 mL), rinse the pipette tip 2 times in the liquid to be aspirated, and then accurately aspirate 0.50 mL of 10 -2 dilution. If there is bacterial liquid hanging on the outer wall of the pipette tip, wipe the pipette tip with sterile paper, noting that the sterile paper should not touch the pipette tip suction port, and add it to a 10 mL plastic centrifuge tube containing 4.50 mL of sterilized normal saline (the pipette tip does not touch the liquid in the centrifuge tube, and the last drop of bacterial liquid should lean against the wall). Mix it evenly on a vortex mixer to form 10 -3 dilution; replace the sterile pipette tip, use a 1 mL pipette equipped with a sterile pipette tip to rinse the pipette tip 2 times in the liquid to be aspirated, and then accurately aspirate 0.50 mL of 10 -3 dilution, add it to a 10 mL plastic centrifuge tube containing 4.50 mL of sterilized normal saline, replace the sterile paper to wipe the bacterial liquid on the outer wall of the pipette tip, noting that the sterile paper should not touch the pipette tip suction port (the pipette tip does not touch the liquid in the centrifuge tube, and the last drop of bacterial liquid should lean against the wall), and mix it evenly on a vortex mixer to form 10 -4 dilution; and so on, continuously dilute to prepare a series of diluted bacterial liquids such as 10 -5 、10 -6 、10 -7 、10 -8 ; The selection of the dilution degree of the bacteria to be tested is determined according to the sample. The more the number of bacteria to be tested contained in the sample, the higher the dilution degree, and vice versa.

[0053] Prepare the solid plate medium in advance, place it in an incubator and incubate it at 37 °C for more than 24 h. Discard those with contaminated bacteria, and leave the blank plates for standby.

[0054] Use a pipette equipped with a 200 μL sterile pipette tip to accurately aspirate 0.10 mL of the diluted bacterial liquid and inoculate it on different numbered plates in accordance with the numbers; use a sterile spatula to spread the bacterial liquid evenly on the solid medium, and use one sterile spatula to scrape 3 culture plates in parallel for each dilution degree; place the plates upside down in a 37 °C constant temperature incubator, stack at most three together, and incubate for 24 - 36 h and count.

[0055] For petri dishes with the number of colonies between 30 and 100, the relative deviation of parallel measurement values at the same gradient shall not exceed 15.0%, and it shall be calculated according to the following formula as the range for the determination of the total number of colonies.

[0056] Relative deviation = (|Measured value - Average value| / Average value) × 100%

[0057] Petri dishes with the number of colonies not between 30 and 100 and relative deviation exceeding 15.0% shall be discarded. Finally, there shall be no less than 3 parallel petri dishes that meet the counting requirements, otherwise redo. The total number of Enterococcus faecium in 1.0 mL or 1.0 g of the sample shall be calculated according to the following formula:

[0058] N = (C × n) / (m × 0.1)

[0059] In the formula, N --- Total viable count of Enterococcus faecium in the sample, unit is CFU;

[0060] C --- Average viable colony count of effective colonies on several effective petri dishes, unit is CFU;

[0061] m --- Sampling amount of the sample, unit is gram, g;

[0062] 0.1 --- Inoculation volume, unit is milliliter, mL;

[0063] n --- Dilution factor.

[0064] Round the calculated result to two significant figures according to the numerical rounding rules of GB / T 8170. The colony count of Enterococcus faecalis in the sample can also be recorded as 1.0 - 9.9 multiplied by the exponential power of 10. The determination results of parallel samples are expressed by the arithmetic mean, and the relative deviation between parallel samples shall not exceed 15.0%.

[0065] 3.1.2 Moist heat treatment

[0066] Weigh 2.0 g of the test sample and spread it evenly in a petri dish (diameter 9 cm), in triplicate, then place it in a perforated test tube basket, and place the test tube basket in a stable constant temperature and humidity chamber (75 °C, relative humidity 95%), accurately time for 5 min, take it out and cool for later use. Transfer all the sample in the petri dish to a conical flask (record the mass), extract it with 100 mL of sterile normal saline for 30 min. Subsequently, detect the number of Enterococcus faecium according to the above method, and record it as C1.

