High-stability mycotoxin degrading enzyme compound preparation as well as preparation method and application thereof

Abstract

The invention belongs to the field of mycotoxin degrading enzyme preparations, and particularly relates to a high-stability mycotoxin degrading enzyme compound preparation as well as a preparation method and application thereof. The mycotoxin degrading enzyme compound preparation composition comprises the following three components in parts by weight: 0.1-10 parts of mycotoxin degrading enzyme, 0.1-10 parts of a protective agent material bag A and 0.1-10 parts of a stabilizer material bag B, the component A of the protective agent material bag comprises 0.1-52 parts by weight of a protective agent; 0.1-55 parts by weight of a slow release stabilizer; the stabilizer material bag B comprises the following components in parts by weight: 10-80 parts of a diluent; 0.1 to 25 parts by weight of a buffer agent; 0.1-8 parts by weight of a dispersant and 0.1-5 parts by weight of a flavoring agent. The method has the beneficial effects that the enzyme is prevented from being interfered and damaged by external conditions, so that the stability, the activity and the tolerance of the enzyme are remarkably improved; when the mycotoxin degrading enzyme microspheres pass through the stomach and intestine, damage of gastrointestinal digestive juice to enzyme can be effectively prevented, and the mycotoxin degrading enzyme microspheres have good gastrointestinal tract slow-release activity; the device is convenient to use.

Description

Technical Field

[0001] The invention belongs to the field of mycotoxin degrading enzyme preparations, and specifically relates to a highly stable mycotoxin degrading enzyme compound preparation, a preparation method and uses thereof. Background Art

[0002] Mold contamination is a global problem, and the mycotoxins it brings threaten the health of livestock and humans. Mycotoxins are secondary metabolites produced during the growth of molds, and are common in grains, feed, etc. There are more than 500 known mycotoxins, and the ones that are currently being focused on are aflatoxins (AF), zearalenone (ZEN), ochratoxin A (OTA), fumonisins (FB), vomitoxin (DON), and T-2 toxin. Mycotoxins are carcinogenic, mutagenic, and teratogenic, and can cause death in humans and animals. There is a high probability that multiple mycotoxins coexist, which has synergistic toxicity.

[0003] The mycotoxin detoxification methods reported so far mainly include physical, chemical and biological methods. The most commonly used physical method is adsorption with attapulgite, montmorillonite, zeolite, glucomannan, activated carbon, plant-based compounds, PVPP, etc., and toxins can be destroyed or eliminated by heat treatment, microwave, γ-ray, ultraviolet light, water washing and other methods. Physical methods are simple and easy to use, but they are not selective, and there are nutrient destruction and secondary hazards. Chemical methods include acid-base treatment, redox treatment, etc., and there are also problems with chemical reagent residues. Biological detoxification methods include microbial methods and enzyme methods. The former is to degrade mycotoxins by adding microorganisms to the material and using enzymes produced by microbial adsorption and / or metabolism. The enzyme method is to purify the enzymes produced by microorganisms, add them to the material, and convert the toxins into low-toxic or non-toxic products through enzymatic reactions. Enzymatic detoxification has mild conditions, high efficiency, component removal, low nutrient loss and environmental friendliness, and is considered to be the best method for mycotoxin detoxification.

[0004] In the prior art, the use of mycotoxin degrading enzymes to reduce or eliminate mycotoxins is an emerging detoxification technology. Although there have been ideas for biological detoxification in previous studies, due to factors such as the small number of enzymes and poor enzyme tolerance, most of the prior art uses the method of adding live bacteria, which limits the application of this idea. In recent years, with the progress of genetic engineering, enzyme protein evolution and synthetic biology, the number of mycotoxin degrading enzymes discovered has increased, and the properties of enzymes such as heat resistance, acid and alkali resistance, salt resistance, and half-life have been improved in an orderly manner. The idea of ​​using pure enzymes for detoxification has gradually become possible. Among them, pure enzymes avoid the problems of complex components of live bacteria and easy inactivation and deterioration. However, the application of pure enzymes is also subject to the problem that enzymes are easily inactivated or their activity is inhibited in complex application environments. Some pharmaceutical preparation technologies have been directly transplanted to protect enzyme activity, such as the method described in Chinese patent CN111493229, which makes the enzyme into a pellet core and makes two layers of enteric and heat-resistant coating on the outside. However, this enteric coating method requires special equipment, complex preparation process, high cost, and difficulty in increasing production. The pharmaceutical preparation method is suitable for high-value drugs, but not for relatively low-priced products such as food and feed. Mycotoxins are mainly produced in the moldy process of feed and its raw materials, food and its raw materials, so how to realize the application of mycotoxin degrading enzymes for food and feed has become a difficult problem that needs to be solved urgently in this field.

