Preparation method of enzyme preparation microcapsule with intestinal tract slow release function

Abstract

The invention discloses a preparation method of an enzyme preparation microcapsule with an intestinal tract slow release function. According to the method, coating wall materials in a specific proportion are adopted, the coating wall materials comprise 10-12 parts of Arabic gum, 4-8 parts of gelatin, 3-6 parts of whey protein, 8-16 parts of beta-cyclodextrin and 5-10 parts of trehalose, the coating wall materials are mixed with 10-15 parts of an enzyme agent, and the enzyme preparation microcapsule is prepared through a spray drying technology. The enzyme preparation microcapsule has excellent thermal stability and gastric juice tolerance, can maintain more than 90% of enzyme activity under the condition of 90 DEG C, and still maintains more than 65% of enzyme activity after being treated in simulated gastric juice for 4 hours. In addition, the enzyme preparation microcapsule has an intestinal directional slow-release function, the enzyme activity is quickly released after entering the intestinal tract for 15 minutes, and the peak value is reached after the enzyme preparation microcapsule enters the intestinal tract for 60 minutes. The method effectively improves the stability and bioavailability of the enzyme preparation in feed processing and animal digestive tract environment, and has good application prospect and market value.

Description

Technical Field

[0001] The present invention belongs to the field of feed additives, and particularly relates to a preparation method of enzyme preparation microcapsules with intestinal sustained release function. Background Art

[0002] Enzyme preparations have broad application prospects in the feed industry due to their advantages such as safety, effectiveness, almost no residue, no toxic effects on animals, and almost no drug resistance in the body. However, the chemical nature of feed enzyme preparations is protein, which is susceptible to the influence of external environmental factors. During the feed processing process, factors such as temperature, moisture, and pressure will significantly reduce the activity of enzyme preparations, thereby affecting their application effects. In addition, enzyme preparations are expensive and are easily inactivated by factors such as surfactants, heavy metal ions, and pH values, resulting in economic losses in industrial production.

[0003] At present, the existing enzyme preparation protection technologies cannot effectively solve the above problems. Especially in high-temperature processing environments and gastric acid conditions, the activity loss of enzyme preparations is still relatively serious. Therefore, developing an enzyme preparation protection technology with high temperature resistance, gastric acid resistance, and the ability to achieve intestinal targeted release has become the key research direction to solve this industry problem. Summary of the Invention

[0004] Aiming at the problem of easy inactivation of enzyme preparations in the prior art, the present invention provides a preparation method of enzyme preparation microcapsules with intestinal sustained release function. The microcapsules can effectively resist the influence of high-temperature environments and gastric juice digestion, protect the activity of enzyme preparations, and achieve sustained release in the intestine, improving the utilization efficiency of enzyme preparations.

[0005] The technical solution of the present invention is as follows:

[0006] The present invention uses a specific combination of coating wall materials and realizes the encapsulation protection of enzyme preparations through a spray drying microencapsulation process, and endows it with the sustained release characteristics in the intestinal environment.

[0007] A preparation method of enzyme preparation microcapsules with intestinal sustained release function is as follows:

[0008] Step 1: Weigh 15 - 30 g of coating wall material, add 50 - 100 mL of deionized water, and stir at 60°C to fully dissolve it.

[0009] Step 2: Weigh 10 - 20 g of enzyme preparation, add 25 - 50 mL of deionized water, and perform ultrasonic treatment to fully dissolve it.

[0010] Step 3: Centrifuge the dissolved enzyme preparation at 10000 r / min for 3 min to obtain a supernatant solution.

[0011] Step 4: Mix the obtained enzyme supernatant solution with the wall material and stir magnetically for 30 min to obtain a mixture. The obtained solution is immediately spray-dried under the conditions of an inlet air temperature of 120 - 135 °C and a feeding peristaltic pump speed of 100 - 200 r / min to obtain enzyme preparation microcapsules.

[0012] Furthermore, the coating wall material, by mass parts, comprises 10 - 12 parts of gum arabic, 4 - 8 parts of gelatin, 3 - 6 parts of whey protein, 8 - 16 parts of β-cyclodextrin, and 5 - 10 parts of trehalose.

