A high-stable mycotoxin-degrading enzyme compound preparation, a preparation method and use thereof

Abstract

The present application belongs to the field of mycotoxin-degrading enzyme preparation, and particularly relates to a high-stability mycotoxin-degrading enzyme compound preparation, a preparation method thereof and use thereof. The mycotoxin-degrading enzyme compound preparation composition comprises three components of mycotoxin-degrading enzyme, protective agent package A and stabilizer package B, wherein the mycotoxin-degrading enzyme is 0.1-10 parts by weight; the protective agent package A comprises the following components: protective agent 0.1-52 parts by weight; slow-release stabilizer 0.1-55 parts by weight; the stabilizer package B comprises the following components: diluent 10-80 parts by weight; buffer 0.1-25 parts by weight; dispersant 0.1-8 parts by weight; and flavoring agent 0.1-5 parts by weight. The present application has the following beneficial effects: the present application protects the enzyme from external condition interference and damage, thereby significantly improving the stability, activity and tolerance of the enzyme; the mycotoxin-degrading enzyme microspheres in the present application can effectively prevent the damage of gastrointestinal digestive juice to the enzyme during the gastrointestinal process, and have good gastrointestinal slow-release activity; and the present application is convenient to use.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of mycotoxin degrading enzyme preparation, and particularly relates to a high-stability mycotoxin degrading enzyme compound preparation as well as a preparation method and application thereof. BACKGROUND

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

[0003] The currently reported mycotoxin detoxification methods mainly include physical methods, chemical methods, and biological methods. The most commonly used method in physical methods is adsorption with attapulgite, montmorillonite, zeolite, glucomannan, activated carbon, plant-based compounds, PVPP, etc., and there are also methods such as heat treatment, microwave, gamma-ray, ultraviolet light, and water washing to destroy or eliminate toxins. Physical methods are simple and easy to implement, but they are not selective, and there are problems of destruction of nutritional components and secondary hazards. Chemical methods include acid-base treatment, oxidation-reduction treatment, etc., and there is also the problem of chemical reagent residues. Biological detoxification methods include microbial methods and enzyme methods, the former is to add microorganisms to the material, and use the enzymes produced by the microorganisms to adsorb and / or metabolize mycotoxins. Enzyme method is to purify the enzymes produced by microorganisms and add them to the material, and through enzyme reaction, the toxins are converted into low-toxic or non-toxic products. Enzyme method is considered to be the best mycotoxin detoxification method because it has mild reaction conditions, high efficiency, clear composition, no easy loss of nutrients, and environmental friendliness.

[0004] In the prior art, the application of mycotoxin-degrading enzymes to reduce or eliminate mycotoxins is an emerging detoxification technology. Although there were ideas of biological detoxification in previous studies, the existing technology mostly adopts the method of adding live bacteria due to the limitations of few enzyme types and poor enzyme tolerance, which limits the application of this idea. In recent years, with the progress of genetic engineering, enzyme protein evolution and synthetic biology, the types of mycotoxin-degrading enzymes discovered have increased, and the performance of enzymes such as heat resistance, acid and alkali resistance, salt tolerance, and half-life has been improved in an orderly manner, making it possible to gradually realize the idea of detoxification using pure enzymes. Among them, pure enzymes avoid the problems of complex composition of live bacteria and easy loss of activity and deterioration. However, the application of pure enzymes is also limited by the problem that enzymes are easily inactivated or their activity is inhibited in complex application environments. Some pharmaceutical preparation technologies are directly transplanted to protect enzyme activity, such as the method described in Chinese Patent CN111493229, which makes the enzyme into a core, and then makes two layers of enteric and heat-resistant coatings outside. However, this enteric coating method requires special equipment, the preparation process is complex, the cost is high, and it is difficult to scale up the production. Pharmaceutical preparation methods are suitable for high-value drugs, but not for relatively low-value products such as food and feed. Therefore, how to realize the application of mycotoxin-degrading enzymes to food and feed has become a difficult problem that needs to be solved in the field.

