A fermented feed containing 3-hydroxybutyric acid, and a preparation method and application thereof

Abstract

The application belongs to the technical field of animal feed, and specifically provides a preparation method of a 3-hydroxybutyric acid-containing fermented feed, which comprises the following steps: (1) destroying the structure of lignocellulose in straw to obtain pretreated raw materials; (2) enzymatically hydrolyzing the pretreated raw materials; and (3) adding Kluyveromyces marxianus to the enzymatically hydrolyzed raw materials to perform mixed fermentation, thereby obtaining the 3-hydroxybutyric acid-containing fermented feed. The preparation method of the 3-hydroxybutyric acid-containing fermented feed provided by the application is low in cost and simple in operation, and can produce functional fermented feed containing 3-hydroxybutyric acid by treating cheap feed raw materials through pretreatment, chemical method, enzymatic method and fermentation method. In the feed, only 0.5-1% of the functional fermented feed needs to be additionally added to play a health care role, and the use cost is reduced by 5-10 times compared with synthetic additives.

Description

Technical Field

[0001] This invention belongs to the field of animal feed technology, specifically relating to a fermented feed containing 3-hydroxybutyric acid, its preparation method, and its application. Background Technology

[0002] In recent years, people have paid increasing attention to reducing the use of antibiotics in feed, which has led to an increase in the incidence of animal diseases and poor growth. Currently, a common solution is to add appropriate health-promoting ingredients to feed. 3-Hydroxybutyric acid (HHB) and its derivatives, as a novel type of fermented feed for health purposes, have attracted widespread attention.

[0003] 3-Hydroxybutyric acid (HHBA) and its derivatives can improve animal growth performance and immune function, increase the efficiency of nutrient absorption and utilization, reduce the incidence of diarrhea, and improve the adaptability of animals to the environment. In addition, HHBA and its derivatives can also enhance the antioxidant capacity of animals, improve their immune function, and improve the composition of animal gut microbiota.

[0004] However, the feed industry is quite sensitive to the cost of additives and raw materials. The cost of biosynthesizing or chemically synthesizing 3-hydroxybutyric acid alone is too high, making it impossible to use on a large scale. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of high cost and complex operation in the biosynthesis or chemical synthesis of 3-hydroxybutyric acid in the prior art.

[0006] Therefore, the present invention provides a method for preparing fermented feed containing 3-hydroxybutyric acid, comprising the following steps:

[0007] (1) The structure of lignin cellulose in straw is destroyed to obtain pretreated raw materials;

[0008] (2) Enzymatic hydrolysis pretreatment of raw materials;

[0009] (3) Add Max Kluyveromyces to the enzymatically hydrolyzed raw material for mixed fermentation to obtain fermented feed containing 3-hydroxybutyric acid.

[0010] Specifically, step (1) involves crushing the straw, adding baking soda, glycerin, Tween 20 and fermented soybean meal, mixing them together, controlling the moisture content at 25-40%, and placing them at 25-30℃ for 1-4 days to obtain pretreated raw materials.

[0011] Specifically, based on the weight of straw, the amount of baking soda added is 1-3%, the amount of glycerol added is 0.5-1%, the amount of Tween 20 added is 0.1-0.5%, and the amount of fermented soybean meal added is 1-5%.

[0012] Specifically, step (2) involves adding 0.5-1% sulfuric acid, 100-500 U / g phytase, 200-1000 U / g cellulase, and 100-500 U / g xylanase to the pretreated raw materials for enzymatic hydrolysis, based on the mass of the pretreated raw materials.

[0013] Specifically, step (2) above also includes adding 0.01-0.05% calcium pantothenate, 0.01-0.05% nicotinic acid, and 0.5-1% ammonium sulfate, based on the mass of the pretreated raw materials.

[0014] Specifically, in step (2) above, the enzymatic hydrolysis temperature is 30-50℃, the enzymatic hydrolysis time is 24-48h, and the pH is maintained between 3 and 6.

[0015] Specifically, in step (3) above, the viable count of Kluyveromyces martensii is 10 based on the mass of the raw material after enzymatic hydrolysis. 6 -10 7 cfu / g.

[0016] Specifically, in step (3) above, the fermentation temperature is 25-35℃ and the fermentation time is 5-10 days.

