Microbial compound feed additive for grass carp and preparation method thereof

Abstract

The invention relates to a microbial compound feed additive for grass carps and a preparation method, and belongs to the technical field of feed additives. The preparation method comprises the following steps: mixing oil tea meal and moringa seed meal, crushing, sieving, carrying out ultraviolet sterilization, adding deionized water, stirring and mixing, adding a complex microbial inoculant, mixing, fermenting, sterilizing, carrying out freeze drying, adding an ethanol solution and glutamic acid, mixing, and carrying out freeze drying to obtain the microbial compound feed additive for grass carps. Adding a composite cross-linking agent, heating and stirring, centrifuging to remove supernate, washing, drying in vacuum, ultrasonically mixing with a chitosan oligosaccharide solution, adding a catalyst, heating, centrifuging, and freeze-drying to obtain the chitosan oligosaccharide. The camellia oleifera meal and the moringa seed meal are fermented through the microbial inoculum, the content of polyphenol, polypeptide and other antioxidant substances in a fermentation product is increased, and the stability and oxidation resistance of the feed additive are remarkably improved by combining organic coating modification of glutamic acid, protocatechualdehyde, citronellal and chitosan oligosaccharide.

Description

Technical Field

[0001] The invention belongs to the technical field of feed additives and relates to a microbial composite feed additive for grass carp and a preparation method thereof. Background Art

[0002] Grass carp is a widely cultivated freshwater fish, named because it mainly feeds on aquatic plants and plant-based feed. In recent years, with the continuous development of aquaculture, traditional feed can no longer fully meet the growth needs of grass carp, and it also brings about problems such as environmental pollution. Therefore, microbial feed, as a new type of environmentally friendly feed, has gradually attracted people's attention.

[0003] Microbial feed refers to feed produced using microbial fermentation technology, and its main ingredients include beneficial microorganisms, fermentation products, and some natural plant raw materials. This feed can not only provide the nutrients needed for grass carp growth, but also improve intestinal health and enhance immunity, thereby increasing the growth rate and survival rate of grass carp. The advantages of microbial feed include: environmental protection, because it does not produce harmful substances during production; rich in nutrients, because fermentation increases the protein and vitamin content; improves immunity by regulating intestinal flora; promotes growth and improves digestion and absorption. This feed not only meets the nutritional needs of grass carp, but also improves the breeding environment, and has broad prospects.

[0004] In the process of producing microbial feed, in order to ensure that the feed is not prone to corruption and oxidation during storage and use, a large amount of artificial synthetic antioxidants are usually added to the feed. However, the use of these antioxidants not only significantly reduces the safety of food, but also has a negative impact on the meat quality of grass carp. Specifically, these chemical additives may cause the meat of grass carp to become less tender and the taste to deteriorate. In addition, the presence of these chemicals may also reduce the palatability of the feed, resulting in a decrease in the grass carp's food intake, which in turn affects its growth and development and overall health. Therefore, these factors must be carefully considered when selecting and using microbial feed to ensure the health of grass carp and food safety. Summary of the invention

[0005] The purpose of the present invention is to provide a microbial composite feed additive for grass carp and a preparation method thereof. The present invention ferments tea oil cake and moringa seed cake by using a bacterial agent to increase the content of polyphenols, polypeptides and other antioxidant substances in the fermentation product, thereby significantly improving the antioxidant performance of the feed additive; at the same time, glutamic acid and a composite cross-linking agent are used to form a chemical graft, a Schiff base structure with a strong free radical scavenging ability is introduced, and protocatechuic aldehyde and citronellal are compounded, which can significantly improve the stability and antioxidant ability of the feed additive, and an unsaturated double bond is introduced to be grafted with chitosan oligosaccharide, thereby further improving the stability of the feed additive and the ABTS+ free radical scavenging ability.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] A method for preparing a microbial composite feed additive for grass carp comprises the following steps:

[0008] S1, mixing camellia oleifera meal and moringa seed meal, crushing, sieving and ultraviolet sterilizing, adding deionized water and stirring to obtain fermentation base liquid;

[0009] S2, mixing the fermentation base liquid and the composite bacterial agent and fermenting them, sterilizing them and freeze-drying them to obtain a fermentation product;

[0010] S3, mixing the fermentation product, ethanol solution and glutamic acid, adding a composite cross-linking agent, heating and stirring, centrifuging to remove the supernatant, washing, and vacuum drying to obtain an intermediate product;

[0011] S4. Ultrasonic mixing of the intermediate product and the chitosan oligosaccharide solution, adding a catalyst for heating treatment, centrifuging, and freeze-drying to obtain the product.

