Composite mildew preventive for feed and preparation method of composite mildew preventive

Abstract

The invention discloses a composite mildew preventive for feed and a preparation method of the composite mildew preventive, and belongs to the technical field of preparation of composite mildew preventive. Comprising the following steps: carrying out centrifugal separation on beer yeast paste to remove impurities so as to obtain solid yeast, carrying out ultrasonic oscillation and two-stage enzymolysis on the solid yeast, carrying out vacuum drying so as to obtain a yeast extract, and pretreating the yeast extract so as to obtain an alkaline yeast extract solution; the preparation method comprises the following steps: mixing an alkaline yeast extract solution and sodium bromoacetate, stirring and reacting to obtain a modified yeast extract solution, adjusting the pH value of the modified yeast extract solution, filtering to obtain a modified yeast extract, mixing the modified yeast extract, cinnamyl aldehyde, ammonium propionate and montmorillonite K10, and crushing to obtain the composite mildew preventive for feed. By synergistically reducing the number of mould and the content of mycotoxin in the feed, the effect of the composite mildew preventive for the feed is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of composite mildew inhibitor preparation, and particularly relates to a composite mildew inhibitor for feed and a preparation method thereof. Background Art

[0002] Feed mold and mycotoxicosis are common and serious problems in livestock and poultry production worldwide. Once moldy, feed loses its value. Severely moldy feed not only significantly reduces its value but can also cause mycotoxicosis in livestock and poultry, even leading to death and significant economic losses. For nearly half a century, extensive research has been conducted worldwide on the application of mold inhibitors, resulting in the development of numerous established products, many of which have become world-renowned brand names. However, the vast majority of these agents focus solely on their mold inhibition properties, failing to consider the potential for mycotoxicosis in livestock and poultry caused by the release of large amounts of mycotoxins during the mold inhibition process.

[0003] Patent CN101984863B discloses a feed mold inhibitor composed of the following components in the following mass percentages: 10.0-30.0% sodium dehydroacetate, 5.0-10.0% cinnamon oil, 2.0-5.0% sodium ethylparaben, 2.0-5.0% sodium propylparaben, 1.0-10.0% white carbon black, and 40.0-80.0% bentonite. Compared with conventional feed mold inhibitors, this feed mold inhibitor has the advantages of being non-irritating and non-corrosive, having minimal impact on feed palatability, requiring a smaller dosage, and being low in cost. This product can be added to all animal feeds, and its advantages are particularly evident when added to milk and piglet feeds, as well as shrimp and crab feeds.

[0004] It can be seen from the above invention that the feed mildew inhibitor does not take into account the negative impact that mycotoxins produced during the inhibition of mold reproduction may have on livestock and poultry.

[0005] Patent CN115067458A discloses a method for biodegrading mycotoxins in feed raw materials, belonging to the field of feed additives. A mycotoxin adsorbent and a biodegrader are added to the feed raw materials to prepare feed or feed precursors, wherein the mycotoxin adsorbent includes yeast cell walls; the biodegrader includes probiotics and a complex enzyme preparation. The present invention uses yeast cell walls as the adsorption material and a probiotic complex enzyme preparation as the biodegradation material, and improves the biodegradation efficiency of mycotoxins through adsorption enrichment based on the biodegradation method.

[0006] It can be seen from the above invention that this method does not take into account the linkage between mold reproduction and mycotoxin production, and the solubility and antioxidant properties of yeast cell walls are poor, which greatly affects the adsorption effect and limits its application in the field of feed additives.

[0007] Therefore, the research on a composite antifungal agent that can be prepared by using materials such as brewer's yeast mud and can synergistically reduce the number of molds and mycotoxin content in feed has broad application prospects. Summary of the Invention

[0008] To address the shortcomings of the existing technology, the present invention utilizes the porous adsorption structure of montmorillonite K10 to aggregate mold and mycotoxins in feed. By using a compound of cinnamaldehyde and ammonium propionate, mold growth and development are inhibited while ensuring safety. Furthermore, by using brewer's yeast slurry as the raw material for the modified yeast extract, the modified yeast extract is able to better adsorb and bind mycotoxins produced by mold, thereby resolving the technical problems raised in the background art. Specifically, the technical solutions of the present invention include the following: A composite mildew inhibitor for feed is obtained by mixing modified yeast extract, cinnamaldehyde, ammonium propionate and montmorillonite K10.