[0017] The present invention also provides a fermented feed containing 3-hydroxybutyric acid, wherein the fermented feed is prepared by any of the preparation methods described above.

[0018] Specifically, the fermented feed containing 3-hydroxybutyric acid mentioned above can be used to prepare functional feed.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0020] The method for preparing fermented feed containing 3-hydroxybutyric acid provided by this invention processes inexpensive feed raw materials through pretreatment, chemical methods, enzymatic methods, and fermentation methods. The production of functional fermented feed containing 3-hydroxybutyric acid is low-cost and easy to operate. Only 0.5-1% of functional fermented feed needs to be added to the feed to achieve health benefits. The cost is 5-10 times lower than that of synthetic additives. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Although representative embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0022] This invention provides a method for preparing fermented feed containing 3-hydroxybutyric acid, comprising the following steps:

[0023] (1) Destroy the structure of lignin cellulose in straw to obtain pretreated raw materials; rice straw, corn straw or wheat straw can be selected according to the actual situation.

[0024] Specifically, straw raw materials are crushed, then baking soda, glycerin, Tween 20, and fermented soybean meal are added and mixed. The moisture content is controlled at 25-40%, and the mixture is placed at 25-30℃ for 1-4 days to break down the structure of lignocellulose, resulting in pretreated raw materials for subsequent fermentation. Fermented soybean meal can cross-link with lignin, reducing the inhibitory effect of lignin on enzymes in subsequent processes.

[0025] Based on the weight of straw, the addition amount of baking soda is 1-3%, glycerol is 0.5-1%, Tween 20 is 0.1-0.5%, and fermented soybean meal is 1-5%.

[0026] (2) Pretreatment of raw materials by a combination of chemical and enzymatic methods;

[0027] Specifically, based on the mass of the pretreated raw materials, add 0.5-1% sulfuric acid, 100-500 U / g phytase, 200-1000 U / g cellulase, and 100-500 U / g xylanase to the pretreated raw materials, control the moisture content to 40-50%, and enzymatically hydrolyze at 30-50℃ for 24-48 hours to allow the enzyme preparation to react fully and maintain the pH between 3 and 6.

[0028] To promote the production of 3-hydroxybutyric acid by Kluyveromyces martensii fermentation of straw during subsequent fermentation, the following ingredients were added to the pretreated raw materials in sequence: 0.5-1% sulfuric acid, 0.01-0.05% calcium pantothenate, 0.01-0.05% nicotinic acid, 0.5-1% ammonium sulfate, 100-500 U / g phytase, 200-1000 U / g cellulase, and 100-500 U / g xylanase.

[0029] (3) Add Max Kluyveromyces to the enzymatically hydrolyzed raw material for mixed fermentation to maintain the viable cell count in the raw material at 10. 6 -10 7 Fermented feed containing 3-hydroxybutyric acid is obtained by controlling the moisture content to 50-60% and fermenting at 25-35℃ for 5-10 days with cfu / g.

[0030] To facilitate the storage of fermented feed and extend its shelf life, the final samples were dried at 50-60℃.

[0031] The effects of the fermented feed containing 3-hydroxybutyric acid and its preparation method of the present invention will be studied through specific embodiments below.

[0032] Example 1:

[0033] This embodiment provides a fermented feed containing 3-hydroxybutyric acid, which is prepared using the following steps:

[0034] (1) Raw material pretreatment

[0035] Rice straw raw material is crushed, then mixed with baking soda, glycerin, Tween 20, and fermented soybean meal. The moisture content is controlled at 25%, and the mixture is left at 25°C for 2 days to break down the structure of lignocellulose, resulting in pretreated raw material for subsequent fermentation. Fermented soybean meal can cross-link with lignin, reducing the inhibitory effect of lignin on enzymes in subsequent processes.

[0036] Based on the weight of straw, the addition amounts are: 1% baking soda, 0.5% glycerol, 0.1% Tween 20, and 1% fermented soybean meal.

[0037] (2) Chemical method combined with enzymatic method treatment

[0038] Based on the mass of the pretreated raw materials, add 0.5% sulfuric acid, 0.01% calcium pantothenate, 0.01% nicotinic acid, 0.5% ammonium sulfate, 100 U / g phytase, 200 U / g cellulase, and 100 U / g xylanase to the pretreated raw materials in the following order. Control the moisture content to 40%, and enzymatically hydrolyze at 30℃ for 24 hours to ensure complete enzyme reaction and maintain the pH at 4.

