Fermented feed containing 3-hydroxybutyric acid as well as preparation method and application of fermented feed

Abstract

The invention belongs to the technical field of animal feeds, and particularly provides a preparation method of a 3-hydroxybutyric acid-containing fermented feed, which comprises the following steps: (1) destroying the structure of lignocellulose in straws to obtain a pretreated raw material; (2) performing enzymolysis pretreatment on the raw materials; and (3) adding Kluyveromyces marxianus into the raw materials subjected to enzymolysis, and carrying out mixed fermentation to obtain the fermented feed containing 3-hydroxybutyric acid. According to the preparation method of the fermented feed containing 3-hydroxybutyric acid, cheap feed raw materials are treated through raw material pretreatment, a chemical method, an enzyme method and a fermentation method, the production cost of the functional fermented feed containing 3-hydroxybutyric acid is low, operation is easy and convenient, the health care effect can be achieved only by additionally adding 0.5-1% of the functional fermented feed into the feed, and the production cost of the functional fermented feed containing 3-hydroxybutyric acid is low. And the use cost is reduced by 5-10 times compared with that of a synthetic additive.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal feed, and particularly relates to a fermented feed containing 3-hydroxybutyric acid, a preparation method thereof, and an application thereof. Background Art

[0002] In recent years, people have paid more and more attention to reducing the use of antibiotics in feed. Subsequently, problems such as the increasing incidence of animal diseases and the incidence of poor growth states have emerged. Currently, the commonly used solution is to add appropriate health care components to the feed. 3-Hydroxybutyric acid and its derivatives, as a new type of health care fermented feed, have attracted wide attention.

[0003] 3-Hydroxybutyric acid and its derivatives can improve the growth performance and immune function of animals, increase the absorption and utilization efficiency of nutrients by animals, reduce the incidence of diarrhea, and improve the adaptability of animals to the environment. In addition, 3-hydroxybutyric acid and its derivatives can also enhance the antioxidant capacity of animals, improve their immune function, and improve the intestinal microbial composition of animals.

[0004] However, the feed industry is sensitive to the costs of additives and raw materials. The cost of separately biosynthesizing or chemically synthesizing 3-hydroxybutyric acid is relatively high, and it cannot be applied on a large scale. Summary of the Invention

[0005] The object of the present 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] To this end, the present invention provides a preparation method of a fermented feed containing 3-hydroxybutyric acid, comprising the following steps:

[0007] (1) Destroy the structure of lignocellulose in straw to obtain a pretreated raw material;

[0008] (2) Enzymatically hydrolyze the pretreated raw material;

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

[0010] Specifically, the above step (1) is specifically that after the straw is crushed, baking soda, glycerol, Tween 20, and fermented soybean meal are added and mixed, the water content is controlled at 25-40%, and it is placed at 25-30 °C for 1-4 days to obtain a pretreated raw material.

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

[0012] Specifically, step (2) above is specifically to 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 for enzymatic hydrolysis based on the mass of the pretreated raw materials.

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

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

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

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

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

[0018] Specifically, the fermented feed containing 3 - hydroxybutyric acid described 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 preparation method of the fermented feed containing 3 - hydroxybutyric acid provided by the present invention processes cheap feed raw materials through raw material pretreatment, chemical method, enzymatic method, and fermentation method. The production of the functional fermented feed containing 3 - hydroxybutyric acid has low cost and simple operation. Only an additional 0.5 - 1% of the functional fermented feed needs to be added to the feed to play a health - care role, and the use cost is reduced by 5 - 10 times compared with synthetic additives. Detailed Embodiments

[0021] The technical solutions in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Although the representative embodiments of the present invention have been described in detail, those of ordinary skill in the technical field to which the present invention belongs 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] The present invention provides a method for preparing a fermented feed containing 3-hydroxybutyric acid, comprising the following steps:

[0023] (1) Destroy the structure of lignocellulose in the straw to obtain a pretreated raw material; the straw can be selected from rice straw, corn straw, and wheat straw according to actual conditions.

[0024] Specifically, the straw raw material is pulverized, and baking soda, glycerol, Tween 20, and fermented soybean meal are added thereto and mixed, and the moisture is controlled at 25-40%, and it is placed at 25-30 °C for 1-4 days to destroy the structure of lignocellulose to obtain a pretreated raw material for subsequent fermentation. The fermented soybean meal can crosslink with lignin to reduce the inhibitory effect of lignin on enzymes in subsequent processes.