[0013] Furthermore, the enzyme agent is 10 - 15 parts by mass.

[0014] Furthermore, the particle size of the enzyme preparation microcapsules is 150 - 180 μm.

[0015] Furthermore, the enzyme preparation microcapsules can maintain more than 90% of the enzyme activity under the high temperature condition of 90 °C.

[0016] Furthermore, after the enzyme preparation microcapsules are treated in gastric juice for 4 h, the enzyme activity retention rate can still reach more than 65%.

[0017] Furthermore, the enzyme preparation microcapsules rapidly release enzyme activity within 15 min after entering intestinal juice, reach a peak value of 629.45 U / g at 60 min, and then the enzyme activity slightly decreases but still remains at a relatively high level.

[0018] Advantages of the present invention:

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] By microencapsulating the enzyme preparation, the present invention significantly improves its stability and delivery efficiency in high temperature and gastric juice environments. The enzyme preparation microcapsules have good thermal stability and can maintain more than 90% of the enzyme activity under the high temperature condition of 90 °C, with basically no loss of enzyme activity. At the same time, the microcapsule structure can effectively resist the digestion of gastric juice. After being treated in gastric juice for 4 h, the enzyme activity retention rate can still reach more than 65%, which is significantly better than the unembedded enzyme preparation, ensuring that the enzyme preparation can smoothly reach the intestine and achieve effective release, and improving its bioavailability at the target site.

[0021] The product of the present invention has outstanding acid and heat resistance and fully adapts to the high temperature granulation process in feed processing, solving the technical problem that traditional enzyme preparations are easily inactivated in acidic and high temperature environments. By realizing the targeted delivery and stable release of the enzyme preparation, the present invention significantly improves the application efficiency of the enzyme preparation, provides an innovative solution for the feed additive industry, and has good economic benefits and broad market prospects. Brief Description of the Drawings

[0022] Figure 1 For the release of enzyme preparation microcapsules in a simulated intestinal fluid environment. Specific implementation manners

[0023] The present invention will be further described below in conjunction with specific embodiments. The following implementation manners are only for illustrative purposes and do not limit the scope of the present invention. Unless otherwise specified, the reagents used in the present invention are all purchased from biochemical stores; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0024] Example 1: Analysis of the tolerance of enzyme preparation in microcapsules to high temperature

[0025] (1) Preparation of buffer solution

[0026] Weigh 20 g of pancreatin and add it to 100 mL of acetic acid - sodium acetate solution and mix well. Adjust the pH of the solution to 6.02 with acetic acid or sodium acetate to prepare a buffer solution for standby.

[0027] (2) Sample treatment

[0028] Weigh 0.5 g (accurate to 0.0001 g) of unembedded enzyme preparation (control group) and embedded enzyme preparation respectively and add them to different beakers. Add 10 mL of acetic acid - sodium acetate buffer solution, mix well and transfer to a 50 mL volumetric flask. Rinse the beaker three times with acetic acid - sodium acetate buffer solution, add the rinsing solution to the volumetric flask, and finally make up to the scale and shake well for standby.

[0029] (3) High - temperature tolerance test

[0030] Set the water bath to 37°C, 80°C, 85°C, and 90°C respectively. After the water temperature is stable, take 1.5 mL of the solution of the unembedded enzyme preparation group and the embedded enzyme preparation group respectively with a 2 mL centrifuge tube and place them in the corresponding water bath temperature for incubation for 5 min. After the incubation, the unembedded enzyme preparation group directly detects the enzyme activity according to the national standard method. For the embedded enzyme preparation group, take 1 mL of the solution and add it to 9 mL of buffer solution, oscillate and react at 38°C and 180 r / min for 15 min, and then detect the enzyme activity according to the national standard method.

[0031] Table 1 Tolerance of enzyme preparation microcapsules to high temperature

[0032] Note: Compared with the enzyme activity of the unembedded enzyme preparation group and the embedded enzyme preparation group at the same temperature, ** indicates P < 0.01.