[0012] As a preferred technical solution of the present invention, in step S1, the sieving is through a 40-mesh sieve; and the stirring and mixing is stirring at a speed of 120-150 r / min for 8-10 min.

[0013] As a preferred technical solution of the present invention, in step S1, the mass ratio of the camellia oleifera meal, the moringa seed meal and the deionized water is 28:10-12:50-55.

[0014] As a preferred technical solution of the present invention, in step S2, the fermentation time is 68-72 hours; the mass ratio of the fermentation base liquid to the composite bacterial agent is 40-45:25.

[0015] As a preferred technical solution of the present invention, in step S2, the composite bacterial agent includes the following components in parts by weight: 2.2-2.5 parts of Bacillus subtilis, 2.4-3.0 parts of Rhizopus oryzae B2, 1.5-1.8 parts of Aspergillus niger B3, 3-5 parts of glucose, 0.9-1.4 parts of potassium sorbate, 1-2 parts of sodium selenite, 1.3-1.8 parts of fructose and 52-60 parts of deionized water; the fructose is a mixture of cane pentasaccharide and cane hexasaccharide in a mass ratio of 1:1.3.

[0016] As a preferred technical solution of the present invention, in step S3, the heating and stirring is stirring at a temperature of 70-75°C for 6-8h; the washing is washing three times with anhydrous ethanol; and the vacuum drying is vacuum drying at a temperature of 72°C to constant weight.

[0017] As a preferred technical solution of the present invention, in step S3, the mass ratio of the fermentation product, the ethanol solution, the glutamic acid and the composite cross-linking agent is 13-15:80-90:1.8-2.1:2.5-3.0; the concentration of the ethanol solution is 70wt%; and the composite cross-linking agent is formed by mixing protocatechuic aldehyde and citronellal in a mass ratio of 2.1:1.2-1.3.

[0018] As a preferred technical solution of the present invention, in step S4, the ultrasonic mixing is performed at a power of 600-800 W for 30-40 min; and the heating treatment is performed at a temperature of 40-45° C. for 4-5 h.

[0019] As a preferred technical solution of the present invention, in step S4, the mass ratio of the intermediate product, chitosan oligosaccharide solution and catalyst is 15-18:48-50:0.07-0.08; the chitosan oligosaccharide solution is formed by mixing chitosan oligosaccharide, acetic acid and deionized water in a mass ratio of 2.1-2.4:0.1-0.2:50; and the catalyst is ammonium cerium nitrate.

[0020] The invention discloses a microbial composite feed additive for grass carp prepared by the preparation method.

[0021] Beneficial effects of the present invention:

[0022] (1) The present invention ferments camellia oleifera meal and moringa seed meal by using a bacterial agent, thereby increasing the content of polyphenols, polypeptides and other antioxidant substances in the fermentation products, thereby significantly improving the antioxidant properties of the feed additive.

[0023] (2) The present invention utilizes glutamic acid and a composite cross-linking agent to form a chemically grafted organically coated modified fermentation product, introduces a Schiff base structure with a strong free radical scavenging ability, and compounded protocatechuic aldehyde and citronellal. Protocatechuic aldehyde has an o-bisphenol structure and a high grafting rate, which can significantly improve the stability and antioxidant capacity of the feed additive. Citronellal has a hydrophobic carbon chain, which reduces the bacterial attachment and consumption of antioxidant components, and it also has a certain free radical scavenging ability. The compounding of protocatechuic aldehyde and citronellal improves the stability of each other, while introducing an unsaturated double bond that can be grafted with chitosan oligosaccharides, further improving the stability of the feed additive and the ABTS+ free radical scavenging ability. DETAILED DESCRIPTION

[0024] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation methods, structures, features and effects of the present invention are described in detail below in combination with the embodiments.

[0025] Example 1

[0026] A method for preparing a microbial composite feed additive for grass carp comprises the following steps:

[0027] S1, after mixing the camellia oleifera meal and the moringa seed meal, crushing, sieving and ultraviolet sterilizing, adding deionized water and stirring and mixing to obtain a fermentation base liquid; the sieving is through a 40-mesh sieve; the stirring and mixing is stirring at a speed of 120r / min for 8min; the mass ratio of the camellia oleifera meal, the moringa seed meal and the deionized water is 28:10:50;

