A method for preparing a gastric transit-stable enzyme preparation

Abstract

The application belongs to the technical field of enzyme preparation processing, and specifically provides a preparation method of a gastric-stable enzyme preparation, which comprises the following steps: (1) placing enzyme liquid in a stirrer, adding stabilizers and carriers into the enzyme liquid, uniformly stirring, then adding emulsifiers and emulsion stabilizers, uniformly stirring again, obtaining enzyme mixture, and heating and keeping warm; (2) heating hot-melt type packaging materials to completely melt into liquid state, and keeping the packaging materials in liquid state; (3) starting the stirrer, slowly pouring the liquid hot-melt type packaging materials into the stirrer while stirring, and obtaining a material liquid; (4) spray drying the material liquid, and obtaining the gastric-stable enzyme preparation. The preparation method provided by the application effectively avoids the destruction of the prepared enzyme preparation by pepsin after the enzyme preparation enters the stomach of an animal, improves the enzyme activity retention rate of the enzyme preparation product after passing through the stomach, and further improves the stability of the enzyme preparation in the storage process, especially the storage stability in a humid environment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of enzyme preparation, and particularly relates to a preparation method of a gastric-stable enzyme preparation. BACKGROUND

[0002] Feed enzyme preparation includes digestive enzymes, non-starch polysaccharide enzymes and other functional enzyme preparations. The digestive enzymes mainly refer to protease, amylase and lipase, etc., the non-starch polysaccharide enzymes mainly refer to cellulase, hemicellulase, phytase, pectinase, etc., in addition, there are glucose oxidase, lysozyme and other functional enzyme preparations. According to different types and different physiological stages of livestock and poultry and the characteristics of specific feed raw materials, the required enzyme preparation is added into the feed, so that the utilization rate of the feed by the livestock and poultry is improved, the use of non-conventional feed resources is expanded, and the feed cost is reduced.

[0003] As known, the essence of enzyme is a protein, when it enters the stomach of the animal together with the feed, due to the strong acidic environment of the stomach, the activity of the enzyme is seriously lost, and at the same time, due to the existence of a large amount of pepsin in the stomach of the animal, other enzyme proteins are decomposed to cause further inactivation of the enzyme. Therefore, in order to ensure that the enzyme added in the feed can play a role, the feed enzyme preparation is usually an enzyme preparation with acid resistance selected, so as to ensure that the enzyme protein entering the acidic environment of the stomach will not be denatured and inactivated due to pH. However, in order to ensure that the enzyme protein of the feed enzyme added in the feed is not destroyed by pepsin, the protease needs to be treated, and the coating technology is usually used for the treatment. The enzyme preparation is usually coated by mixing the dried powdery enzyme preparation with a carrier, a binder and the like, and then granulated by a granulating device to obtain a pill core, and finally, the pill core is coated with a wall material by a coating device, such as the patent CN105265756B which is realized by using the above process. The process steps required by this process are relatively more, and relatively speaking, the process is relatively complex, and the intermediate steps are more likely to cause loss of enzyme activity. SUMMARY

[0004] The purpose of the present application is to provide a method for directly preparing a gastric-stable enzyme preparation microcapsule product from enzyme preparation fermentation liquor.

[0005] To this end, the present application provides a preparation method of a gastric-stable enzyme preparation, comprising the following steps:

[0006] (1) placing the enzyme liquor in a stirrer, adding a stabilizer and a carrier into the enzyme liquor, stirring uniformly, then adding an emulsifier and an emulsion stabilizer, stirring uniformly again, obtaining an enzyme mixture, and heating and keeping warm;

[0007] (2) heating the hot-melt type coating material to completely melt into a liquid state, and keeping the coating material in a liquid state;

[0008] (3) Start the blender, and slowly pour the liquid hot melt type coating material into the blender while stirring, to obtain a material liquid;

[0009] (4) Spray dry the material liquid to obtain a gastric-stable enzyme preparation.

[0010] Specifically, the enzyme protein in the enzyme liquid has certain temperature resistance, and the enzyme activity retention rate is > 90% after water bath at 60℃ for 1h.

[0011] Specifically, the amount of the stabilizer added in step (1) is 2-5% by mass of the enzyme liquid, the amount of the carrier added is 15-25%, the amount of the emulsifier added is 2-5%, and the amount of the emulsification stabilizer added is 0.2-2%.

[0012] Specifically, the mass ratio of the hot melt type coating material to the enzyme liquid is 1:(5-20).

