A processing method capable of improving the heat and acid resistance of enzyme preparations and its application

Abstract

The present invention belongs to the technical field of enzyme preparations for feed, and discloses a processing method and application for improving the heat and acid resistance of enzyme preparations. The processing method of the present invention is as follows: the biofilm material is melted, stirred evenly and ultrasonicated to obtain a liposome film; an enzyme preparation and an emulsifier are added to the liposome film, fully mixed and ultrasonicated to obtain an enzyme preparation-liposome mixed solution; the temperature of the obtained enzyme preparation-liposome mixed solution is suddenly reduced to room temperature, and high-pressure centrifugal spray is performed to obtain a powdered enzyme preparation-liposome. The present invention utilizes a method for encapsulating the enzyme preparation with unilamellar liposomes to ensure that the enzyme preparation is isolated from the external environment, prevents it from oxidative deterioration, prolongs its active time, can improve the adaptability of the enzyme preparation to the environment, enhances the tolerance of the enzyme preparation to temperature and gastric acid, and is suitable for the processing and production of enzyme preparations for feed.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme preparations for feed, and in particular to a processing method and application thereof capable of improving the heat and acid resistance of enzyme preparations. Background Art

[0002] Enzyme preparations, as biologically active natural catalysts, bind to substrates, lowering the activation energy required for reactions and catalyzing the breakdown of macromolecules (such as proteins, fats, and polysaccharides) into easily absorbed smaller molecules, such as amino acids, oligopeptides, fatty acids, and glucose. Based on their mode of action, they can be divided into two categories: exogenous digestive enzymes and non-digestive enzymes. Non-digestive enzymes are enzymes that are not naturally secreted into the digestive tract by animals. These enzymes can digest indigestible substances or degrade anti-nutritional factors, primarily including cellulases, xylanases, β-glucanases, and phytases. Exogenous digestive enzymes are amylases, proteases, and lipases that are naturally secreted by animals but are mostly derived from microorganisms and plants. As new, highly effective feed additives, enzyme preparations provide an effective means for developing new feed ingredients and reducing feed production costs. They can also improve animal performance and reduce waste pollution, ensuring and enabling sustainable development of the feed and animal husbandry industries, including high efficiency, grain conservation, and environmental protection.

[0003] However, since enzyme preparations are usually very sensitive to conditions such as strong acids, strong alkalis, high temperatures and organic solvents, and are easily inactivated, their further research and development and promotion and application are hindered. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a processing method and application that can improve the heat and acid resistance of enzyme preparations.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a processing method for improving the heat and acid resistance of an enzyme preparation, comprising the following steps:

[0007] (1) Melting the biomembrane material, stirring it evenly, and then ultrasonicating it to obtain a liposome membrane;

[0008] (2) adding an enzyme preparation and an emulsifier to the liposome membrane, mixing thoroughly, and then ultrasonicating to obtain an enzyme preparation-liposome mixture;

[0009] (3) The temperature of the obtained enzyme preparation-liposome mixture is suddenly reduced to room temperature, and high-pressure centrifugal spray is performed to obtain a powdered enzyme preparation-liposome.

[0010] The present invention utilizes a method of encapsulating the enzyme preparation in a single-layer liposome, which has good fusion and sustained-release properties with the cell membrane, ensures that the enzyme preparation is isolated from the external environment, prevents it from oxidative deterioration, prolongs its activity time, improves the adaptability of the enzyme preparation to the environment, enhances the enzyme preparation's tolerance to temperature and gastric acid, improves the stability of the enzyme preparation, and improves the effect of the enzyme preparation.

[0011] As a preferred embodiment of the processing method for improving the heat and acid resistance of the enzyme preparation described in the present invention, in the step (1), the biofilm material includes 50%-68% palmitic acid glyceryl ester, 30%-45% stearic acid glyceryl ester and 2%-5% cholesterol in terms of mass percentage.

[0012] As a preferred embodiment of the processing method for improving the heat and acid resistance of enzyme preparations according to the present invention, in the step (1), the melting temperature is 45°C-65°C.

[0013] As a preferred embodiment of the processing method for improving the heat and acid resistance of the enzyme preparation described in the present invention, in the step (1), the stirring speed is 5000r / min-12000r / min, and the time is 5min-10min; the ultrasonic frequency is 30000Hz-40000Hz, and the ultrasonic time is 5min-10min.