[0039] (3) Fermentation

[0040] Add 20 billion CFU / g of Kluyveromyces martensii (CGMCC 10621) yeast powder to the enzymatically hydrolyzed raw material and mix it with the raw material to maintain the viable cell count in the raw material at 10. 6 The sample was fermented at 30°C for 5 days with a cfu / g concentration and a moisture content of 50%. The final sample was dried at 50°C, and the 3-hydroxybutyric acid content was determined to be 1.2% by high performance liquid chromatography.

[0041] Example 2:

[0042] This embodiment provides a fermented feed containing 3-hydroxybutyric acid, which is prepared using the following steps:

[0043] (1) Raw material pretreatment

[0044] Rice straw raw material is crushed, then mixed with baking soda, glycerin, Tween 20, and fermented soybean meal. The moisture content is controlled at 40%, and the mixture is placed at 30°C for 2 days to break down the structure of lignocellulose, resulting in pretreated raw material for subsequent fermentation. Fermented soybean meal can cross-link with lignin, reducing the inhibitory effect of lignin on enzymes in subsequent processes.

[0045] Based on the weight of straw, the addition amounts are: 1% baking soda, 0.5% glycerol, 0.1% Tween 20, and 1% fermented soybean meal.

[0046] (2) Chemical method combined with enzymatic method treatment

[0047] Based on the mass of the pretreated raw materials, add 0.5% sulfuric acid, 0.01% calcium pantothenate, 0.01% nicotinic acid, 0.5% ammonium sulfate, 100 U / g phytase, 200 U / g cellulase, and 100 U / g xylanase to the pretreated raw materials in the following order. Control the moisture content to 50%, and enzymatically hydrolyze at 50℃ for 24 hours to ensure complete enzyme reaction and maintain the pH at 5.

[0048] (3) Fermentation

[0049] Add 20 billion CFU / g of Kluyveromyces martensii (CGMCC 10621) yeast powder to the enzymatically hydrolyzed raw material and mix it with the raw material to maintain the viable cell count in the raw material at 10. 7 The sample was fermented at 30°C for 10 days with a cfu / g concentration and a moisture content of 60%. The final sample was dried at 60°C, and the 3-hydroxybutyric acid content was determined to be 2.05% by high performance liquid chromatography.

[0050] Example 3:

[0051] This embodiment provides a fermented feed containing 3-hydroxybutyric acid, which is prepared using the following steps:

[0052] (1) Raw material pretreatment

[0053] Rice straw raw material is crushed, then mixed with baking soda, glycerin, Tween 20, and fermented soybean meal. The moisture content is controlled at 40%, and the mixture is placed at 30°C for 2 days to break down the structure of lignocellulose, resulting in pretreated raw material for subsequent fermentation. Fermented soybean meal can cross-link with lignin, reducing the inhibitory effect of lignin on enzymes in subsequent processes.

[0054] Based on the weight of straw, the addition amounts are: 1% baking soda, 0.5% glycerol, 0.1% Tween 20, and 1% fermented soybean meal.

[0055] (2) Chemical method combined with enzymatic method treatment

[0056] Based on the mass of the pretreated raw materials, add 0.5% sulfuric acid, 100 U / g phytase, 200 U / g cellulase, and 100 U / g xylanase sequentially to the pretreated raw materials. Control the moisture content to 50%, and enzymatically hydrolyze at 50℃ for 24 hours to allow the enzyme preparation to react fully and maintain the pH at 5.

[0057] (3) Fermentation

[0058] Add 20 billion CFU / g of Kluyveromyces martensii (CGMCC 10621) yeast powder to the enzymatically hydrolyzed raw material and mix it with the raw material to maintain the viable cell count in the raw material at 10. 7 The sample was fermented at 30℃ for 10 days with a cfu / g concentration and a moisture content of 60%. The final sample was dried at 60℃, and the 3-hydroxybutyric acid (3-HYBA) content was determined to be 0.08% by high-performance liquid chromatography (HPLC). The production efficiency of 3-HYBA was reduced due to the absence of added nutrients such as calcium pantothenate, nicotinic acid, and ammonium sulfate during fermentation.