[0005] The degradation enzymes used in the enzymatic method mainly include oxidases (such as laccase, ammonia oxidase, peroxidase, multi-copper oxidase, etc.), DON dehydrogenase, glycosyltransferase, DON hydrolase, ZEN lactone hydrolase ZHD101, carboxypeptidase hydrolase (such as esterase), etc. Establishing an efficient degradation system for enzymatic degradation of mycotoxins is the key to biological detoxification technology. Enzymes are proteins, and there are problems such as poor stability and difficulty in expression. Due to the complexity of materials, such as dryness, viscosity, and complex ingredients (such as feed, food, etc.), enzymes are often denatured and inactivated or their activity is inhibited. Especially in products for oral use, most toxin-degrading enzymes are difficult to withstand gastrointestinal digestion and lose their function. Summary of the invention

[0006] The purpose of the present invention is to solve the problems that mycotoxin degrading enzymes are easily affected by the environment during storage and use, have low stability, are easily denatured and inactivated, and are easily digested by gastrointestinal digestive juices.

[0007] In order to achieve the above object, the specific technical scheme of the present invention is as follows: The first aspect of the present invention provides a highly stable mycotoxin degrading enzyme compound preparation, comprising three components: mycotoxin degrading enzyme, protective agent package A and stabilizer package B, wherein: mycotoxin degrading enzyme 0.1-10 parts by weight; protective agent package A comprises: protective agent 0.1-52 parts by weight; sustained-release stabilizer 0.1-55 parts by weight; stabilizer package B comprises: diluent 10-80 parts by weight; buffer 0.1-25 parts by weight; dispersant 0.1-8 parts by weight and flavoring 0.1-5 parts by weight.

[0008] In another preferred embodiment, the mycotoxin degrading enzyme includes one or more combinations of zearalenone hydrolase, aflatoxin B1 degrading enzyme, laccase, vomitoxin dehydrogenase, vomitoxin hydrolase, ammonia oxidase, peroxidase, multi-copper oxidase, glycosyltransferase, etc.; preferably, the mycotoxin degrading enzyme is one or more combinations of zearalenone hydrolase, laccase, vomitoxin dehydrogenase, and vomitoxin hydrolase.

[0009] In another preferred embodiment, the protective agent is selected from one or more combinations of salts such as hyaluronic acid, trehalose, NaCl, MgCl2, ZnSO4, CaCl2, etc.; preferably, it is a combination of hyaluronic acid, trehalose, NaCl, MgCl2, ZnSO4, and CaCl2; the sustained-release stabilizer is one or more combinations of substances with gelling properties such as xanthan gum, gum arabic, alginate, carrageenan, guar gum, konjac gum, gellan gum, etc.; preferably, it is a combination of xanthan gum, gum arabic, alginate and carrageenan.

[0010] In another preferred embodiment, the diluent is one or more combinations of dextrin and glucose; preferably, the diluent is a combination of dextrin and glucose; the buffer is one or more combinations of amino acids such as baking soda, citrate, phosphate, arginine, glycine, lysine, and alanine; preferably, it is a combination of baking soda, citrate, phosphate, and arginine or a combination of baking soda, citrate, and arginine.

[0011] In another preferred embodiment, the dispersant is polyethylene glycol (PEG); and the flavoring agent is sodium glutamate.

[0012] In another preferred embodiment, the compound preparation is a sustained-release preparation, and the preferred proportion of the compound preparation is 0.1wt% to 90wt% of the total weight of the preparation composition, and the rest is an acceptable carrier, such as conventional excipients, fillers, binders, disintegrants, absorption enhancers, surfactants, adsorption carriers, etc.