[0014] As a preferred embodiment of the processing method for improving the heat and acid resistance of the enzyme preparation described in the present invention, in the step (2), the enzyme preparation is at least one of protease, xylanase, β-glucanase, β-mannanase, α-galactosidase, and amylase.

[0015] As a preferred embodiment of the processing method for improving the heat and acid resistance of the enzyme preparation according to the present invention, in the step (2), the mass ratio of the enzyme preparation to the emulsifier is 1:(5-10); the mass ratio of the enzyme preparation to the liposome is 1:(1-3).

[0016] As a preferred embodiment of the processing method for improving the heat and acid resistance of enzyme preparations according to the present invention, in step (2), the ultrasonic frequency is 30,000 Hz-40,000 Hz, and the ultrasonic time is 5 min-10 min.

[0017] As a preferred embodiment of the processing method for improving the heat and acid resistance of enzyme preparations according to the present invention, in step (3), the pressure of the high-pressure centrifugal spray is 0.4Mpa-0.7Mpa, and the rotation speed is 15000r / min-25000r / min.

[0018] In a second aspect, the present invention provides an enzyme preparation-liposome prepared by the processing method capable of improving the heat and acid resistance of the enzyme preparation.

[0019] As a preferred embodiment of the enzyme preparation-liposome of the present invention, the diameter of the enzyme preparation-liposome is 100 nm-500 nm.

[0020] In a third aspect, the present invention applies the processing method and the enzyme preparation-liposome in feed processing.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The processing method of the present invention adopts single-layer liposomes to encapsulate the enzyme preparation, does not require the use of organic solvents, does not require the use of an aqueous phase, and avoids the influence of organic solvents on the enzyme preparation. The enzyme preparation-liposome prepared by the method can better ensure that the enzyme preparation is isolated from the external environment, protect it from the influence of external environmental conditions, prevent it from oxidative deterioration, prolong its activity time, improve its adaptability to the environment, enhance its temperature tolerance, improve the stability of the enzyme preparation, and have high fusion with cell membranes, thereby improving its action effect. The enzyme preparation-liposome prepared by the method is suitable for the processing and production of enzyme preparations for feed. DETAILED DESCRIPTION

[0023] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0024] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are all commercially available unless otherwise specified. The emulsifier used is Tween 80.

[0025] Example 1: A processing method for improving the heat and acid resistance of enzyme preparations

[0026] The processing method comprises the following steps:

[0027] (1) The biomembrane material is melted at 45°C, stirred and then ultrasonicated to obtain a single-layer liposome membrane;

[0028] The biofilm material comprises palmitic acid glyceryl, stearic acid glyceryl and cholesterol, wherein the mass ratio of palmitic acid glyceryl, stearic acid glyceryl and cholesterol is 50%, 45% and 5% respectively;

[0029] The stirring speed is 5000 r / min, and the stirring time is 5 min; the ultrasonic frequency is 40000 Hz and the ultrasonic time is 5 min;

[0030] (2) adding the enzyme preparation and the emulsifier to the liposome membrane, mixing thoroughly and then sonicating to obtain an enzyme preparation-liposome mixture;

[0031] The mass ratio of the enzyme preparation to the emulsifier is 1:5; the mass ratio of the enzyme preparation to the liposome is 1:1;

[0032] The ultrasonic frequency is 40000 Hz and the ultrasonic time is 5 min;

[0033] The enzyme preparation is β-glucanase;

[0034] (3) using liquid nitrogen to flash freeze the enzyme preparation-liposome mixture to reduce the temperature to room temperature, and then spraying it through high-pressure centrifugation to obtain a powdered enzyme preparation-liposome;

[0035] The pressure of the high-pressure centrifugal spray is 0.4 MPa and the rotation speed is 15000 r / min;

[0036] The powdered enzyme preparation-liposome diameter was detected to be 500 nm;

[0037] (4) The activity of the powdered enzyme preparation - liposome enzyme was determined by gastric and wet heat methods.

[0038] The wet heat method is to add 100g of powdered enzyme preparation-liposome to 100g of corn starch containing 20% moisture to make granules, and the original enzyme activity is measured to be 70,000u / g. Then, it is stored in a high temperature environment of 100°C for 5 minutes, and the enzyme activity is measured to be 65,800u / g (a decrease of 6%). The gastric test is to treat the enzyme preparation-liposome in simulated gastric fluid with a pH value of 3 for 2 hours, and the enzyme activity is measured to be 66,500u / g (a decrease of 5%).