[0009] Furthermore, the preparation method of the modified yeast extract comprises the following steps: After the brewer's yeast slurry was centrifuged at 5000 rpm for 10 min, the precipitate was taken to obtain crude yeast; 1 part by weight of crude yeast was mixed with 20 parts by weight of deionized water, and the mixture was centrifuged at 1000 rpm for 30 min. The precipitate was dried at 40°C for 6 h to obtain solid yeast. 1 part by weight of solid yeast and 10 parts by weight of deionized water were mixed, and after sonication at 320 W power for 40 minutes, the precipitate was dried at 60° C. for 2 hours to obtain a primary yeast extract; The primary yeast extract, deionized water, and β-mannanase were mixed and hydrolyzed at 55°C for 1 hour. The precipitate was dried at 60°C for 2 hours to obtain the secondary yeast extract. The secondary yeast extract, deionized water and compound protease were mixed and hydrolyzed at 60°C for 1 hour, and the precipitate was dried at 60°C for 2 hours to obtain the tertiary yeast extract; 1 part by weight of the third-grade yeast extract was washed with 100 parts by weight of a 75% by volume ethanol solution for 10 minutes, and then vacuum-dried at 60° C. for 6 hours to obtain a yeast extract; Yeast extract, 75% by volume ethanol solution, and sodium carbonate were mixed and stirred at 100 rpm for 10 min at 60°C to obtain an alkaline yeast extract solution; The alkaline yeast extract solution and sodium bromoacetate were mixed and stirred at 100 rpm for 4 h at 60° C. to obtain a modified yeast extract solution; The modified yeast extract solution is adjusted in pH and then filtered to obtain a crude modified yeast extract; 1 part by weight of crude modified yeast extract was washed with 100 parts by weight of 75% by volume ethanol solution for 10 minutes, and then vacuum dried at 60° C. for 6 hours to obtain modified yeast extract.

[0010] Furthermore, the mass ratio of the primary yeast extract:deionized water:β-mannanase is 80:120:1.

[0011] Furthermore, the composite protease is composed of papain, neutral protease, alkaline protease and acidic protease mixed in a mass ratio of 3:1:1:1.

[0012] Furthermore, the mass ratio of the secondary yeast extract:deionized water:complex protease is 80:120:1.

[0013] Furthermore, the mass ratio of the yeast extract: the 75% by volume ethanol solution: the sodium carbonate is 1:10:5.

[0014] Furthermore, the mass ratio of the alkaline yeast extract solution to sodium bromoacetate is 16:2.

[0015] Furthermore, the process of filtering the modified yeast extract solution after adjusting the pH includes adjusting the pH to 7 with citric acid and then filtering with a filter membrane with a pore size of 0.45 μm.

[0016] Furthermore, the mass ratio of the modified yeast extract, cinnamaldehyde, ammonium propionate and montmorillonite K10 is 2:38-40:10-12:50.

[0017] A composite antifungal agent for feed is prepared by a method for preparing a composite antifungal agent for feed.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention uses cinnamaldehyde and ammonium propionate as inhibitors, which not only has a good effect on inhibiting mold growth but also ensures the safety of the composite antifungal agent.

[0019] (2) The present invention reduces the comprehensive production cost of the composite antifungal agent and improves industrial linkage and social and economic benefits by using beer yeast mud as the raw material of the modified yeast extract.

[0020] (3) The present invention improves the solubility and antioxidant properties of yeast extract by modifying the yeast extract without using dangerous reagents, so that the yeast extract can better bind to mycotoxins and is more suitable for feed use.

[0021] (4) The present invention uses modified yeast extract as a binder to effectively adsorb and bind mycotoxins, thereby inactivating the mycotoxins and reducing the risk of mycotoxin poisoning in livestock and poultry.