[0013] In another preferred embodiment, the specific percentage components of the compound preparation include: Mycotoxin degrading enzyme 0.1-10%; Among them, sustained-release stabilizers include Xanthan gum 1-10%, Carrageenan 0.1-5%, Alginate 0.1-5%, Gum Arabic 0.1-3%, Guar gum 1%-5%, Konjac gum 1%-5% Gellan gum 0.1%-5%; Wherein, the dispersant is polyethylene glycol 0.1-5%; Among them, the protective agent includes Hyaluronic acid 0.1-3%, NaCl 0.1-3%, MgCl2 0.1-3%, ZnSO4 0.1-3%, CaCl2 0.1-3%, Trehalose 1-35%; Among them, the diluent includes Dextrin 1-90%, Starch 1-90%, Glucose 1-10%; The buffer includes Baking soda 0.1-5%, Citrate 0.1-10%, Phosphate 0.1-5%, Arginine 0.1-5%, Glycine 0.1-5%, Lysine 0.1-5%, Alanine 0.1-5%; Wherein, the flavoring agent is monosodium glutamate 0.1-5%.

[0014] In another preferred embodiment, the dosage form of the compound preparation includes tablets, capsules, and granules; preferably, the dosage form of the compound preparation is granules.

[0015] In another preferred embodiment, the protective agent package A is in colloidal form and is used to encapsulate and fix the mycotoxin degrading enzyme. After spray drying, the particles in the protective agent package A will aggregate together, encapsulating the mycotoxin degrading enzyme inside, and then mix with the stabilizer package B to form microspheres with the mycotoxin degrading enzyme fixedly encapsulated inside.

[0016] In a second aspect of the present invention, a method for preparing a highly stable mycotoxin degrading enzyme compound preparation is provided, comprising the following steps: S1: Add the mycotoxin decomposing enzyme to the colloidal protective agent package A, and stir and homogenize it with a blender until it is completely dissolved and evenly becomes a liquid that is easy to flow; S2: spray drying the dissolved enzyme solution to prepare enzyme powder; S3: Evenly mix the enzyme powder and stabilizer package B in a ratio of 1:1-1:10 to obtain the final microsphere enzyme preparation; preferably, the ratio is 1:1~1:5.

[0017] In the third aspect of the present invention, there is provided a use of a highly stable mycotoxin degrading enzyme compound preparation, wherein the compound preparation is used to prevent, remove or reduce the contamination of mycotoxin degraders or detoxifying agents and other products in the production process of raw materials such as feed or food.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention has developed a novel mycotoxin degrading enzyme protective preparation composition for the first time through extensive and in-depth research and a large number of screenings.

[0019] (1) The compound preparation composition of the present invention fixes and encapsulates the mycotoxin degrading enzyme in microspheres and supplements them with sufficient enzyme activity protectants and stabilizers to protect the enzyme from interference and damage from external conditions, thereby significantly improving the stability, activity and tolerance of the enzyme.

[0020] (2) The mycotoxin degrading enzyme microspheres in the compound preparation composition of the present invention can effectively prevent the gastrointestinal digestive juice from destroying the enzyme during the process of passing through the gastrointestinal tract due to the barrier effect of the microspheres. At the same time, when the microspheres slowly disintegrate in the gastrointestinal juice, the protective agent in the preparation is released together with the enzyme to effectively protect and maintain the enzyme activity, and has good gastrointestinal sustained-release activity.

[0021] (3) The compound oral preparation composition of the present invention is easy to use.

[0022] (4) The compound preparation composition of the present invention also has a sustained release effect in a liquid environment other than the gastrointestinal environment, thereby improving the enzyme activity effect through sustained release.