[0013] Specifically, the hot melt type coating material is one of long-chain fatty acids, fatty acid esters, hydrogenated oil, and plant wax.

[0014] Specifically, the stabilizer includes glycerol, sorbitol, aspartic acid, mannitol, and vitamin E, and the mass ratio of the components is preferably (10-30):(5-10):(1-10):(1-5):(5-10).

[0015] Specifically, the carrier includes corn starch and malt dextrin, and the mass ratio of the components is preferably (5-8):(1-4).

[0016] Specifically, the emulsifier is a water-in-oil type emulsifier, and the HLB value is ≤ 6.0.

[0017] Specifically, the emulsifier includes sorbitan fatty acid ester, sucrose fatty acid ester, and glycerol monostearate, and the mass ratio of the components is preferably (0-5):(1-5):(4-10).

[0018] Specifically, the emulsification stabilizer includes guar gum, xanthan gum, casein, and carboxymethyl cellulose, and the mass ratio of the components is preferably (1-5):(0-3):(1-5):(1-2).

[0019] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0020] The method for preparing a gastric-stable enzyme preparation provided by this invention involves directly preparing a gastric-stable microencapsulated enzyme preparation from enzyme fermentation broth. This effectively avoids the enzyme preparation being destroyed by pepsin after entering the animal's stomach, improving the retention rate of enzyme activity after the feed enzyme preparation passes through the animal's stomach and ensuring that the product can exert its intended effect. Furthermore, the microencapsulation coating method improves the stability of the enzyme preparation during storage, especially its stability under humid conditions. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Although representative embodiments of the present invention have been described in detail, those skilled in the art 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.

[0022] This invention provides a method for preparing a gastric-stable enzyme preparation, comprising the following steps:

[0023] (1) Place the filtered, sterilized and concentrated enzyme solution in a storage tank equipped with a high-speed shear mixer. The enzyme protein in the enzyme solution has a certain temperature resistance. The enzyme activity retention rate is >90% after 1 hour in a water bath at 60℃. Based on the mass of the enzyme solution, add 2-5% stabilizer and 15-25% carrier to the enzyme solution. After stirring at low speed until uniform, add 2-5% emulsifier and 0.2-2% emulsion stabilizer. Stir at low speed again until uniform to obtain an enzyme mixture. Heat the mixture to 50-60℃ and keep it warm.

[0024] The stabilizers include glycerol, sorbitol, aspartic acid, mannitol and vitamin E, with a mass ratio of (10-30):(5-10):(1-10):(1-5):(5-10).

[0025] The carrier consists of corn starch and maltodextrin, with a mass ratio of (5-8):(1-4).

[0026] The emulsifier is a water-in-oil emulsifier, that is, the HLB value of the emulsifier is ≤6.0. The emulsifier includes sorbitan fatty acid ester, sucrose fatty acid ester, and glyceryl monostearate, and the mass ratio of each component is (0-5):(1-5):(4-10); preferably, the emulsifier is composed of sucrose fatty acid ester and glyceryl monostearate in a mass ratio of 1:3.

[0027] The emulsifying stabilizer includes guar gum, xanthan gum, casein, and carboxymethyl cellulose, with the mass ratio of each component being (1-5):(0-3):(1-5):(1-2); preferably, the emulsifying stabilizer is composed of guar gum, casein, and carboxymethyl cellulose in a mass ratio of 2:2:1.

[0028] (2) Heat the hot melt packaging material until it is completely melted into a liquid state, and then cool it down until the packaging material remains in a liquid state; the mass ratio of the hot melt packaging material to the enzyme solution is 1:(5-20), preferably 1:(5-10);

[0029] The hot-melt packaging material is selected from one of the following: long-chain fatty acids, fatty acid esters, hydrogenated oils, and vegetable waxes, with a melting temperature of 50-80℃; furthermore, the hot-melt packaging material is one of the following: stearic acid, glyceryl stearate, hydrogenated palm oil, and palm wax.

[0030] (3) Turn on the high-speed shear mixer and adjust the speed to 1500-3000 rpm. While stirring, slowly pour the liquid hot melt packaging material into the mixer. After the hot melt packaging material is completely poured in, continue shearing and stirring for 15-30 minutes to obtain the liquid material. Throughout the process, keep the liquid temperature in the mixer tank at 50-60℃.