[0028] S2, the fermentation base liquid and the composite bacterial agent are mixed and fermented, and freeze-dried after sterilization to obtain a fermentation product; the fermentation time is 68h; the mass ratio of the fermentation base liquid and the composite bacterial agent is 40:25; the composite bacterial agent comprises the following components in weight: 2.2 parts of Bacillus subtilis, 2.4 parts of Rhizopus oryzae B2, 1.5 parts of Aspergillus niger B3, 3 parts of glucose, 0.9 parts of potassium sorbate, 1 part of sodium selenite, 1.3 parts of fructose and 52 parts of deionized water; the fructose is mixed with cane pentasaccharide and cane hexasaccharide in a mass ratio of 1:1.3;

[0029] S3, after mixing the fermentation product, ethanol solution and glutamic acid, adding a composite cross-linking agent, heating and stirring, centrifuging to remove the supernatant, washing, vacuum drying, and obtaining an intermediate product; the heating and stirring is stirring at 70°C for 6 hours; the washing is washing three times with anhydrous ethanol; the vacuum drying is vacuum drying at 72°C to constant weight; the mass ratio of the fermentation product, ethanol solution, glutamic acid and composite cross-linking agent is 13:80:1.8:2.5; the concentration of the ethanol solution is 70wt%; the composite cross-linking agent is a mixture of protocatechuic aldehyde and citronellal in a mass ratio of 2.1:1.2;

[0030] S4. Ultrasonic mixing of the intermediate product and the chitosan oligosaccharide solution, adding a catalyst for heat treatment, centrifuging, and freeze-drying to obtain the product; the ultrasonic mixing is performed at a power of 600 W for 30 minutes; the heat treatment is performed at a temperature of 40° C. for 4 hours; the mass ratio of the intermediate product, the chitosan oligosaccharide solution, and the catalyst is 15:48:0.07; the chitosan oligosaccharide solution is prepared by mixing chitosan oligosaccharide, acetic acid, and deionized water in a mass ratio of 2.1:0.1:50; and the catalyst is ammonium cerium nitrate.

[0031] Example 2

[0032] A method for preparing a microbial composite feed additive for grass carp comprises the following steps:

[0033] S1, after mixing the camellia oleifera meal and the moringa seed meal, crushing, sieving and ultraviolet sterilizing, adding deionized water and stirring and mixing to obtain a fermentation base liquid; the sieving is through a 40-mesh sieve; the stirring and mixing is stirring at a speed of 135r / min for 9min; the mass ratio of the camellia oleifera meal, the moringa seed meal and the deionized water is 28:11:52;

[0034] S2, the fermentation base liquid and the composite bacterial agent are mixed and fermented, and freeze-dried after sterilization to obtain a fermentation product; the fermentation time is 70h; the mass ratio of the fermentation base liquid and the composite bacterial agent is 42:25; the composite bacterial agent comprises the following components in weight: 2.4 parts of Bacillus subtilis, 2.7 parts of Rhizopus oryzae B2, 1.6 parts of Aspergillus niger B3, 4 parts of glucose, 1.2 parts of potassium sorbate, 1.5 parts of sodium selenite, 1.6 parts of fructose and 56 parts of deionized water; the fructose is mixed by sucrose pentasaccharide and sucrose hexasaccharide in a mass ratio of 1:1.3;

[0035] S3, after mixing the fermentation product, ethanol solution and glutamic acid, adding a composite cross-linking agent, heating and stirring, centrifuging to remove the supernatant, washing, vacuum drying, and obtaining an intermediate product; the heating and stirring is stirring at 72°C for 7h; the washing is washing three times with anhydrous ethanol; the vacuum drying is vacuum drying at 72°C to constant weight; the mass ratio of the fermentation product, ethanol solution, glutamic acid and composite cross-linking agent is 14:85:2:2.8; the concentration of the ethanol solution is 70wt%; the composite cross-linking agent is a mixture of protocatechuic aldehyde and citronellal in a mass ratio of 2.1:1.25;

[0036] S4. Ultrasonic mixing of the intermediate product and the chitosan oligosaccharide solution, adding a catalyst for heat treatment, centrifuging, and freeze-drying to obtain the product; the ultrasonic mixing is performed at a power of 700 W for 35 minutes; the heat treatment is performed at a temperature of 42° C. for 4.5 hours; the mass ratio of the intermediate product, the chitosan oligosaccharide solution, and the catalyst is 16:49:0.075; the chitosan oligosaccharide solution is prepared by mixing chitosan oligosaccharide, acetic acid, and deionized water in a mass ratio of 2.2:0.15:50; and the catalyst is ammonium cerium nitrate.