[0023] (5) The compound preparation of the present invention can be used for mammals, including humans and animals, preferably in the form of oral preparations (such as powders and tablets). The optimal dose should be determined according to the specific use. Usually, the dose is started from a small dose and gradually increased until the most suitable dose is found. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the release rate of each formulation in simulated gastric and intestinal fluids; wherein A is simulated gastric fluid, B is simulated intestinal fluid, a is formulation 1, b is formulation 2, c is formulation 3, d is formulation 4, e is formulation 5, and f is formulation 6; Figure 2The morphological changes of the preparation of formula 1 when dissolved in simulated gastric juice and intestinal juice within 1-2 hours; A is simulated gastric juice for 60 minutes, B is simulated gastric juice for 90 minutes, C is simulated gastric juice for 120 minutes, D is simulated intestinal juice for 60 minutes, E is simulated intestinal juice for 90 minutes, and F is simulated intestinal juice for 120 minutes. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0026] Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial channels.

[0027] Example 1 Example 1 According to the components listed in Table 1, the sustained-release stabilizer and protective agent were accurately weighed and mixed for 10 minutes to form a protective agent package A; the diluent, buffer, dispersant and flavoring agent were accurately weighed and mixed for 10 minutes to form a stabilizer package B.

[0028]

[0029] Example 2 Take 100 ml of zearalenone hydrolase ZHD101 enzyme solution (enzyme activity concentration is 2000 U / ml, solid content is 5 mg / ml), add 15 grams of various colloidal protective agents A prepared in Example 1 at a ratio of 15% (w / v), and use a stirrer to homogenize for 10-60 minutes at a speed of 1000 r / min until it is completely dissolved and uniformly becomes an easy-flowing liquid. Spray dry the dissolved enzyme solution to prepare enzyme powder I; according to the formula of each group in Table 1, weigh 45 g of stabilizer B according to 3 times the amount of A powder, mix enzyme powder I and stabilizer B evenly, and obtain the final microsphere enzyme preparation with an enzyme activity content of ~3000 U / g.

[0030] Example 3 1. Loading capacity and encapsulation efficiency detection Take an appropriate amount of microspheres and grind them into powder. Weigh 10 mg of powder and add it to 50 ml of phosphate buffer (pH 7.4). Ultrasound to fully dissolve it. Filter out the particles with ordinary filter paper, take the filtrate and dilute it appropriately, and measure the OD280 value by spectrophotometry. According to the molar extinction coefficient of ZHD101 enzyme of 1.426, calculate the enzyme content (OD280 / 1.423, unit is mg / ml). Calculate the drug loading and encapsulation efficiency according to the following formula:

[0031] The enzyme loading and encapsulation efficiency of enzyme preparations with different formulations are shown in Table 2. The loading and encapsulation efficiency of formulation 1 are the best.

[0032]

[0033] 2. ZHD101 enzyme release test See also Figure 1 Two release media were tested, namely simulated gastric fluid A (0.05 mol / L HCL, pH 1.2) and simulated intestinal fluid B (0.05 mol / L phosphate buffer, pH 7.4). Two portions of the six enzyme preparations prepared in Example 2 were weighed, each with 100 mg, and placed in 100 ml of different release media, respectively, and stirred at 37 ° C and 100 r / min. 5 ml of samples were taken at regular intervals and supplemented with the same volume of fresh release liquid. After the sample was centrifuged in a high-speed centrifuge for 20 minutes, the supernatant was taken to detect the protein concentration in the release liquid and calculate the cumulative release rate.

[0034] The sampling time is: 30min, 60min, 90min, 120min for gastric juice; 30min, 60min, 90min, 120min, 240min for intestinal juice. The release test results of each formula are shown in Table 3, which shows that the gastric juice protection performance and intestinal juice sustained release effect of formula 1 are both good.

[0035]

[0036] For the sample of formula 1 above, samples were taken at 60min, 90min, and 120min, and the changes in the microsphere morphology were observed under a microscope. Figure 2 , indicating that: in the simulated gastric fluid, the microspheres of the sample of preparation 1 showed a shrunken morphology, with intact structure and no breakage, and there was no difference in morphology at 60min, 90min, and 120min; in the simulated intestinal fluid, as time went on, the interface between the edge of the microspheres and the simulated intestinal fluid became less obvious, and finally became indistinguishable from the intestinal fluid. A large number of microspheres were broken within 1 hour, and the breakage rate reached more than 50% in another preferred example. After 2 hours, the microspheres were completely broken, with only some small fragments in the field of view, which shows that the preparation of formula 1 showed good protection and loading functions.