[0031] (4) Turn on the spray dryer to spray dry the liquid material to obtain a gastric-stable enzyme preparation. The spray dryer tower wall and the receiving section are equipped with a cooling system, which can quickly reduce the outlet material temperature to room temperature.

[0032] The effects of the method for preparing the gastric-stable enzyme preparation of the present invention are studied below through specific embodiments. In the embodiments, the enzymes used all exhibited an enzyme activity retention rate greater than 90% after being in a water bath at 60°C for 1 hour.

[0033] Example 1:

[0034] This embodiment provides a method for preparing a gastric-stable enzyme preparation, including the following steps:

[0035] (1) Place 200kg of filtered, sterilized and concentrated enzyme solution into a storage tank equipped with a high-speed shear mixer. Add 2% stabilizer and 15% carrier by weight of enzyme solution. Stir at low speed until uniform. Then add 2% emulsifier and 0.2% emulsion stabilizer. Stir at low speed until uniform to obtain enzyme mixture. Heat the mixture to 50°C and keep it warm.

[0036] The stabilizer is composed of glycerol, sorbitol, aspartic acid, mannitol and vitamin E, with a mass ratio of 10:5:1:1:5.

[0037] The carrier is composed of corn starch and maltodextrin, with a mass ratio of 5:1.

[0038] The emulsifier is composed of sucrose fatty acid ester and glyceryl monostearate, with a mass ratio of 1:4.

[0039] The emulsifying stabilizer is composed of guar gum, casein, and carboxymethyl cellulose, with a mass ratio of 1:1:1.

[0040] (2) Heat 10 kg of glyceryl stearate in the packaging material until it is completely melted into a liquid state, and then cool it down to 60°C. The packaging material remains in a liquid state.

[0041] (3) Adjust the speed of the high-speed shear mixer to 1500 rpm, slowly pour the liquid packaging material into the storage tank, and continue shearing and mixing for 15 minutes after the packaging material has been completely poured in to obtain the liquid to be spray dried. Keep the liquid temperature in the storage tank at 50°C throughout the process.

[0042] (4) Turn on the spray dryer to dry the liquid in step (3) to obtain a gastric stable enzyme preparation.

[0043] In this embodiment, the above-described method was used to prepare gastric-stable cellulase preparations, gastric-stable xylanase preparations, gastric-stable mannanase preparations, and gastric-stable phytase preparations, respectively, using cellulase, xylanase, mannanase, and phytase as initial enzyme solutions.

[0044] Example 2:

[0045] This embodiment provides a method for preparing a gastric-stable enzyme preparation, including the following steps:

[0046] (1) Place 200kg of filtered, sterilized and concentrated enzyme solution into a storage tank equipped with a high-speed shear mixer. Add 5% stabilizer and 25% carrier by weight of enzyme solution. Stir at low speed until uniform. Then add 5% emulsifier and 2% emulsion stabilizer. Stir at low speed until uniform. Heat the solution to 60℃ and keep it warm.

[0047] The stabilizer is composed of glycerol, sorbitol, aspartic acid, mannitol and vitamin E, with a mass ratio of 30:10:10:5:10.

[0048] The carrier is composed of corn starch and maltodextrin, with a mass ratio of 8:4.

[0049] The emulsifier is composed of sorbitan fatty acid ester, sucrose fatty acid ester, and glyceryl monostearate, with a mass ratio of 5:5:10.

[0050] The emulsifying stabilizer is composed of guar gum, xanthan gum, casein, and carboxymethyl cellulose, with a mass ratio of 5:3:5:2.

[0051] (2) Heat 40 kg of stearic acid for the packaging material until it is completely melted into a liquid state, and then cool it down to 60°C. The packaging material remains in a liquid state.

[0052] (3) Adjust the speed of the high-speed shear mixer to 3000 rpm, slowly pour the liquid packaging material into the storage tank, and continue shearing and mixing for 30 minutes after the packaging material is completely poured in to obtain the liquid to be spray dried. Keep the liquid temperature in the storage tank at 60℃ throughout the process.

[0053] (4) Turn on the spray dryer to dry the liquid in step (3) to obtain a gastric stable enzyme preparation.

[0054] In this embodiment, the above-described method was used to prepare gastric-stable cellulase preparations, gastric-stable xylanase preparations, gastric-stable mannanase preparations, and gastric-stable phytase preparations, respectively, using cellulase, xylanase, mannanase, and phytase as initial enzyme solutions.