[0022] (5) The present invention uses montmorillonite K10 as the base material of the composite mildew inhibitor and utilizes the porous adsorption structure of montmorillonite K10 to aggregate molds and mycotoxins, allowing them to fully contact with inhibitors and binders to improve the effect of the composite mildew inhibitor. DETAILED DESCRIPTION

[0023] The following will clearly and completely describe the technical solutions of the present invention through the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0024] Unless otherwise specified, the raw materials and reagents used in the present invention are commercially available or can be prepared by known methods.

[0025] Montmorillonite K10, β-mannanase, papain, neutral protease, alkaline protease, and acidic protease were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0026] Ammonium propionate was purchased from Jinan Anmu Biotechnology Co., Ltd.

[0027] Preparation Example 1: The preparation of modified yeast extract includes the following steps: After centrifuging 1 kg of brewer's yeast puree at 5000 rpm for 10 minutes, the precipitate was collected to obtain crude yeast. One part by weight of crude yeast and 20 parts by weight of deionized water were placed in a beaker and stirred at 50 rpm for 10 minutes. After centrifuging at 1000 rpm for 30 minutes, the precipitate was dried in a constant temperature drying oven at 40°C for 6 hours to obtain solid yeast. One part by weight of solid yeast and 10 parts by weight of deionized water were placed in a beaker and stirred at 50 rpm for 10 minutes. After ultrasonication at 320 W for 40 minutes, the precipitate was dried in a constant temperature drying oven at 60°C for 2 hours to obtain a primary yeast extract. 80 parts by weight of primary yeast extract, 120 parts by weight of deionized water, and 1 part by weight of β-mannanase were placed in a beaker, stirred at 50 rpm for 10 minutes, and then placed in a water bath. After hydrolysis at 55°C for 1 hour, the precipitate was dried in a constant temperature drying oven at 60°C for 2 hours to obtain a secondary yeast extract. 3 parts by weight of papain, 1 part by weight of neutral protease, 1 part by weight of alkaline protease, and 1 part by weight of acid protease were placed in a beaker, stirred at 50 rpm for 10 minutes to obtain a composite protease. 80 parts by weight of secondary yeast extract, 120 parts by weight of deionized water, and 1 part by weight of composite protease were placed in a beaker, stirred at 50 rpm for 10 minutes, and then placed in a water bath. After hydrolysis at 60°C for 1 hour, the precipitate was dried in a constant temperature drying oven at 60°C for 2 hours to obtain a tertiary yeast extract. After rinsing 1 part by weight of tertiary yeast extract with 100 parts by volume of 75% ethanol solution for 10 minutes, the mixture was vacuum dried in a vacuum drying oven at 60°C for 6 hours to obtain a yeast extract. After placing 1 part by weight of yeast extract, 10 parts by volume of 75% ethanol solution, and 5 parts by weight of sodium carbonate in a beaker, the beaker was placed in a water bath and stirred at 100 rpm at 60°C for 4 hours to obtain a modified yeast extract solution. Citric acid was added to the modified yeast extract solution to adjust its pH to 7. Before each pH test, the modified yeast extract solution was stirred at 100 rpm for 5 minutes. The modified yeast extract solution at pH 7 was filtered through a 0.45 μm pore size filter membrane, and the solids were collected to obtain a crude modified yeast extract. After rinsing 1 part by weight of the crude modified yeast extract with 100 parts by volume of 75% ethanol solution for 10 minutes, the mixture was vacuum dried in a vacuum drying oven at 60°C for 6 hours to obtain a modified yeast extract.