[0039] Example 2: A processing method for improving the heat and acid resistance of enzyme preparations

[0040] The processing method comprises the following steps:

[0041] (1) The biofilm material is melted at 55°C, stirred and then ultrasonicated to obtain a single-layer liposome membrane;

[0042] The biofilm material comprises palmitic acid glyceryl, stearic acid glyceryl and cholesterol, wherein the mass ratio of palmitic acid glyceryl, stearic acid glyceryl and cholesterol is 60%, 35% and 5% respectively;

[0043] The stirring speed is 9000 r / min, and the stirring time is 10 min; the ultrasonic frequency is 40000 Hz and the ultrasonic time is 10 min;

[0044] (2) adding the enzyme preparation and the emulsifier to the liposome membrane, mixing thoroughly and then sonicating to obtain an enzyme preparation-liposome mixture;

[0045] The mass ratio of the enzyme preparation to the emulsifier is 1:5; the mass ratio of the enzyme preparation to the liposome is 1:2;

[0046] The ultrasonic frequency is 40000 Hz and the ultrasonic time is 5 min;

[0047] The enzyme preparation is a protease;

[0048] (3) using liquid nitrogen to flash freeze the enzyme preparation-liposome mixture to reduce the temperature to room temperature, and then spraying it through high-pressure centrifugation to obtain a powdered enzyme preparation-liposome;

[0049] The pressure of the high-pressure centrifugal spray is 0.5 MPa and the rotation speed is 18000 r / min;

[0050] The powdered enzyme preparation-liposome diameter was detected to be 300 nm;

[0051] (4) The activity of the powdered enzyme preparation - liposome enzyme was determined by gastric and wet heat methods.

[0052] The wet heat method is to add 100g of powdered enzyme preparation-liposome to 100g of corn starch containing 20% moisture to make granules, and the original enzyme activity is measured to be 82000u / g, and then stored in a high temperature environment of 110°C for 5 minutes, and the enzyme activity is measured to be 75400u / g (decreased by 8%). The gastric test is to treat the enzyme preparation-liposome in simulated gastric fluid with a pH value of 3 for 2h, and the enzyme activity is measured to be 77900u / g (decreased by 5%).

[0053] Example 3: A processing method for improving the heat and acid resistance of enzyme preparations

[0054] The processing method comprises the following steps:

[0055] (1) The biofilm material is melted at 65°C, stirred and then ultrasonicated to obtain a single-layer liposome membrane;

[0056] The biofilm material comprises palmitic acid glyceryl, stearic acid glyceryl and cholesterol, wherein the mass ratio of palmitic acid glyceryl, stearic acid glyceryl and cholesterol is 65%, 30% and 5% respectively;

[0057] The stirring speed is 12000 r / min, and the stirring time is 10 min; the ultrasonic frequency is 40000 Hz and the ultrasonic time is 10 min;

[0058] (2) adding the enzyme preparation and the emulsifier to the liposome membrane, mixing thoroughly and then sonicating to obtain an enzyme preparation-liposome mixture;

[0059] The mass ratio of the enzyme preparation to the emulsifier is 1:10; the mass ratio of the enzyme preparation to the liposome is 1:2;

[0060] The ultrasonic frequency is 40000 Hz and the ultrasonic time is 5 min;

[0061] The enzyme preparation is amylase;

[0062] (3) using liquid nitrogen to flash freeze the enzyme preparation-liposome mixture to reduce the temperature to room temperature, and then spraying it through high-pressure centrifugation to obtain a powdered enzyme preparation-liposome;

[0063] The pressure of the high-pressure centrifugal spray is 0.7 MPa and the rotation speed is 25000 r / min;

[0064] The powdered enzyme preparation-liposome diameter was detected to be 100 nm;

[0065] (4) The activity of the powdered enzyme preparation - liposome enzyme was determined by gastric and wet heat methods.

[0066] The wet heat method is to add 100g of powdered enzyme preparation-liposome to 100g of corn starch containing 20% moisture to make granules, and the original enzyme activity is measured to be 90,000u / g. Then, it is stored in a high temperature environment of 120°C for 5 minutes, and the enzyme activity is measured to be 85,500u / g (a decrease of 5%). The gastric test is to treat the enzyme preparation-liposome in simulated gastric fluid with a pH value of 3 for 2 hours, and the enzyme activity is measured to be 84,600u / g (a decrease of 6%).