[0005] The degrading enzymes used in enzyme methods mainly include oxidases (such as laccase, ammonia oxidase, peroxidase, multicopper oxidase, etc.), DON dehydrogenase, glycosyltransferase, DON hydrolytic enzyme, ZEN lactone hydrolytic enzyme ZHD101, carboxypeptidase hydrolytic enzyme (such as esterase), etc. Establishing a high-efficiency degradation system for degrading mycotoxins by enzymes is the key to biological detoxification technology. Enzymes are proteins, and there are problems such as poor stability and difficult expression. Due to the complexity of materials, such as drying, sticking, and complex ingredients (such as feed and food), enzymes are often denatured and inactivated or their activity is inhibited. Especially in orally used products, most toxin-degrading enzymes are difficult to tolerate gastrointestinal digestion and lose function. SUMMARY

[0006] The purpose of the present application is to solve the problems of mycotoxin-degrading enzymes being easily affected by the environment, low stability, easy denaturation and inactivation, and easy digestion by gastrointestinal juices during storage and use.

[0007] To achieve the above-mentioned purpose, the specific technical solutions of the present application are as follows:

[0008] The first aspect of the present application provides a high stable mycotoxin degrading enzyme compound preparation, which comprises 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; slow-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 agent 0.1-5 parts by weight.

[0009] In another preferred embodiment, the mycotoxin degrading enzyme comprises one or more combinations of zearalenone hydrolase, aflatoxin B1 degrading enzyme, laccase, vomitoxin dehydrogenase, vomitoxin hydrolase, amine oxidase, peroxidase, multicopper oxidase, glycosyltransferase, etc.; preferably, the mycotoxin degrading enzyme is one or more combinations of zearalenone hydrolase, laccase, vomitoxin dehydrogenase, vomitoxin hydrolase.

[0010] In another preferred embodiment, the protective agent is selected from one or more combinations of hyaluronic acid, trehalose, NaCl, MgCl2, ZnSO4, CaCl2 and the like; preferably, it is a combination of hyaluronic acid, trehalose, NaCl, MgCl2, ZnSO4 and CaCl2; the slow-release stabilizer is one or more combinations of xanthan gum, gum arabic, alginate, carrageenan, guar gum, konjac gum, gellan gum and the like having gel-forming properties; preferably, it is a combination of xanthan gum, gum arabic, alginate and carrageenan.

[0011] 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 soda powder, citrate, phosphate, arginine, glycine, lysine, alanine and the like; preferably, it is a combination of soda powder, citrate, phosphate and arginine or a combination of soda powder, citrate and arginine.

[0012] In another preferred embodiment, the dispersant is polyethylene glycol (PEG); the flavoring agent is sodium glutamate.

[0013] In another preferred embodiment, the compound preparation is a slow-release preparation, and the preferred ratio of the compound preparation is 0.1wt%-90wt% of the total weight of the preparation composition, and the remaining part is an acceptable carrier, such as conventional excipients, fillers, binders, disintegrants, absorption promoters, surfactants, adsorption carriers and the like.

[0014] In another preferred embodiment, the specific percentage components of the compound preparation include:

[0015] Mycotoxin degrading enzyme 0.1-10%;

[0016] Among them, sustained-release stabilizers include

[0017] Xanthan gum 1-10%,

[0018] Carrageenan 0.1-5%,

[0019] Alginate 0.1-5%,

[0020] Gum Arabic 0.1-3%,

[0021] Guar gum 1%-5%,

[0022] Konjac gum 1%-5%

[0023] Gellan gum 0.1%-5%;

[0024] Wherein, the dispersant is polyethylene glycol 0.1-5%;

[0025] Among them, protective agents include

[0026] Hyaluronic acid 0.1-3%,

[0027] NaCl 0.1-3%,

[0028] MgCl2 0.1-3%,

[0029] ZnSO4 0.1-3%,

[0030] CaCl2 0.1-3%,

[0031] Trehalose 1-35%;

[0032] Among them, diluents include

[0033] Dextrin 1-90%,

[0034] Starch 1-90%,

[0035] Glucose 1-10%;

[0036] Among them, the buffer includes

[0037] Baking soda 0.1-5%,

[0038] Citrate 0.1-10%,

[0039] Phosphate 0.1-5%,

[0040] Arginine 0.1-5%,

[0041] Glycine 0.1-5%,

[0042] Lysine 0.1-5%,

[0043] Alanine 0.1-5%;

[0044] Among them, the flavoring agent is sodium glutamate 0.1-5%.

[0045] In another preferred embodiment, the dosage form of the compound preparation includes tablets, capsules, granules; preferably, the dosage form of the compound preparation is granules.