[0059] Example 4:

[0060] This example provides a fermented feed containing 3-hydroxybutyric acid, prepared using the following steps:

[0061] (1) Raw material pretreatment

[0062] After rice straw raw material is crushed, baking soda, glycerin and Tween 20 are added and mixed together, the moisture content is controlled at 40%, and it is placed at 30℃ for 2 days.

[0063] Based on the weight of straw, the addition amount of baking soda is 1%, glycerol is 0.5%, and Tween 20 is 0.1%.

[0064] (2) Chemical method combined with enzymatic method treatment

[0065] Based on the mass of the pretreated raw materials, add 0.5% sulfuric acid, 0.01% calcium pantothenate, 0.01% nicotinic acid, 0.5% ammonium sulfate, 100 U / g phytase, 200 U / g cellulase, and 100 U / g xylanase to the pretreated raw materials in the following order. Control the moisture content to 50%, and enzymatically hydrolyze at 50℃ for 24 hours to ensure complete enzyme reaction and maintain the pH at 5.

[0066] (3) Fermentation

[0067] Add 20 billion CFU / g of Kluyveromyces martensii (CGMCC 10621) yeast powder to the enzymatically hydrolyzed raw material and mix it with the raw material to maintain the viable cell count in the raw material at 10. 7 The sample was fermented at 30℃ for 10 days with a cfu / g concentration and a moisture content of 60%. The final sample was dried at 60℃, and the 3-hydroxybutyric acid (3-HYBA) content was determined to be 0.1% by high-performance liquid chromatography (HPLC). Unfermented soybean meal cross-links with lignin in straw, affecting enzyme activity and preventing the degradation of straw fiber into fermentable sugars, thus impacting the efficiency of 3-HYBA production during fermentation.

[0068] Example 5:

[0069] This embodiment provides a fermented feed containing 3-hydroxybutyric acid, which is prepared using the following steps:

[0070] (1) Raw material pretreatment

[0071] Rice straw raw material is crushed, then mixed with baking soda, glycerin, Tween 20, and fermented soybean meal. The moisture content is controlled at 25%, and the mixture is left at 25°C for 2 days to break down the structure of lignocellulose, resulting in pretreated raw material for subsequent fermentation. Fermented soybean meal can cross-link with lignin, reducing the inhibitory effect of lignin on enzymes in subsequent processes.

[0072] Based on the weight of straw, the addition amounts are: 1% baking soda, 0.5% glycerol, 0.1% Tween 20, and 1% fermented soybean meal.

[0073] (2) Chemical method combined with enzymatic method treatment

[0074] Based on the mass of the pretreated raw materials, add 0.5% sulfuric acid, 0.01% calcium pantothenate, 0.01% nicotinic acid, 0.5% ammonium sulfate, 100 U / g phytase, 200 U / g cellulase, and 100 U / g xylanase to the pretreated raw materials in the following order. Control the moisture content to 40%, and enzymatically hydrolyze at 30℃ for 24 hours to ensure complete enzyme reaction and maintain the pH at 4.

[0075] (3) Fermentation

[0076] Add 20 billion CFU / g of Kluyveromyces martensii (CICC 1953) yeast powder to the enzymatically hydrolyzed raw material and mix it with the raw material to maintain the viable cell count in the raw material at 10. 6 The sample was fermented at 30°C for 5 days with a cfu / g concentration and a moisture content of 50%. The final sample was dried at 50°C, and the 3-hydroxybutyric acid content was determined to be 0.08% by high performance liquid chromatography.

[0077] Comparative Example 1:

[0078] This comparative example provides a fermented feed prepared using the following steps:

[0079] (1) Raw material pretreatment

[0080] Rice straw raw material is crushed, then mixed with baking soda, glycerin, Tween 20, and fermented soybean meal. The moisture content is controlled at 25%, and the mixture is left at 25°C for 2 days to break down the structure of lignocellulose, resulting in pretreated raw material for subsequent fermentation. Fermented soybean meal can cross-link with lignin, reducing the inhibitory effect of lignin on enzymes in subsequent processes.