[0055] Example 3:

[0056] This embodiment provides a method for preparing a gastric-stable enzyme preparation, including the following steps:

[0057] (1) Place 200 kg of filtered, sterilized and concentrated enzyme solution into a storage tank equipped with a high-speed shear mixer. Add 2% stabilizer and 20% carrier by weight of enzyme solution. Stir at low speed until uniform. Then add 3% emulsifier and 1% emulsion stabilizer. Stir at low speed until uniform. Heat the solution to 50°C and keep it warm.

[0058] The stabilizer is composed of glycerol, sorbitol, aspartic acid, mannitol and vitamin E, with a mass ratio of 20:5:5:1:5.

[0059] The carrier is composed of corn starch and maltodextrin, with a mass ratio of 5:2.

[0060] The emulsifier is composed of sorbitan fatty acid ester, sucrose fatty acid ester, and glyceryl monostearate, with a mass ratio of 1:2:5.

[0061] The emulsifying stabilizer is composed of guar gum, xanthan gum, casein, and carboxymethyl cellulose, with a mass ratio of 2:1:2:1.

[0062] (2) Heat 20 kg of hydrogenated palm oil for packaging until it is completely melted into a liquid state, and then cool it down to 60°C. The packaging material remains in a liquid state.

[0063] (3) Adjust the speed of the high-speed shear mixer to 2000 rpm, slowly pour the liquid packaging material into the storage tank, and continue shearing and mixing for 20 minutes after the packaging material has been completely poured in to obtain the liquid to be spray dried. Keep the liquid temperature in the storage tank at 55℃ throughout the process.

[0064] (4) Turn on the spray dryer to dry the liquid in step (3) to obtain a gastric stable enzyme preparation.

[0065] In this embodiment, the above-described method was used to prepare gastric-stable cellulase preparations, gastric-stable xylanase preparations, gastric-stable mannanase preparations, and gastric-stable phytase preparations, respectively, using cellulase, xylanase, mannanase, and phytase as initial enzyme solutions.

[0066] Example 4:

[0067] This embodiment provides a method for preparing a gastric-stable enzyme preparation, including the following steps:

[0068] (1) Place 200 kg of filtered, sterilized and concentrated enzyme solution into a storage tank equipped with a high-speed shear mixer. Add 2.5% stabilizer and 20% carrier by weight of enzyme solution. Stir at low speed until uniform. Then add 2.5% emulsifier and 1.5% emulsion stabilizer. Stir at low speed until uniform. Heat the solution to 55°C and keep it warm.

[0069] The stabilizer is composed of glycerol, sorbitol, aspartic acid, mannitol and vitamin E, with a mass ratio of 15:5:1:1:10.

[0070] The carrier is composed of corn starch and maltodextrin, with a mass ratio of 6:2.

[0071] The emulsifier is composed of sucrose fatty acid ester and glyceryl monostearate, with a mass ratio of 1:3.

[0072] The emulsifying stabilizer is composed of guar gum, casein, and carboxymethyl cellulose, with a mass ratio of 2:2:1.

[0073] (2) Heat 30 kg of glyceryl stearate in the packaging material until it is completely melted into a liquid state, and then cool it down to 60°C. The packaging material remains in a liquid state.

[0074] (3) Adjust the speed of the high-speed shear mixer to 2500 rpm, slowly pour the liquid packaging material into the storage tank, and continue shearing and mixing for 25 minutes after the packaging material is completely poured in to obtain the liquid to be spray dried. Keep the liquid temperature in the storage tank at 57℃ throughout the process.

[0075] (4) Turn on the spray dryer to dry the liquid material in step (3).

[0076] In this embodiment, the above-described method was used to prepare gastric-stable cellulase preparations, gastric-stable xylanase preparations, gastric-stable mannanase preparations, and gastric-stable phytase preparations, respectively, using cellulase, xylanase, mannanase, and phytase as initial enzyme solutions.

[0077] Comparative Example 1:

[0078] This comparative example provides a method for preparing an enzyme preparation, which differs from Example 3 in that no emulsifier and emulsion stabilizer are added in step (1).

[0079] In this comparative example, the cellulase preparation, xylanase preparation, mannanase preparation, and phytase preparation were prepared using the above method with cellulase, xylanase, mannanase, and phytase as initial enzyme solutions, respectively.

[0080] Example 5:

[0081] In this embodiment, the enzyme preparation prepared by direct loading and spray drying of raw enzyme solution was used as a control sample to study the storage stability and sustained-release properties of the enzyme preparations and raw enzyme solution prepared in Examples 1-4 and Comparative Example 1.