[0046] In another preferred embodiment, the protective agent package A is colloidal, used to wrap and fix the mycotoxin degrading enzyme, after spray drying, the microparticles in the protective agent package A will gather together, wrapping the mycotoxin degrading enzyme inside, and then mixed with the stabilizer package B, forming the microspheres with the mycotoxin degrading enzyme fixed and wrapped inside.

[0047] In the second aspect of the present application, a preparation method of a high-stability mycotoxin degrading enzyme compound preparation is provided, including the following steps:

[0048] S1: adding the mycotoxin degrading enzyme into the colloidal protective agent package A, and homogenizing with a blender until completely dissolved and uniformly into a flowable liquid;

[0049] S2: spray drying the dissolved enzyme liquid to prepare enzyme powder;

[0050] S3: mixing the enzyme powder with the stabilizer package B according to a ratio of 1:1-1:10 to obtain the final microspherical enzyme preparation; preferably, the ratio is 1:1-1:5.

[0051] In the third aspect of the present application, a use of a high-stability mycotoxin degrading enzyme compound preparation is provided, which is used for preventing, removing or reducing the pollution of mycotoxin degrading agents or detoxification agents and the like products in the production process of raw material products for feeding or eating.

[0052] Compared with the prior art, the present application has the following beneficial effects:

[0053] Through extensive and in-depth research, and through a large number of screening, the present application first develops a novel protective preparation composition of mycotoxin degrading enzyme.

[0054] (1) The compound preparation composition of the present application fixes and wraps the mycotoxin degrading enzyme in the microspheres, and is supplemented with sufficient enzyme activity protective agents and stabilizers, so that the enzyme is protected from external conditions interference and damage, thereby significantly improving the stability, activity and tolerance of the enzyme.

[0055] (2) The mycotoxin-degrading enzyme microspheres in the compound preparation composition of the present application can effectively prevent the destruction of the enzyme by the gastric and intestinal digestive juice due to the barrier effect of the microspheres during the gastrointestinal process, and the protective agent and the enzyme in the preparation are released together when the microspheres slowly disintegrate in the gastric and intestinal juice, which can effectively protect and maintain the enzyme activity, and has good gastrointestinal sustained-release activity.

[0056] (3) The compound oral preparation composition of the present application is convenient to use.

[0057] (4) The compound preparation composition of the present application also has a sustained-release effect in a liquid environment other than the gastrointestinal environment, and the enzyme activity effect is improved by sustained release.

[0058] (5) The compound preparation of the present application can be used for mammals, including humans and animals, and is preferably an oral preparation (such as a powder and a tablet). The optimal dose should be determined according to the specific use. Generally, it starts from a small dose and gradually increases the dose until the optimal dose is found. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 The release rates of the respective preparation formulations in simulated gastric and intestinal juice; wherein A is simulated gastric juice, B is simulated intestinal juice, 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;

[0060] Figure 2 The morphological changes of the formulation 1 in simulated gastric and intestinal juice within 1-2 hours; wherein A is simulated gastric juice for 60 min, B is simulated gastric juice for 90 min, C is simulated gastric juice for 120 min, D is simulated intestinal juice for 60 min, E is simulated intestinal juice for 90 min, and F is simulated intestinal juice for 120 min. DETAILED DESCRIPTION

[0061] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods in the following examples are not specified, which are usually carried out according to the conventional conditions or the conditions recommended by the manufacturers. Unless otherwise specified, percentages and parts are calculated by weight.

[0062] The experimental materials and reagents used in the following examples can be obtained from the market unless otherwise specified.

[0063] Example 1

[0064] Example 1: Accurately weigh the sustained-release stabilizer and the protective agent according to the components listed in Table 1, mix for 10 min to form a protective agent package A; accurately weigh the diluent, buffer, dispersant and flavoring agent, mix for 10 min to form a stabilizer package B.

[0065]

[0066] Example 2

[0067] Take 100 ml of zearalenone lactonase ZHD101 enzyme solution (enzyme activity concentration is 2000 U / ml, solid content is 5 mg / ml), add 15 grams of various colloidal protectants A prepared in Example 1 at a proportion of 15% (w / v), homogenize with a blender at a speed of 1000 r / min for 10-60 min until completely dissolved and evenly into a flowable liquid. The dissolved enzyme solution is spray dried to produce enzyme powder I; according to the formulations of each group in Table 1, weigh 45 g of stabilizer B at 3 times the amount of A powder, mix the enzyme powder I with the stabilizer B evenly to obtain the final microspherical enzyme preparation, with an enzyme activity content of ~3000 U / g.