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

[0026] (2) Treat the pretreated raw material by a chemical method combined with an enzymatic method;

[0027] Specifically, based on the mass of the pretreated raw material, 0.5-1% sulfuric acid, 100-500 U / g phytase, 200-1000 U / g cellulase, and 100-500 U / g xylanase are added to the pretreated raw material, the moisture is controlled at 40-50%, and enzymatic hydrolysis is carried out at 30-50 °C for 24-48 hours to make the enzyme preparation fully react and maintain the pH between 3 and 6.

[0028] In order to promote the fermentation of straw by Kluyveromyces marxianus to produce 3-hydroxybutyric acid in the subsequent fermentation process, 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 are sequentially added to the pretreated raw material.

[0029] (3) Add Kluyveromyces marxianus to the enzymatically hydrolyzed raw material for mixed fermentation to keep the viable bacteria count in the raw material at 10 6 -10 7 cfu / g, control the moisture at 50-60%, and ferment at 25-35 °C for 5-10 days to obtain a fermented feed containing 3-hydroxybutyric acid.

[0030] For the convenience of storing the fermented feed and increasing the storage period, the final sample is dried at 50-60 °C.

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

[0032] Example 1:

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

[0034] (1) Pretreatment of raw materials

[0035] After the rice straw raw material is pulverized, baking soda, glycerol, Tween 20 and fermented soybean meal are added thereto and mixed, and the moisture is controlled at 25%, and it is placed at 25 °C for 2 days to destroy the structure of lignocellulose to obtain a pretreated raw material for subsequent fermentation. Fermented soybean meal can produce a cross-linking effect with lignin, reducing the inhibitory effect of lignin on enzymes in subsequent processes.

[0036] Based on the mass of the straw, the addition amount of baking soda is 1%, the addition amount of glycerol is 0.5%, the addition amount of Tween 20 is 0.1%, and the addition amount of fermented soybean meal is 1%.

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

[0038] Based on the mass of the pretreated raw material, 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 are sequentially added to the pretreated raw material. The moisture is controlled at 40%, and enzymatic hydrolysis is carried out at 30 °C for 24 hours to enable the enzyme preparation to fully react and maintain the pH at 4.

[0039] (3) Fermentation

[0040] 20 billion cfu / g of Kluyveromyces marxianus (CGMCC 10621) bacterial powder is added to the enzymatically hydrolyzed raw material and mixed with the raw material to keep the viable bacteria count in the raw material at 10 6 cfu / g, control the moisture at 50%, ferment at 30 °C for 5 days, and finally dry the sample at 50 °C. The content of 3-hydroxybutyric acid in it is detected by high performance liquid chromatography to be 1.2%.

[0041] Example 2:

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

[0043] (1) Pretreatment of raw materials

[0044] After the rice straw raw material is pulverized, baking soda, glycerol, Tween 20 and fermented soybean meal are added thereto and mixed, and the moisture is controlled at 40%, and it is placed at 30 °C for 2 days to destroy the structure of lignocellulose to obtain a pretreated raw material for subsequent fermentation. Fermented soybean meal can produce a cross-linking effect with lignin, reducing the inhibitory effect of lignin on enzymes in subsequent processes.

[0045] Based on the straw quality, the addition amount of sodium bicarbonate is 1%, the addition amount of glycerol is 0.5%, the addition amount of Tween 20 is 0.1%, and the addition amount of fermented soybean meal is 1%.

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

[0047] Based on the mass of the pretreated raw material, 0.5% sulfuric acid, 0.01% calcium pantothenate, 0.01% niacin, 0.5% ammonium sulfate, 100 U / g phytase, 200 U / g cellulase, and 100 U / g xylanase are sequentially added to the pretreated raw material. Control the moisture content to be 50%, and carry out enzymatic hydrolysis at 50 °C for 24 hours to make the enzyme preparation fully react and maintain the pH at 5.

[0048] (3) Fermentation

[0049] Add the bacterial powder of Kluyveromyces marxianus (CGMCC 10621) at 20 billion cfu / g to the enzymatically hydrolyzed raw material and mix it with the raw material to keep the viable bacteria count in the raw material at 10 7 cfu / g, control the moisture content to be 60%, ferment at 30 °C for 10 days, and finally dry the sample at 60 °C. The content of 3-hydroxybutyric acid in it is detected by high performance liquid chromatography to be 2.05%.

[0050] Example 3:

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

[0052] (1) Raw material pretreatment

[0053] After the rice straw raw material is pulverized, sodium bicarbonate, glycerol, Tween 20 and fermented soybean meal are added to it and mixed. Control the moisture content to be 40%, and place it at 30 °C for 2 days to break the structure of lignocellulose to obtain the pretreated raw material for subsequent fermentation. Fermented soybean meal can produce a cross-linking effect with lignin to reduce the inhibitory effect of lignin on enzymes in subsequent processes.