[0082] 1. Comparison of storage stability

[0083] The enzyme preparations prepared in Examples 1-4 and Comparative Example 1, along with the control sample, were placed at 45°C and 85% humidity for 3 months. Samples were taken at 0, 1, 2, and 3 months to detect enzyme activity and calculate enzyme activity retention rate. The results are shown in Table 1.

[0084] Table 1 Enzyme activity and retention rate at different storage times

[0085]

[0086]

[0087] The data in the table show that the storage stability of various enzyme activities in the samples prepared using the method provided by this invention is significantly improved compared to samples dried using conventional methods. However, the results of Comparative Example 1 show that, although no emulsification step was performed during the preparation process, the storage stability of the enzyme preparation was improved compared to samples dried using conventional methods, but significantly lower than that of the enzyme preparation prepared in the examples of this invention.

[0088] 2. Comparison of sustained-release properties

[0089] The enzyme preparations prepared in Examples 1-4 and Comparative Example 1, along with the control sample, were subjected to in vitro simulated digestion experiments. After digestion in a gastric environment (pH 2.0, containing pepsin) for 2 hours, the undigested samples were filtered through filter paper, dried at low temperature, and then the enzyme activity was measured. The enzyme activity before and after gastric digestion was compared to obtain the gastric retention rate of the samples, and the results are shown in Table 2.

[0090] Table 2. Enzyme activity retention rate after gastric passage.

[0091]

[0092] The data in the table show that after treatment in a simulated gastric environment, most of the enzyme activity of the samples prepared using the method provided by this invention is effectively preserved. This avoids the destruction of these enzymes in the stomach, ensuring that most enzyme preparations can smoothly enter the intestinal stage and function better. The results of Comparative Example 1 show that, without an emulsification step during preparation, the gastric retention rate of the enzyme preparation is higher than that of samples dried using conventional methods, but significantly lower than that of the enzyme preparations prepared in the embodiments of this invention.

[0093] The above examples are merely illustrative of the present invention and do not constitute a limitation on the scope of protection of the present invention. All designs that are the same as or similar to the present invention are within the scope of protection of the present invention.

Claims

1. A method for preparing a gastric-stable enzyme preparation, characterized in that, Includes the following steps: (1) Place the enzyme solution in a mixer, add stabilizer and carrier to the enzyme solution, stir evenly, add emulsifier and emulsion stabilizer, stir evenly again to obtain enzyme mixture, heat and keep warm; the emulsifier includes sorbitan fatty acid ester, sucrose fatty acid ester, and glyceryl monostearate; the emulsion stabilizer includes guar gum, xanthan gum, casein, and carboxymethyl cellulose; (2) Heat the hot melt packaging material until it is completely melted into a liquid state, and keep the packaging material in a liquid state; (3) Turn on the mixer and slowly pour the liquid hot melt packaging material into the mixer while stirring to obtain the liquid material; (4) Spray dry the liquid to obtain a gastric-stable enzyme preparation.

2. The method for preparing the gastric-stable enzyme preparation as described in claim 1, characterized in that: The enzyme protein in the enzyme solution has an enzyme activity retention rate of >90% after being bathed in a water bath at 60°C for 1 hour.

3. The method for preparing the gastric-stable enzyme preparation as described in claim 1, characterized in that: Based on the mass of the enzyme solution, the amount of stabilizer added in step (1) is 2-5%, the amount of carrier added is 15-25%, the amount of emulsifier added is 2-5%, and the amount of emulsion stabilizer added is 0.2-2%.

4. The method for preparing the gastric-stable enzyme preparation as described in claim 1, characterized in that: The mass ratio of the hot-melt packaging material to the enzyme solution is 1:(5-20).

5. The method for preparing the gastric-stable enzyme preparation as described in claim 1, characterized in that: The hot-melt packaging material is one of long-chain fatty acids, fatty acid esters, hydrogenated oils, and vegetable waxes.

6. The method for preparing the gastric-stable enzyme preparation as described in claim 1, characterized in that: The stabilizers include glycerol, sorbitol, aspartic acid, mannitol, and vitamin E.

7. The method for preparing the gastric-stable enzyme preparation as described in claim 1, characterized in that: The carrier includes corn starch and maltodextrin.

8. A gastric-stable enzyme preparation prepared by the preparation method according to any one of claims 1-7.

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