[0081] Based on the weight of straw, the addition amounts are: 1% baking soda, 0.5% glycerol, 0.1% Tween 20, and 1% fermented soybean meal.

[0082] (2) Chemical method combined with enzymatic method treatment

[0083] Based on the mass of the pretreated raw materials, add 0.5% sulfuric acid, 0.01% calcium pantothenate, 0.01% nicotinic acid, 0.5% ammonium sulfate, 100 U / g phytase, 200 U / g cellulase, and 100 U / g xylanase to the pretreated raw materials in the following order. Control the moisture content to 40%, and enzymatically hydrolyze at 30℃ for 24 hours to ensure complete enzyme reaction and maintain the pH at 4.

[0084] (3) Fermentation

[0085] Add 20 billion CFU / g of Kluyveromyces martensii (CICC 1765) yeast powder to the enzymatically hydrolyzed raw material and mix it with the raw material to maintain the viable cell count in the raw material at 10. 6 The sample was fermented at 30°C for 5 days with a cfu / g concentration and a moisture content of 50%. The final sample was dried at 50°C, and no 3-hydroxybutyric acid was detected by high performance liquid chromatography.

[0086] The above examples are merely illustrative of the present invention and do not constitute a limitation on the scope of protection of the present invention. All designs that are the same as or similar to the present invention are within the scope of protection of the present invention.

Claims

1. A method for preparing a fermented feed containing 3-hydroxybutyric acid, characterized in that, Includes the following steps: (1) The structure of lignocellulose in straw is destroyed to obtain pretreated raw materials; (2) Enzymatic hydrolysis pretreatment of raw materials; (3) Add Max Kluyveromyces to the enzymatically hydrolyzed raw material for mixed fermentation to obtain fermented feed containing 3-hydroxybutyric acid; The Kluyveromyces masculinus includes at least one of the following: Kluyveromyces masculinus with accession number CGMCC 10621 and Kluyveromyces masculinus with accession number CICC 1953.

2. The method for preparing fermented feed containing 3-hydroxybutyric acid as described in claim 1, characterized in that: The specific step (1) involves crushing the straw, adding baking soda, glycerin, Tween 20 and fermented soybean meal, mixing them together, controlling the moisture content at 25-40%, and placing them at 25-30℃ for 1-4 days to obtain pretreated raw materials.

3. The method for preparing fermented feed containing 3-hydroxybutyric acid as described in claim 2, characterized in that: Based on the weight of straw, the amount of baking soda added is 1-3%, the amount of glycerol added is 0.5-1%, the amount of Tween 20 added is 0.1-0.5%, and the amount of fermented soybean meal added is 1-5%.

4. The method for preparing fermented feed containing 3-hydroxybutyric acid as described in claim 1, characterized in that: Step (2) specifically involves adding 0.5-1% sulfuric acid, 100-500 U / g phytase, 200-1000 U / g cellulase, and 100-500 U / g xylanase to the pretreated raw materials based on their mass for enzymatic hydrolysis.

5. The method for preparing fermented feed containing 3-hydroxybutyric acid as described in claim 4, characterized in that: Step (2) further includes adding 0.01-0.05% calcium pantothenate, 0.01-0.05% nicotinic acid, and 0.5-1% ammonium sulfate, based on the mass of the pretreated raw materials.

6. The method for preparing fermented feed containing 3-hydroxybutyric acid as described in claim 4, characterized in that: In step (2), the enzymatic hydrolysis temperature is 30-50℃, the enzymatic hydrolysis time is 24-48h, and the pH is maintained between 3 and 6.

7. The method for preparing fermented feed containing 3-hydroxybutyric acid as described in claim 1, characterized in that: In step (3), the viable count of *Kluyveromyces martensii* is 10 based on the mass of the raw material after enzymatic hydrolysis. 6 -10 7 cfu / g.

8. The method for preparing fermented feed containing 3-hydroxybutyric acid as described in claim 1, characterized in that: The fermentation temperature in step (3) is 25-35℃ and the fermentation time is 5-10 days.

9. A fermented feed containing 3-hydroxybutyric acid, characterized in that: The fermented feed is prepared using the preparation method described in any one of claims 1-8.

10. The application of the fermented feed containing 3-hydroxybutyric acid as described in claim 9 in the preparation of functional feed.