[0033] Temperature (°C) 37 80 85 90 Enzyme activity of the unembedded enzyme preparation group (U / g) 786.92±85.42 471.66±43.96 208.20±26.72 0 Enzyme activity of the embedded enzyme preparation group (U / g) 733.63±74.16 715.53±61.75** 707.87±76.12** 687.48±65.14**

[0034] Note: Compared with the enzyme activity of the unembedded enzyme preparation group and the embedded enzyme preparation group at the same temperature, ** indicates P < 0.01.

[0035] In this embodiment, the enzyme preparation treated by the microcapsule embedding technology exhibits remarkable high-temperature tolerance. As shown in the test results in Table 1, in the temperature range of 37 °C to 90 °C, the enzyme activity of the unembedded enzyme preparation decreases significantly with the increase of temperature and is completely inactivated at 90 °C; while the enzyme preparation embedded in microcapsules maintains stable activity under the same temperature conditions and still maintains more than 90% of the enzyme activity even at 90 °C. This indicates that the microcapsule embedding technology can effectively protect the enzyme preparation from the influence of high temperature, improve its thermal stability, and is beneficial to expanding the application of the enzyme preparation in high-temperature industrial environments.

[0036] Example 2 Analysis of the Tolerance of Enzyme Preparation in Microcapsules in a Simulated Gastric Juice Environment

[0037] (1)Preparation of in vitro simulated gastric juice

[0038] Weigh 0.9 g of pepsin and add it to 10 mL of gastric electrolyte solution, and mix well. Use 1 mol / L hydrochloric acid solution to adjust the pH of the solution to 2.94 to obtain the simulated gastric juice.

[0039] (2)Sample treatment

[0040] Take 9 mL of the prepared simulated gastric juice in a beaker and place it in a 37 °C water bath to equilibrate for 10 min. Weigh 0.5 g of the unembedded enzyme preparation and the embedded enzyme preparation (accurate to 0.0001 g) in different beakers respectively, and then add 10 mL of acetic acid-sodium acetate buffer and mix well. Transfer the mixture to a 50 mL volumetric flask, rinse the beaker 3 times with acetic acid-sodium acetate buffer, pour the rinsing solution into the volumetric flask together, and finally make up the volume to the scale with acetic acid-sodium acetate buffer and shake well for standby.

[0041] (3)Simulated gastric juice tolerance test

[0042] Take 1 mL of the unembedded enzyme preparation and the embedded enzyme preparation solution respectively, add them to the above simulated gastric juice, mix well and place them in a constant temperature shaker at 39 °C and 180 r / min for incubation for 4 h. At 0 h, 1 h, 1.5 h and 4 h during the incubation process, take 1 mL of the reaction solution for enzyme activity determination. The samples in the unembedded enzyme preparation group can be directly detected according to the national standard method; the samples in the embedded enzyme preparation group need to add 9 mL of buffer first, oscillate and react for 15 min at 38 °C and 180 r / min, and then determine the enzyme activity according to the national standard method.

[0043] Table 2 Tolerance of Enzyme Preparation Microcapsules in a Simulated Gastric Juice Environment

[0044] Time (h) 0 1 1.5 4 Enzyme activity of the unembedded enzyme preparation group (U / g) 786.92±72.56 0 0 0 Enzyme activity of the embedded enzyme preparation group (U / g) 778.07±59.72 701.52±80.19** 682.36±70.57** 507.62±60.73**

[0045] Note: Compared with the enzyme activity of the unembedded enzyme preparation group and the embedded enzyme preparation group at the same time, ** indicates P < 0.01.

[0046] In this example, the enzyme preparation treated by the microcapsule embedding technology showed significant tolerance in the simulated gastric juice environment. As shown in the test results of Table 2, the unembedded enzyme preparation was completely inactivated in the simulated gastric juice in 1 h; while the enzyme preparation embedded in the microcapsule retained about 90.2%, 87.7% and 65.2% of the enzyme activity after 1 h, 1.5 h and 4 h in the simulated gastric juice respectively. It shows that the microcapsule embedding technology can effectively protect the enzyme preparation from the influence of the acidic environment of gastric juice, significantly improve its stability in the simulated gastric juice, and provide a strong guarantee for the targeted release and function exertion of the enzyme preparation.