[0067] Example 4: A processing method for improving the heat and acid resistance of enzyme preparations

[0068] The processing method comprises the following steps:

[0069] (1) The biofilm material is melted at 65°C, stirred and then ultrasonicated to obtain a single-layer liposome membrane;

[0070] The biofilm material comprises palmitic acid glyceryl, stearic acid glyceryl and cholesterol, wherein the mass ratio of palmitic acid glyceryl, stearic acid glyceryl and cholesterol is 68%, 30% and 2% respectively;

[0071] The stirring speed is 12000 r / min, and the stirring time is 10 min; the ultrasonic frequency is 40000 Hz and the ultrasonic time is 10 min;

[0072] (2) adding the enzyme preparation and the emulsifier to the liposome membrane, mixing thoroughly and then sonicating to obtain an enzyme preparation-liposome mixture;

[0073] The mass ratio of the enzyme preparation to the emulsifier is 1:10; the mass ratio of the enzyme preparation to the liposome is 1:2;

[0074] The ultrasonic frequency is 40000 Hz and the ultrasonic time is 5 min;

[0075] The enzyme preparation is a mixed enzyme preparation of protease and xylanase;

[0076] (3) using liquid nitrogen to flash freeze the enzyme preparation-liposome mixture to reduce the temperature to room temperature, and then spraying it through high-pressure centrifugation to obtain a powdered enzyme preparation-liposome;

[0077] The pressure of the high-pressure centrifugal spray is 0.7 MPa and the rotation speed is 25000 r / min;

[0078] The powdered enzyme preparation-liposome diameter was detected to be 100 nm;

[0079] (4) The activity of the powdered enzyme preparation - liposome enzyme was determined by gastric and wet heat methods.

[0080] The wet heat method is used to measure the powdered enzyme preparation-liposomes prepared, and its protease activity is 95,000 u / g, and its xylanase activity is 200,000 u / g. Then, after being stored in a high temperature environment of 120°C for 5 minutes, its protease activity is 92,150 u / g (a decrease of 3%), and its xylanase activity is 186,000 u / g (a decrease of 7%). The gastric test is to treat the enzyme preparation-liposomes in simulated gastric fluid with a pH value of 3 for 2 hours, and its protease activity is 91,200 u / g (a decrease of 4%), and its xylanase activity is 188,000 u / g (a decrease of 6%). Comparative Example 1: A processing method that can improve the heat and acid resistance of enzyme preparations

[0081] Compared with Example 3, the difference is:

[0082] In the step (1), the mass ratios of palmitic acid glyceryl, stearic acid glyceryl, and cholesterol are 40%, 55%, and 5%;

[0083] The wet heat method was used to determine the prepared powdered enzyme preparation-liposome, whose original enzyme activity was 75,000 u / g. The powdered enzyme preparation-liposome was then stored in a high temperature environment at 120°C for 5 minutes, and its enzyme activity was determined to be 63,000 u / g (a decrease of 16%). The gastric transgastric experiment was used to treat the enzyme preparation-liposome in simulated gastric fluid with a pH value of 3 for 2 hours, and its enzyme activity was determined to be 67,500 u / g (a decrease of 10%).

[0084] Comparative Example 2: A processing method for improving the heat and acid resistance of enzyme preparations

[0085] Compared with Example 3, the difference is:

[0086] In the step (1), the mass ratio of palmitic acid glyceryl, stearic acid glyceryl, and cholesterol is 80%, 10%, and 10%;

[0087] The wet heat method was used to determine the prepared powdered enzyme preparation-liposome, whose original enzyme activity was 65,000 u / g. It was then stored in a high temperature environment at 120°C for 5 minutes, and its enzyme activity was determined to be 50,050 u / g (a decrease of 23%). The gastric transgastric experiment was used to treat the enzyme preparation-liposome in simulated gastric fluid with a pH value of 3 for 2 hours, and its enzyme activity was determined to be 55,250 u / g (a decrease of 15%).

[0088] Comparative Example 3: A processing method for improving the heat and acid resistance of enzyme preparations

[0089] Compared with Example 3, the difference is:

[0090] In the step (3), the mass ratio of the enzyme preparation to the emulsifier is 1:2;

[0091] The wet heat method was used to determine the prepared powdered enzyme preparation-liposome, whose original enzyme activity was 85,000 u / g. The powdered enzyme preparation-liposome was then stored in a high temperature environment at 120°C for 5 minutes, and its enzyme activity was determined to be 79,900 u / g (a decrease of 16%). The gastric transgastric experiment was used to treat the enzyme preparation-liposome in simulated gastric fluid with a pH value of 3 for 2 hours, and its enzyme activity was determined to be 74,800 u / g (a decrease of 12%).