[0067] 3.1.3 Temperature resistance retention rate in the hot and humid box

[0068] K1 = (C1 / C0) × 100%

[0069] In the formula, K1: Temperature resistance retention rate; C0: Original bacterial count; C1: Temperature-resistant bacterial count.

[0070] The detection results of the temperature resistance retention rate of each test group are shown in Table 1.

[0071] 3.2 Evaluation of gastric acid resistance

[0072] 3.2.1 Detection of the initial number of Enterococcus faecalis

[0073] Detect the initial number of bacteria according to the method in 3.1.1, and record it as C0.

[0074] 3.2.2 Gastric acid resistance treatment

[0075] Pepsin: Dissolve pepsin with hydrochloric acid solution at pH 2.5 to prepare a 0.1 mg / mL pepsin solution, sigma7012.

[0076] Weigh 0.2 - 1.0 g of Enterococcus faecalis into a conical flask, extract it with sterile normal saline for 30 min, and control the bacterial concentration at 10 6 CFU / mL.

[0077] Bacterial solution: Pepsin solution = 4:1. Take 8 mL of the test bacterial solution and mix it with 2 mL of pepsin in a 30 mL glass centrifuge tube, and incubate it in a water bath shaker at 37 °C for 1 hour.

[0078] 3.2.3 Determination of the gastric acid-resistant bacterial solution

[0079] After the incubation is completed, cool the bacterial solution to room temperature, dilute it with sterile normal saline, and at the same time adjust the pH value to the normal bacterial solution determination condition. Detect the number of gastric acid-resistant bacteria according to the above method, and record it as C2.

[0080] 3.2.4 Calculation of the gastric acid resistance retention rate:

[0081] K2 = (C2 / C0) × 100%

[0082] In the formula, K2: gastric acid resistance retention rate; C0: initial number of bacteria; C2: number of gastric acid-resistant bacteria

[0083] The detection results of the gastric acid resistance retention rate of each test group are shown in Table 1.

[0084] Table 1 Detection results of the temperature resistance and gastric acid resistance retention rate of Enterococcus faecalis

[0085]

[0086]

[0087] As can be seen from Table 1, after being mixed and treated with the composition provided by the present invention, the survival rates of Enterococcus faecium at 75°C and in gastric acid at pH 2.5 are different. With the increase in the concentration of locust bean gum, the survival rates at high temperature and in gastric acid increase to a certain extent, but the improvement effect is not significant, both within 15%. When the pH of the mixed solution is adjusted to 5.0, the survival rates at high temperature and in gastric acid both increase significantly. When the composition is 12% starch, 0.8% locust bean gum, 1% succinic acid, and 0.05% sodium citrate, the survival rates at high temperature and in gastric acid both reach the highest.

[0088] 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 thermotolerant and acid-resistant Enterococcus faecium preparation, characterized in that, The thermotolerant and acid-resistant Enterococcus faecium preparation is prepared by the following steps: (1) Prepare a composition for improving the stress resistance of Enterococcus faecium; by mass percentage, the composition includes 1% succinic acid, 0.05% sodium citrate, 0.4 - 1.2% locust bean gum, 12% starch, and the balance is water; (2) Mix the composition and the Enterococcus faecium fermentation broth in a volume ratio of 1:1 to obtain a composite fermentation broth; (3) Adjust the pH of the composite fermentation broth, and dry it while stirring to obtain the thermotolerant and acid-resistant Enterococcus faecium preparation.

2. The thermotolerant and acid-resistant Enterococcus faecium preparation according to claim 1, characterized in that: In step (3), the pH of the composite fermentation broth is adjusted to 4.0 - 5.

0.

3. Use of the thermotolerant and acid-resistant Enterococcus faecium preparation according to any one of claims 1 - 2 in feed additives.

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