[0068] Example 3

[0069] 1. Drug loading and encapsulation efficiency detection

[0070] Take an appropriate amount of microspheres and grind them, weigh 10 mg of the powder into 50 ml of phosphate buffer (pH 7.4), and ultrasonicate to fully erode. Filter out the particles with ordinary filter paper, dilute the filtrate as appropriate, and measure the OD280 value by spectrophotometry. Calculate the enzyme content according to the molar extinction coefficient of ZHD101 enzyme, which is 1.426 (OD280 / 1.423, unit: mg / ml). Calculate the drug loading and encapsulation efficiency according to the following formula:

[0071]

[0072] The enzyme loading and encapsulation efficiency of enzyme preparations of different formulations can be seen from Table 2, and the loading and encapsulation efficiency of Formula 1 are the best.

[0073]

[0074] 2. ZHD101 enzyme release test

[0075] Referring to Figure 1 Two release media were tested, namely simulated gastric juice A (0.05 mol / L HCL, pH 1.2) and simulated intestinal juice B (0.05 mol / L phosphate buffer, pH 7.4). Two 100 mg samples of the six enzyme preparations prepared in Example 2 were weighed and placed in 100 ml of different release media, and stirred at 37°C and 100 r / min. Every certain time, 5 ml of sample was taken and supplemented with the same volume of fresh release medium. After centrifugation in a high-speed centrifuge for 20 min, the supernatant was detected to calculate the cumulative release rate.

[0076] The sampling time is: gastric juice 30 min, 60 min, 90 min, 120 min; intestinal juice 30 min, 60 min, 90 min, 120 min, 240 min. The release rate determination results of each formula are shown in Table 3, and it can be seen that the gastric juice protection performance and intestinal juice sustained release effect of formula 1 are good.

[0077]

[0078] For the above formula 1 sample, samples were taken at 60 min, 90 min, and 120 min, and the microsphere morphology was observed under a microscope. The microscopic results are shown in Table 3, and it can be seen that the gastric juice protection performance and intestinal juice sustained release effect of formula 1 are good. Figure 2 , which shows that: in the simulated gastric juice, the microspheres of sample 1 present a shriveled morphology, the structure is intact, and there is no fragmentation, and the morphology at 60 min, 90 min, and 120 min is not different; in the simulated intestinal juice, with the extension of time, the interface between the edge of the microspheres and the simulated intestinal juice becomes not obvious, and finally becomes indistinguishable from the intestinal juice, and within 1 h, a large number of microspheres are damaged, and the damage rate is more than 50% in another preferred example. After 2 h, the microspheres have been completely damaged, and there are only some small fragments in the field of view, and it can be seen that formula 1 formulation shows good protection and loading function.

[0079] Example 4: Enzyme activity protection effect test

[0080] Experimental group: 20 mg of ZHD101 enzyme preparation of formula 1 (enzyme activity content is 3 U / mg) is added to 980 mg of montmorillonite to make 60 U / g of ZHD101 complex mycotoxin removal product. 10 mg of the product (0.6 U) is weighed and added to 10 ml of a solution containing 3 ppm of zearalenone (pH 6.5), and incubated at 35°C for 120 min. The supernatant is separated by centrifugation, and the ZHD101 enzyme activity retention is detected by HPLC. At the same time, a positive control group and a test control group are set.

[0081] Positive control group: ZHD101 enzyme solution (2000 U / ml) is diluted 60 times with PBS (pH 6.5) to a concentration of 33.3 U / ml, and 18 μl of the diluted enzyme solution (0.6 U) is added to 10 ml of a solution containing 3 ppm of zearalenone (pH 6.5), and incubated at 35°C for 120 min. The supernatant is separated by centrifugation, and the zearalenone degradation is detected by HPLC method, and the enzyme activity retention is calculated.

[0082] Test control group: ZHD101 enzyme solution (2000 U / ml) was diluted 60 times with PBS (pH 6.5) to a concentration of 33.3 U / ml, and 18 μl of the diluted enzyme solution (0.6 U) was added to 8 mg of montmorillonite and 10 ml of a mixture containing 3 ppm of zearalenone (pH 6.5), which was then incubated at 35°C for 120 min. The supernatant was obtained by centrifugation, and the zearalenone degradation was detected by HPLC to calculate the enzyme activity.