[0092] Comparative Example 4: A processing method for improving the heat and acid resistance of enzyme preparations

[0093] Compared with Example 3, the difference is:

[0094] In the step (3), the mass ratio of the enzyme preparation to the emulsifier is 1:15;

[0095] The wet heat method was used to determine the prepared powdered enzyme preparation-liposome, whose original enzyme activity was 96000u / g. It was then stored in a high temperature environment of 120°C for 5 minutes, and its enzyme activity was determined to be 86880u / g (a decrease of 9.5%). The gastric transgastric experiment was used to treat the enzyme preparation-liposome in simulated gastric fluid with a pH value of 3 for 2 hours, and its enzyme activity was determined to be 87360u / g (a decrease of 9%).

[0096] Comparative Example 5: A processing method for improving the heat and acid resistance of enzyme preparations

[0097] Compared with Example 3, the difference is:

[0098] In the step (3), the mass ratio of the enzyme preparation to the liposome is 1:0.5;

[0099] The wet heat method was used to determine the prepared powdered enzyme preparation-liposome, whose original enzyme activity was 70,000 u / g. The enzyme activity was then determined to be 55,300 u / g (a decrease of 21%) after being stored in a high temperature environment of 120°C for 5 minutes. The gastric transgastric experiment was used to treat the enzyme preparation-liposome in simulated gastric fluid with a pH value of 3 for 2 hours, and the enzyme activity was determined to be 59,500 u / g (a decrease of 15%).

[0100] Comparative Example 6: A processing method for improving the heat and acid resistance of enzyme preparations

[0101] Compared with Example 3, the difference is:

[0102] In the step (3), the mass ratio of the enzyme preparation to the liposome is 1:5;

[0103] The wet heat method was used to determine the prepared powdered enzyme preparation-liposome, whose original enzyme activity was 98,000 u / g. It was then stored in a high temperature environment at 120°C for 5 minutes, and its enzyme activity was determined to be 86,240 u / g (a decrease of 12%). The gastric transgastric experiment was used to treat the enzyme preparation-liposome in simulated gastric fluid with a pH value of 3 for 2 hours, and its enzyme activity was determined to be 89,180 u / g (a decrease of 9%).

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A processing method for improving the heat and acid resistance of an enzyme preparation, characterized in that: The following steps are involved: (1) Melt the biomembrane material, stir it evenly, and then ultrasonicate it to obtain the liposome membrane; Calculated by mass percentage, the biofilm material comprises 50%-68% palmitic acid glyceryl, 30%-45% stearic acid glyceryl and 2%-5% cholesterol; (2) adding an enzyme preparation and an emulsifier to the liposome membrane, mixing thoroughly, and then sonicating to obtain an enzyme preparation-liposome mixture; The enzyme preparation is at least one of protease, xylanase, β-glucanase, β-mannanase, α-galactosidase, and amylase; The mass ratio of the enzyme preparation to the emulsifier is 1:(5-10); the mass ratio of the enzyme preparation to the liposome is 1:(1-3); (3) The temperature of the obtained enzyme preparation-liposome mixture is suddenly reduced to room temperature, and high-pressure centrifugal spray is performed to obtain a powdered enzyme preparation-liposome.

2. The processing method for improving the heat and acid resistance of an enzyme preparation according to claim 1, characterized in that: In the step (1), the melting temperature is 45°C-65°C.

3. The processing method for improving the heat and acid resistance of an enzyme preparation according to claim 1, characterized in that: In the step (1), the stirring speed is 5000 r / min-12000 r / min, and the time is 5 min-10 min; the frequency of the ultrasound is 30000 Hz-40000 Hz, and the time of the ultrasound is 5 min-10 min.

4. The processing method for improving the heat and acid resistance of an enzyme preparation according to claim 1, characterized in that: In step (3), the pressure of the high-pressure centrifugal spray is 0.4 MPa-0.7 MPa, and the rotation speed is 15000 r / min-25000 r / min.

5. Enzyme preparation-liposome prepared by the processing method capable of improving the heat and acid resistance of enzyme preparation according to any one of claims 1 to 4.

6. The enzyme preparation-liposome according to claim 5, characterized in that The diameter of the enzyme preparation-liposome is 100nm-500nm.

7. The processing method according to any one of claims 1 to 4, and use of the enzyme preparation-liposome according to claim 5 or 6 in feed processing.