[0028] Preparation Example 2: The preparation of yeast extract includes the following steps: After centrifuging 1 kg of brewer's yeast puree at 5000 rpm for 10 minutes, the precipitate was collected to obtain crude yeast. One part by weight of crude yeast and 20 parts by weight of deionized water were placed in a beaker and stirred at 50 rpm for 10 minutes. After centrifuging at 1000 rpm for 30 minutes, the precipitate was dried in a constant temperature drying oven at 40°C for 6 hours to obtain solid yeast. One part by weight of solid yeast and 10 parts by weight of deionized water were placed in a beaker and stirred at 50 rpm for 10 minutes. After ultrasonication at 320 W for 40 minutes, the precipitate was dried in a constant temperature drying oven at 60°C for 2 hours to obtain a primary yeast extract. 80 parts by weight of primary yeast extract, 120 parts by weight of deionized water, and 1 part by weight of β-mannanase were placed in a beaker, stirred at 50 rpm for 10 minutes, and then placed in a water bath. After hydrolysis at 55°C for 1 hour, the precipitate was dried in a constant temperature drying oven at 60°C for 2 hours to obtain a secondary yeast extract. 3 parts by weight of papain, 1 part by weight of neutral protease, 1 part by weight of alkaline protease, and 1 part by weight of acid protease were placed in a beaker, stirred at 50 rpm for 10 minutes to obtain a composite protease. 80 parts by weight of secondary yeast extract, 120 parts by weight of deionized water, and 1 part by weight of composite protease were placed in a beaker, stirred at 50 rpm for 10 minutes, and then placed in a water bath. After hydrolysis at 60°C for 1 hour, the precipitate was dried in a constant temperature drying oven at 60°C for 2 hours to obtain a tertiary yeast extract. One part by weight of the third-grade yeast extract was washed with 100 parts by weight of a 75% by volume ethanol solution for 10 minutes, and then vacuum-dried in a vacuum drying oven at 60° C. for 6 hours to obtain a yeast extract.

[0029] Example 1: A method for preparing a composite mildew inhibitor for feed, specifically comprising the following steps: 2 parts by weight of the modified yeast extract of Preparation Example 1, 38 parts by weight of cinnamaldehyde, 10 parts by weight of ammonium propionate, and 50 parts by weight of montmorillonite K10 were placed in a beaker and stirred at 50 rpm for 10 minutes to allow complete adsorption and mixing. The mixture was then dried at low temperature until the weight remained constant, crushed in a powder grinder, and sieved through a 20-mesh sieve until the particles could pass through a sieve with an aperture of 0.85 mm to obtain a composite antifungal agent for feed.

[0030] Example 2: A method for preparing a composite mildew inhibitor for feed, specifically comprising the following steps: 2 parts by weight of the modified yeast extract of Preparation Example 1, 40 parts by weight of cinnamaldehyde, 10 parts by weight of ammonium propionate, and 50 parts by weight of montmorillonite K10 were placed in a beaker and stirred at 50 rpm for 10 minutes to allow complete adsorption and mixing. The mixture was then dried at low temperature until the weight remained constant, crushed in a powder grinder, and sieved through a 20-mesh sieve to obtain a composite mildew inhibitor for feed.

[0031] Example 3: A method for preparing a composite mildew inhibitor for feed, specifically comprising the following steps: 2 parts by weight of the modified yeast extract of Preparation Example 1, 38 parts by weight of cinnamaldehyde, 12 parts by weight of ammonium propionate, and 50 parts by weight of montmorillonite K10 were placed in a beaker and stirred at 50 rpm for 10 minutes to allow complete adsorption and mixing. The mixture was then dried at low temperature until the weight remained constant, crushed in a powder grinder, and sieved through a 20-mesh sieve to obtain a composite mildew inhibitor for feed.

[0032] Example 4: A method for preparing a composite mildew inhibitor for feed, specifically comprising the following steps: 2 parts by weight of the modified yeast extract of Preparation Example 1, 40 parts by weight of cinnamaldehyde, 12 parts by weight of ammonium propionate, and 50 parts by weight of montmorillonite K10 were placed in a beaker and stirred at 50 rpm for 10 minutes to allow complete adsorption and mixing. The mixture was then dried at low temperature until the weight remained constant, crushed in a powder grinder, and sieved through a 20-mesh sieve to obtain a composite mildew inhibitor for feed.

[0033] Comparative Example 1: A method for preparing a composite mildew inhibitor for feed, specifically comprising the following steps: The modified yeast extract of Preparation Example 1 in Example 3 was replaced with the yeast extract of Preparation Example 2, and the other conditions remained the same as in Example 3.