[0047] Example 3 Analysis of the release of the enzyme preparation in the microcapsule in the simulated intestinal juice environment

[0048] (1)Preparation of in vitro simulation fluid

[0049] Preparation of in vitro simulated gastric juice: Weigh 0.9 g of pepsin and add it to 10 mL of gastric electrolyte solution, and mix well. Use 1 mol / L hydrochloric acid solution to adjust the pH of the solution to 2.94 to obtain the simulated gastric juice.

[0050] Preparation of in vitro simulated intestinal juice: First, dissolve 5.4 g of sodium chloride, 0.65 g of potassium chloride and 0.33 g of calcium chloride in 1000 mL of water to make intestinal electrolyte solution, then add 20 g of pancreatin to 100 mL of intestinal electrolyte solution, mix well, and adjust the pH value to 6.02 with 1mol / L sodium hydroxide solution for standby.

[0051] (2)Simulated intestinal juice release test

[0052] Weigh 0.1 g of the enzyme preparation microcapsule sample and add it to a preheated test tube (9.9 mL / tube) containing simulated gastric juice. Place the test tube in a constant temperature shaker at 39℃ and 180 r / min and oscillate and incubate for 4 h. After the incubation is over, take 1 mL of the mixed solution after gastric juice incubation from each tube and transfer it to 9 mL of preheated simulated intestinal juice, and continue to incubate for 2 h at the same temperature and rotation speed.

[0053] During the incubation, take 1 tube of sample every 15 min (the corresponding time points are 0, 15, 30, 60 and 120 min), and measure the enzyme activity at each time point according to the national standard method to analyze the release behavior of the microcapsule in the intestinal environment.

[0054] As Figure 1As shown, the enzyme preparation microcapsules rapidly released enzyme activity within 15 minutes after being transferred into simulated intestinal fluid, reached a peak of 629.45 U / g at 60 minutes, and still maintained a relatively high enzyme activity level at 120 minutes. This indicates that the microcapsules have the characteristics of gastrointestinal targeted delivery and can effectively protect the enzyme preparation to release the active ingredient after reaching the intestine through the stomach.

Claims

1. A method for preparing enzyme preparation microcapsules with intestinal sustained release function, characterized in that, It includes the following steps: Step 1: Weigh 15 - 30 g of the coated wall material, add 50 - 100 mL of deionized water, and stir at 60 °C to fully dissolve it. Step 2: Weigh 10 - 20 g of the enzyme agent, add 25 - 50 mL of deionized water, and perform ultrasonic treatment to fully dissolve it. Step 3: Centrifuge the dissolved enzyme agent at 10000 r / min for 3 min to obtain the supernatant solution. Step 4: Mix the obtained enzyme agent supernatant solution with the wall material, and magnetically stir for 30 min to obtain a mixed solution. Immediately spray-dry the obtained solution under the conditions of an inlet air temperature of 120 - 135 °C and a feeding peristaltic pump speed of 100 - 200 r / min to obtain enzyme preparation microcapsules.

2. The preparation method of an enzyme preparation microcapsule with an intestinal sustained release function according to claim 1, wherein, The coated wall material, by mass fraction, includes 10 - 12 parts of arabic gum, 4 - 8 parts of gelatin, 3 - 6 parts of whey protein, 8 - 16 parts of β-cyclodextrin, and 5 - 10 parts of trehalose.

3. The preparation method of an enzyme preparation microcapsule with an intestinal sustained release function according to claim 1, wherein The enzyme agent is 10 - 15 parts by mass fraction.

4. The enzyme preparation microcapsules prepared by the method according to any one of claims 1 - 3.

5. The enzyme preparation microcapsule according to claim 4, wherein The particle size of the enzyme preparation microcapsules is 150 - 180 μm.

6. The enzyme preparation microcapsule according to claim 4, wherein The enzyme preparation microcapsules maintain more than 90% enzyme activity at a high temperature of 90 °C.

7. The enzyme preparation microcapsule according to claim 4, wherein After being treated in gastric juice for 4 h, the enzyme activity retention rate of the enzyme preparation microcapsules reaches more than 65%.

8. The enzyme preparation microcapsule according to claim 4, characterized in that, The enzyme preparation microcapsules have an intestinal sustained-release function.

9. The application of an enzyme preparation microcapsule as described in claim 4 in the field of feed processing.

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