[0037] Example 4 Enzyme activity protection effect test Experimental group: Take 20 mg of ZHD101 enzyme preparation (enzyme activity content of 3U / mg) of formula 1, add 980 mg of montmorillonite, mix evenly to make 60U / g ZHD101 composite mycotoxin removal product. Weigh 10 mg of the product (0.6U), add 10 ml of solution containing 3ppm zearalenone (pH6.5), incubate at 35℃ for 120min, centrifuge and take the supernatant, and use HPLC to detect the enzyme activity retention of ZHD101. At the same time, set up a positive control group and a test control group.

[0038] Positive control group: dilute ZHD101 enzyme solution (2000U / ml) 60 times with PBS (pH6.5) to an enzyme activity concentration of 33.3U / ml, take 18µl of diluted enzyme solution (0.6U) and add it to 10ml of solution containing 3ppm zearalenone (pH6.5), incubate at 35℃ for 120min, centrifuge and take the supernatant, detect the degradation of zearalenone by HPLC method, and calculate the enzyme activity.

[0039] Experimental control group: ZHD101 enzyme solution (2000U / ml) was diluted 60 times with PBS (pH6.5) to an enzyme activity concentration of 33.3U / ml. 18µl of the diluted enzyme solution (0.6U) was added to 8mg of montmorillonite and 10ml of a mixed solution containing 3ppm of zearalenone (pH6.5). The mixture was incubated at 35℃ for 120min, and the supernatant was collected by centrifugation. The degradation of zearalenone was detected by HPLC, and the enzyme activity was calculated.

[0040] Negative control group: incubate in 10 ml of a mixed solution (pH 6.5) containing 3 ppm zearalenone at 35°C for 120 min, and centrifuge to obtain the supernatant.

[0041] The high performance liquid chromatography (HPLC) detection method is to detect the reduction of ZEN to characterize the enzyme activity of ZHD101. Take 400 μL of the above reaction solution and add 800 μL of 100% methanol solution to stop the reaction. Take 200 μL of the terminated reaction solution for HPLC detection at an absorbance of 254 nm. The chromatographic conditions are: mobile phase acetonitrile: water = 60:40; flow rate is 1 ml / min; column temperature is 30°C; the chromatography column is a ZORBAX Eclipse XDB-C18 column. The enzyme activity retention rate of ZHD101 is calculated according to the following formula:

[0042] Among them, A x is the ZEN peak area of ​​each experimental group, and A0 is the peak area of ​​the negative control group without enzyme. The test results of enzyme activity retention are shown in Table 4. Formula 1 can retain 95.2% of the enzyme activity, while the enzyme solution without any protective measures has almost lost all the enzyme activity.

[0043]

[0044] Example 5 Effect of different enzyme weights on enzyme activity protection Take the zearalenone hydrolase freeze-dried powder, prepare the protective agent A1 powder and the stabilizer B1 powder according to formula 1, and prepare three compound preparations with the weight parts of mycotoxin decomposing enzyme being 0.1, 5 and 10 parts by weight according to the proportions in Table 5 and the method in Example 2.

[0045] According to the method described in Example 3 and Example 4, the encapsulation efficiency, loading capacity and enzyme activity retention rate of the three enzyme preparations were determined. The results are shown in Table 6. The encapsulation efficiency and enzyme activity retention rate of the three enzyme weight portions of high, medium and low were all greater than 95%, showing good application performance.

[0046]

[0047] In summary, this patent solves the instability problems of enzymes such as inactivation and degradation caused by the influence of material components and gastrointestinal digestive fluid by preparing mycotoxin-degrading enzymes in a preparation composition, and gives the enzymes protection and sustained-release effects, so that the enzyme concentration can be maintained for a longer time and the enzyme activity can be better exerted.

[0048] The above is a detailed description of the embodiments, which is convenient for those skilled in the art to correctly understand and use the present invention. Any improvements or modifications to the technical solutions obtained by those skilled in the art based on the present invention on the basis of the prior art, without innovative work, only through analysis, analogy or limited enumeration, etc., should be within the scope of protection determined by the claims.