[0034] Comparative Example 2: A method for preparing a composite mildew inhibitor for feed, specifically comprising the following steps: 38 parts by weight of cinnamaldehyde, 12 parts by weight of ammonium propionate and 50 parts by weight of montmorillonite K10 were placed in a beaker and stirred at 50 rpm for 10 minutes to allow complete adsorption and mixing. The mixture was then dried at low temperature until the weight remained unchanged, placed in a powder grinder for crushing, and sieved through a 20-mesh sieve to obtain a composite mildew inhibitor for feed.

[0035] Comparative Example 3: A method for preparing a composite mildew inhibitor for feed, specifically comprising the following steps: 2 parts by weight of the modified yeast extract of Preparation Example 1, 38 parts by weight of cinnamaldehyde, 12 parts by weight of propionic acid, and 50 parts by weight of montmorillonite K10 were placed in a beaker and stirred at 50 rpm for 10 minutes to allow complete adsorption and mixing. The mixture was then dried at low temperature until the weight remained constant, crushed in a powder grinder, and sieved through a 20-mesh sieve to obtain a composite mildew inhibitor for feed.

[0036] Comparative Example 4: A method for preparing a composite mildew inhibitor for feed, specifically comprising the following steps: 2 parts by weight of the modified yeast extract of Preparation Example 1, 50 parts by weight of ammonium propionate, and 50 parts by weight of montmorillonite K10 were placed in a beaker and stirred at 50 rpm for 10 minutes to allow complete adsorption and mixing. The mixture was then dried at low temperature until the weight remained constant, crushed in a powder grinder, and sieved through a 20-mesh sieve to obtain a composite mildew inhibitor for feed.

[0037] Comparative Example 5: A method for preparing a composite mildew inhibitor for feed, specifically comprising the following steps: 2 parts by weight of the modified yeast extract of Preparation Example 1, 50 parts by weight of cinnamaldehyde, and 50 parts by weight of montmorillonite K10 were placed in a beaker and stirred at 50 rpm for 10 minutes to allow complete adsorption and mixing. The mixture was then dried at low temperature until the weight remained constant, crushed in a powder grinder, and sieved through a 20-mesh sieve to obtain a composite antifungal agent for feed.

[0038] 1000 g of identical finished pig feed was evenly divided into 10 groups, each containing 100 g. 9 of the groups were added with the composite mold inhibitor for feed obtained in Examples 1 to 4 and Comparative Examples 1 to 5 (the amount of the composite mold inhibitor for feed was 0.1% of the weight of the pig feed) and mixed evenly. The last group was used as a control group and was not treated. The feed was then spread onto 150 mm sterile culture plates and placed in the same mold incubator at a temperature of 30° C. and a humidity of 90%. The culture was carried out for a total of 30 days. Samples were taken on the 10th and 30th days, and mold counts were tested according to GB / T13092-2006 "Determination of the Total Number of Molds in Feed." The test results are shown in Table 1 below. Table 1 Mold count results While testing the mold count on the 10th and 30th days of incubation, a microplate reader was used to detect the content of representative mycotoxins: aflatoxin at absorbance values ​​at 630nm and 450nm. The test results are shown in Table 2 below: Table 2 Aflatoxin content results From the above two data we can see that: (1) The composite antifungal agents for feed of Examples 1 to 4 have good mold inhibition and mycotoxin binding properties, and can effectively prevent feed from mildewing and reduce the toxicity caused by mold.

[0039] (2) It can be seen from Comparative Example 1 that the modified yeast extract has a stronger binding effect on mycotoxins, which can effectively reduce the aflatoxin content in pig feed and play a synergistic role in inhibiting mold reproduction.

[0040] (3) Comparative Example 2 shows that although cinnamaldehyde and ammonium propionate can inhibit mold growth, mold will still release a large amount of mycotoxins during the process of slow growth or death, resulting in the overall aflatoxin content in pig feed not being effectively controlled.

[0041] (4) It can be seen from Comparative Example 3 that the stability of propionic acid is not as good as that of ammonium propionate, which greatly reduces the long-term effect of the composite antifungal agent for feed.

[0042] (5) From Comparative Example 4, it can be seen that while ammonium propionate inhibits mold growth, it also increases the release of mycotoxins. Therefore, the dosage of ammonium propionate needs to be controlled.