[0083] Negative control group: 10 ml of a mixture containing 3 ppm of zearalenone (pH 6.5) was incubated at 35°C for 120 min, and the supernatant was obtained by centrifugation.

[0084] The HPLC detection method is a method for detecting a small amount of ZEN to represent the enzyme activity of ZHD101. 400 μL of the above reaction solution was added with 800 μL of 100% methanol solution to stop the reaction. 200 μL of the terminated reaction solution was used for HPLC detection at 254 nm absorbance, and the chromatographic conditions were as follows: mobile phase acetonitrile: water = 60:40; flow rate 1 ml / min; column temperature 30°C; and chromatographic column ZORBAX Eclipse XDB-C18 column. The enzyme activity retention rate of ZHD101 was calculated according to the following formula:

[0085]

[0086] A0 x A0 is the peak area of the enzyme-free negative control group.

[0087] The enzyme activity retention effect test results are shown in Table 4. The enzyme activity of the formula 1 group can be retained by 95.2%, while the enzyme activity of the enzyme solution without any protective measures is almost lost.

[0088]

[0089] Example 5: Effect of different enzyme weight portions on enzyme activity protection effect

[0090] The zearalenone lactone hydrolase freeze-dried powder was prepared according to formula 1 to prepare the protective agent A1 powder and the stabilizer B1 powder. According to the proportions in Table 5 and the method of Example 2, three kinds of compound preparations with enzyme weight portions of 0.1, 5 and 10 were prepared.

[0091]

[0092] According to the methods described in Examples 3 and 4, the encapsulation efficiency, loading capacity and enzyme activity retention rate of the three enzyme activity preparations were determined, and the results are shown in Table 6. The encapsulation efficiency and enzyme activity retention rate of high, medium and low enzyme weight portions are all greater than 95%, which have good application performance.

[0093]

[0094] In summary, the present patent solves the instability problems of enzyme inactivation and degradation caused by the influence of material composition and gastrointestinal juice by preparing the mycotoxin-decomposing enzyme in a preparation composition, and gives the enzyme protection and slow-release effect, so that it can maintain a longer enzyme concentration and better exert the enzyme activity.

[0095] The above is a detailed description of the embodiments, which facilitates the correct understanding and use of the present application by those skilled in the art. Any improved or modified technical solutions obtained by those skilled in the art on the basis of the prior art without innovative labor, only through analysis, analogy or limited enumeration, etc. should be within the protection scope determined by the claims.

Claims

1. A high-stable mycotoxin-degrading enzyme complex preparation, characterized in that, The mycotoxin-decomposing enzyme, the protective agent package A and the stabilizer package B, wherein: The mycotoxin-decomposing enzyme; The protective agent package A is composed of a protective agent, a slow-release stabilizer; The stabilizer package B is composed of a diluent, a buffer, a dispersant and a flavoring agent; The specific percentage components of the compound preparation include: The mycotoxin-decomposing enzyme 5%; The slow-release stabilizer includes Xanthan gum 1%, Carrageenan 1%, Sodium alginate 1%, Gum arabic 1%; The protective agent includes Hyaluronic acid 3.7%, NaCl 0.9%, MgCl2 0.2%, ZnSO4 0.1%, CaCl2 0.1%, Trehalose 15%; The diluent includes Dextrin 40%, Glucose 21%; The buffer includes Soda powder 1.3%, Citrate 1.3%, Sodium phosphate 2.0%, Arginine 0.4%; The dispersant is polyethylene glycol 2.0%; The flavoring agent is sodium glutamate 3.0%; The preparation method specifically includes the following steps: S1: The mycotoxin-decomposing enzyme is added to the protective agent package A in a colloidal form, and a blender is used for stirring and homogenizing until completely dissolved and uniformly into a flowable liquid; S2: The dissolved enzyme solution is spray-dried to prepare an enzyme powder; S3: The enzyme powder and the stabilizer package B are mixed uniformly at a ratio of 1:1-1:10 to obtain the final microspheric enzyme preparation.

2. A high stable mycotoxin degrading enzyme complex preparation according to claim 1, characterized in that, The compound preparation is a slow-release preparation.

3. The high stable mycotoxin degrading enzyme complex preparation according to claim 1, characterized in that, The dosage form of the compound preparation includes tablets, capsules and granules.

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