[0037] Example 3

[0038] A method for preparing a microbial composite feed additive for grass carp comprises the following steps:

[0039] S1, after mixing the camellia oleifera meal and the moringa seed meal, crushing, sieving and ultraviolet sterilizing, adding deionized water and stirring and mixing to obtain a fermentation base liquid; the sieving is through a 40-mesh sieve; the stirring and mixing is stirring at a speed of 150r / min for 10min; the mass ratio of the camellia oleifera meal, the moringa seed meal and the deionized water is 28:12:55;

[0040] S2, mixing the fermentation base liquid and the composite bacterial agent and fermenting them, sterilizing and freeze-drying them to obtain a fermentation product; the fermentation time is 72h; the mass ratio of the fermentation base liquid and the composite bacterial agent is 45:25; the composite bacterial agent comprises the following components in weight: 2.5 parts of Bacillus subtilis, 3.0 parts of Rhizopus oryzae B2, 1.8 parts of Aspergillus niger B3, 5 parts of glucose, 1.4 parts of potassium sorbate, 2 parts of sodium selenite, 1.8 parts of fructose and 60 parts of deionized water; the fructose is mixed with cane pentasaccharide and cane hexasaccharide in a mass ratio of 1:1.3;

[0041] S3, after mixing the fermentation product, ethanol solution and glutamic acid, adding a composite cross-linking agent, heating and stirring, centrifuging to remove the supernatant, washing, vacuum drying, and obtaining an intermediate product; the heating and stirring is stirring at 75°C for 8 hours; the washing is washing three times with anhydrous ethanol; the vacuum drying is vacuum drying at 72°C to constant weight; the mass ratio of the fermentation product, ethanol solution, glutamic acid and composite cross-linking agent is 15:90:2.1:3.0; the concentration of the ethanol solution is 70wt%; the composite cross-linking agent is a mixture of protocatechuic aldehyde and citronellal in a mass ratio of 2.1:1.3;

[0042] S4. Ultrasonic mixing of the intermediate product and the chitosan oligosaccharide solution, adding a catalyst for heat treatment, centrifuging, and freeze-drying to obtain the product; the ultrasonic mixing is performed at 800 W for 40 minutes; the heat treatment is performed at 45° C. for 5 hours; the mass ratio of the intermediate product, the chitosan oligosaccharide solution, and the catalyst is 18:50:0.08; the chitosan oligosaccharide solution is prepared by mixing chitosan oligosaccharide, acetic acid, and deionized water in a mass ratio of 2.4:0.2:50; and the catalyst is ammonium cerium nitrate.

[0043] Comparative Example 1

[0044] Compared with Example 2, Comparative Example 1 is different in that glutamic acid is not used, and the other components, preparation steps and parameters are the same.

[0045] Comparative Example 2

[0046] Compared with Example 2, the difference in Comparative Example 2 is that citronellal replaces protocatechuic aldehyde, and the other components, preparation steps and parameters are the same.

[0047] Comparative Example 3

[0048] Compared with Example 2, Comparative Example 3 is different in that protocatechuic aldehyde replaces citronellal, and the other components, preparation steps and parameters are the same.

[0049] Comparative Example 4

[0050] Compared with Example 2, Comparative Example 4 is different in that chitosan oligosaccharide is not used, and the remaining components, preparation steps and parameters are the same.

[0051] Comparative Example 5

[0052] Compared with Example 2, the difference in Comparative Example 5 is that ultrasonic mixing is not performed in step S4, and the remaining components, preparation steps and parameters are the same.

[0053] The feed additives prepared in Examples 1-3 and Comparative Examples 1-5 were subjected to ABTS + Free radical scavenging ability test:

[0054] Weigh 6.6 mg of potassium persulfate and 38.5 mg of ABTS, dissolve them in 5 ml of deionized water, mix the two solutions, and react them in a dark environment for 12 hours to use this as the mother solution. Take a small amount from the mother solution and dilute it with methanol until its absorbance at a wavelength of 734 nm reaches the range of 0.70±0.02, which is the working solution. Take 10 μl of the fermentation product to be tested, add 990 μl of the working solution, let it react in the dark for 10 minutes, and then measure the absorbance value of the mixed solution at a wavelength of 734 nm.

[0055] ABTS + Free radical scavenging rate = (A 空白 -A 样品 ) / A 空白 *100%

[0056] Table 1

[0057] Clearance rate (%) Example 1 91.6 Example 2 93.4 Example 3 92.7 Comparative Example 1 78.8 Comparative Example 2 81.6 Comparative Example 3 80.5 Comparative Example 4 83.1 Comparative Example 5 85.7

[0058] From the test results in Table 1, it can be seen that compared with Comparative Examples 1-5, the feed additives prepared in Examples 1-3 of the present invention have excellent ABTS+ free radical scavenging ability.