[0043] (6) From Comparative Example 5, it can be seen that cinnamaldehyde has a better inhibitory effect on mold growth in the early stage than ammonium propionate, and while cinnamaldehyde inhibits mold growth, it does not cause a significant increase in the release of mycotoxins. However, the stability of cinnamaldehyde is not as good as that of ammonium propionate. The use of a single cinnamaldehyde will have a significant impact on the long-term effect of the composite antifungal agent for feed.

[0044] The embodiments described above provide a detailed description of the technical solutions and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A method for preparing a composite antifungal agent for feed, characterized in that: The preparation method comprises the following steps: A composite mildew inhibitor for feed is obtained by mixing modified yeast extract, cinnamaldehyde, ammonium propionate and montmorillonite K10.

2. The method for preparing a composite mildew inhibitor for feed according to claim 1, characterized in that: The preparation method of the modified yeast extract comprises the following steps: After the brewer's yeast slurry was centrifuged at 5000 rpm for 10 min, the precipitate was taken to obtain crude yeast; 1 part by weight of crude yeast was mixed with 20 parts by weight of deionized water, and the mixture was centrifuged at 1000 rpm for 30 min. The precipitate was dried at 40°C for 6 h to obtain solid yeast. 1 part by weight of solid yeast and 10 parts by weight of deionized water were mixed, and after sonication at 320 W power for 40 minutes, the precipitate was dried at 60° C. for 2 hours to obtain a primary yeast extract; The primary yeast extract, deionized water, and β-mannanase were mixed and hydrolyzed at 55°C for 1 hour. The precipitate was dried at 60°C for 2 hours to obtain the secondary yeast extract. The secondary yeast extract, deionized water and compound protease were mixed and hydrolyzed at 60°C for 1 hour, and the precipitate was dried at 60°C for 2 hours to obtain the tertiary yeast extract; 1 part by weight of the third-grade yeast extract was washed with 100 parts by weight of a 75% by volume ethanol solution for 10 minutes, and then vacuum-dried at 60° C. for 6 hours to obtain a yeast extract; Yeast extract, 75% by volume ethanol solution, and sodium carbonate were mixed and stirred at 100 rpm for 10 min at 60°C to obtain an alkaline yeast extract solution; The alkaline yeast extract solution and sodium bromoacetate were mixed and stirred at 100 rpm for 4 h at 60° C. to obtain a modified yeast extract solution; The modified yeast extract solution is adjusted in pH and then filtered to obtain a crude modified yeast extract; 1 part by weight of crude modified yeast extract was washed with 100 parts by weight of 75% by volume ethanol solution for 10 minutes, and then vacuum dried at 60° C. for 6 hours to obtain modified yeast extract.

3. The method for preparing a composite mildew inhibitor for feed according to claim 2, characterized in that: The mass ratio of the primary yeast extract: deionized water: β-mannanase is 80:120:

1.

4. The method for preparing a composite mildew inhibitor for feed according to claim 2, characterized in that: The mass ratio of the secondary yeast extract:deionized water:complex protease is 80:120:

1.

5. A composite mildew inhibitor for feed and a preparation method thereof according to claim 2, characterized in that: The mass ratio of the yeast extract: the 75% ethanol solution by volume: the sodium carbonate is 1:10:

5.

6. A composite mildew inhibitor for feed and a preparation method thereof according to claim 2, characterized in that: The mass ratio of the alkaline yeast extract solution to sodium bromoacetate is 16:

2.

7. The method for preparing a composite mildew inhibitor for feed according to claim 2, characterized in that: The process of filtering the modified yeast extract solution after adjusting the pH includes adjusting the pH to 7 with citric acid and then filtering with a filter membrane with a pore size of 0.45 μm.

8. The method for preparing a composite antifungal agent for feed according to claim 1, characterized in that: The mass ratio of the modified yeast extract, cinnamaldehyde, ammonium propionate and montmorillonite K10 is 2:38-40:10-12:

50.

9. A composite antifungal agent for feed prepared by the method for preparing a composite antifungal agent for feed according to any one of claims 1 to 8.

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