Claims

1. A highly stable mycotoxin degrading enzyme compound preparation, characterized in that: It includes three components: mycotoxin decomposing enzyme, protective agent package A and stabilizer package B, among which: 0.1 to 10 parts by weight of mycotoxin degrading enzyme; The protective agent package A comprises: 0.1 to 52 parts by weight of protective agent; 0.1 to 55 parts by weight of sustained-release stabilizer; The components of stabilizer package B are: 10 to 80 parts by weight of diluent; 0.1 to 25 parts by weight of buffer; 0.1 to 8 parts by weight of dispersant and 0.1 to 5 parts by weight of flavoring agent.

2. A highly stable mycotoxin degrading enzyme compound preparation according to claim 1, characterized in that: The mycotoxin degrading enzyme is one or more combinations of zearalenone hydrolase, aflatoxin B1 degrading enzyme, laccase, vomitoxin dehydrogenase, vomitoxin hydrolase, ammonia oxidase, peroxidase, multi-copper oxidase, and glycosyltransferase.

3. A highly stable mycotoxin degrading enzyme compound preparation according to claim 1, characterized in that: The protective agent is selected from one or more combinations of hyaluronic acid, trehalose, NaCl, MgCl2, ZnSO4, and CaCl2; the sustained-release stabilizer is one or more combinations of xanthan gum, gum arabic, alginate, carrageenan, guar gum, konjac gum, and gellan gum.

4. A highly stable mycotoxin degrading enzyme compound preparation according to claim 1, characterized in that: The diluent is one or more combinations of dextrin and glucose; the buffer is one or more combinations of baking soda, citrate, phosphate, arginine, glycine, lysine, and alanine; the dispersant is polyethylene glycol (PEG); and the flavoring agent is sodium glutamate.

5. A highly stable mycotoxin degrading enzyme compound preparation according to claim 1, characterized in that: The compound preparation is a sustained-release preparation.

6. A highly stable mycotoxin degrading enzyme compound preparation according to claim 1, characterized in that: The specific percentage components of the compound preparation include: Mycotoxin degrading enzyme 0.1-10%; Among them, sustained-release stabilizers include Xanthan gum 1-10%, Carrageenan 0.1-5%, Alginate 0.1-5%, Gum Arabic 0.1-3%, Guar gum 1%-5%, Konjac gum 1%-5% Gellan gum 0.1%-5%; Wherein, the dispersant is polyethylene glycol 0.1-5%; Among them, the protective agent includes Hyaluronic acid 0.1-3%, NaCl 0.1-3%, MgCl2 0.1-3%, ZnSO4 0.1-3%, CaCl2 0.1-3%, Trehalose 1-35%; Among them, the diluent includes Dextrin 1-90%, Starch 1-90%, Glucose 1-10%; The buffer includes Baking soda 0.1-5%, Citrate 0.1-10%, Phosphate 0.1-5%, Arginine 0.1-5%, Glycine 0.1-5%, Lysine 0.1-5%, Alanine 0.1-5%; Wherein, the flavoring agent is monosodium glutamate 0.1-5%.

7. A highly stable mycotoxin degrading enzyme compound preparation according to claim 1, characterized in that: The dosage forms of the compound preparation include tablets, capsules and granules.

8. The highly stable mycotoxin degrading enzyme compound preparation according to claim 1, characterized in that: The protective agent package A is in colloidal form and is used to encapsulate and fix the mycotoxin degrading enzyme. After spray drying, the particles in the protective agent package A will gather together to encapsulate the mycotoxin degrading enzyme inside, and then mix with the stabilizer package B to form microspheres with the mycotoxin degrading enzyme fixed inside.

9. A method for preparing the highly stable mycotoxin degrading enzyme compound preparation according to any one of claims 1 to 8, characterized in that: The following steps are involved: S1: Add the mycotoxin decomposing enzyme to the colloidal protective agent package A, and stir and homogenize it with a blender until it is completely dissolved and evenly becomes a liquid that is easy to flow; S2: spray drying the dissolved enzyme solution to prepare enzyme powder; S3: The enzyme powder and stabilizer package B are mixed evenly in a ratio of 1:1-1:10 to obtain the final microsphere enzyme preparation.

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