[0059] The present invention utilizes glutamic acid and a composite cross-linking agent to form a chemical graft, introduces a Schiff base structure with a strong free radical scavenging ability, and compounding protocatechuic aldehyde and citronellal. Protocatechuic aldehyde has an o-bisphenol structure and a high grafting rate, and can significantly improve the stability and antioxidant capacity of feed additives. Citronellal has a hydrophobic carbon chain, reduces bacterial attachment and consumption of antioxidant components, and itself also has a certain free radical scavenging ability. Protocatechuic aldehyde and citronellal are compounded to improve each other's stability, and at the same time, an unsaturated double bond is introduced to be grafted with chitosan oligosaccharide, thereby further improving the stability of the feed additive and the ABTS+ free radical scavenging ability.

[0060] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any indirect modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a microbial compound feed additive for grass carp, characterized in that: The preparation method comprises the following steps: S1, mixing camellia oleifera meal and moringa seed meal, crushing, sieving and ultraviolet sterilizing, adding deionized water and stirring to obtain fermentation base liquid; S2, mixing the fermentation base liquid and the composite bacterial agent and fermenting them, sterilizing them and freeze-drying them to obtain a fermentation product; S3, mixing the fermentation product, ethanol solution and glutamic acid, adding a composite cross-linking agent, heating and stirring, centrifuging to remove the supernatant, washing, and vacuum drying to obtain an intermediate product; S4. Ultrasonic mixing of the intermediate product and the chitosan oligosaccharide solution, adding a catalyst for heating treatment, centrifuging, and freeze-drying to obtain the product.

2. The microbial compound feed additive for grass carp according to claim 1, characterized in that: In step S1, the sieving is through a 40-mesh sieve; and the stirring and mixing is stirring at a speed of 120-150 r / min for 8-10 min.

3. The microbial compound feed additive for grass carp according to claim 1, characterized in that: In step S1, the mass ratio of the camellia oleifera meal, the moringa seed meal and the deionized water is 28:10-12:50-55.

4. The microbial compound feed additive for grass carp according to claim 1, characterized in that: In step S2, the fermentation time is 68-72 hours; the mass ratio of the fermentation base liquid to the composite bacterial agent is 40-45:

25.

5. The microbial compound feed additive for grass carp according to claim 1, characterized in that: In step S2, the composite bacterial agent includes the following components in parts by weight: 2.2-2.5 parts of Bacillus subtilis, 2.4-3.0 parts of Rhizopus oryzae B2, 1.5-1.8 parts of Aspergillus niger B3, 3-5 parts of glucose, 0.9-1.4 parts of potassium sorbate, 1-2 parts of sodium selenite, 1.3-1.8 parts of fructose and 52-60 parts of deionized water; the fructose is a mixture of cane pentasaccharide and cane hexasaccharide in a mass ratio of 1:1.

3.

6. The microbial compound feed additive for grass carp according to claim 1, characterized in that: In step S3, the heating and stirring is stirring at a temperature of 70-75°C for 6-8h; the washing is washing three times with anhydrous ethanol; and the vacuum drying is vacuum drying at a temperature of 72°C to constant weight.

7. The microbial compound feed additive for grass carp according to claim 1, characterized in that: In step S3, the mass ratio of the fermentation product, ethanol solution, glutamic acid and composite cross-linking agent is 13-15:80-90:1.8-2.1:2.5-3.0; the concentration of the ethanol solution is 70wt%; the composite cross-linking agent is a mixture of protocatechuic aldehyde and citronellal in a mass ratio of 2.1:1.2-1.

3.

8. The microbial compound feed additive for grass carp according to claim 1, characterized in that: In step S4, the ultrasonic mixing is performed at a power of 600-800 W for 30-40 min; and the heating treatment is performed at a temperature of 40-45° C. for 4-5 h.

9. The microbial compound feed additive for grass carp according to claim 1, characterized in that: In step S4, the mass ratio of the intermediate product, chitosan oligosaccharide solution and catalyst is 15-18:48-50:0.07-0.08; the chitosan oligosaccharide solution is prepared by mixing chitosan oligosaccharide, acetic acid and deionized water in a mass ratio of 2.1-2.4:0.1-0.2:50; and the catalyst is ammonium cerium nitrate.

10. A microbial compound feed additive for grass carp prepared by the preparation method according to any one of claims 1 to 9.