[0054] Based on the straw quality, the addition amount of sodium bicarbonate is 1%, the addition amount of glycerol is 0.5%, the addition amount of Tween 20 is 0.1%, and the addition amount of fermented soybean meal is 1%.

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

[0056] Based on the mass of the pretreated raw material, 0.5% sulfuric acid, 100 U / g phytase, 200 U / g cellulase, and 100 U / g xylanase are sequentially added to the pretreated raw material. Control the moisture content to be 50%, and carry out enzymatic hydrolysis at 50 °C for 24 hours to make the enzyme preparation fully react and maintain the pH at 5.

[0057] (3) Fermentation

[0058] Add the Kluyveromyces marxianus (CGMCC 10621) bacterial powder with 20 billion cfu / g to the enzymatically hydrolyzed raw material and mix it with the raw material to keep the viable bacteria count in the raw material at 10 7 cfu / g, control the moisture content to 60%, ferment at 30°C for 10 days, finally dry the sample at 60°C, and detect the 3-hydroxybutyric acid content in it by high performance liquid chromatography, which is 0.08%. Since no nutritional elements such as calcium pantothenate, nicotinic acid, and ammonium sulfate are added during the fermentation process, the production effect of 3-hydroxybutyric acid has decreased.

[0059] Example 4:

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

[0061] (1) Raw material pretreatment

[0062] After the rice straw raw material is pulverized, add baking soda, glycerol, and Tween 20 to it and mix, control the moisture content at 40%, and place it at 30°C for 2 days.

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

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

[0065] Based on the mass of the pretreated raw material, 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 material in sequence. Control the moisture content at 50%, enzymatically hydrolyze at 50°C for 24 hours to make the enzyme preparation fully react and maintain the pH at 5.

[0066] (3) Fermentation

[0067] Add the Kluyveromyces marxianus (CGMCC 10621) bacterial powder with 20 billion cfu / g to the enzymatically hydrolyzed raw material and mix it with the raw material to keep the viable bacteria count in the raw material at 10 7 cfu / g, control the moisture content to 60%, ferment at 30°C for 10 days, finally dry the sample at 60°C, and detect the 3-hydroxybutyric acid content in it by high performance liquid chromatography, which is 0.1%. The cross-linking of lignin in unfermented soybean meal and straw will affect the enzyme activity and prevent the fibers in the straw from being degraded into fermentable sugars, thus affecting the efficiency of fermenting to produce 3-hydroxybutyric acid.

[0068] Example 5:

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

[0070] (1) Raw material pretreatment

[0071] After the rice straw raw material is crushed, baking soda, glycerol, Tween 20 and fermented soybean meal are added thereto and mixed, and the moisture is controlled at 25%, and it is placed at 25°C for 2 days to break the structure of lignocellulose to obtain a pretreated raw material for subsequent fermentation. Fermented soybean meal can produce a cross-linking effect with lignin, reducing the inhibitory effect of lignin on enzymes in subsequent processes.

[0072] Based on the mass of the straw, the addition amount of baking soda is 1%, the addition amount of glycerol is 0.5%, the addition amount of Tween 20 is 0.1%, and the addition amount of fermented soybean meal is 1%.

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

[0074] Based on the mass of the pretreated raw material, 0.5% sulfuric acid, 0.01% calcium pantothenate, 0.01% niacin, 0.5% ammonium sulfate, 100 U / g phytase, 200 U / g cellulase, and 100 U / g xylanase are sequentially added to the pretreated raw material. The moisture is controlled at 40%, and enzymatic hydrolysis is carried out at 30°C for 24 hours to make the enzyme preparation react fully and maintain the pH at 4.

[0075] (3) Fermentation

[0076] The bacterial powder of Kluyveromyces marxianus (CICC 1953) at 20 billion cfu / g is added to the enzymatically hydrolyzed raw material and mixed with the raw material to keep the viable bacteria count in the raw material at 10 6 cfu / g, control the moisture at 50%, ferment at 30°C for 5 days, and finally dry the sample at 50°C. The content of 3-hydroxybutyric acid in it is detected by high performance liquid chromatography to be 0.08%.

[0077] Comparative Example 1:

[0078] This comparative example provides a fermented feed, which is prepared by the following steps:

[0079] (1) Raw material pretreatment

[0080] After the rice straw raw material is crushed, baking soda, glycerol, Tween 20 and fermented soybean meal are added thereto and mixed, and the moisture is controlled at 25%, and it is placed at 25°C for 2 days to break the structure of lignocellulose to obtain a pretreated raw material for subsequent fermentation. Fermented soybean meal can produce a cross-linking effect with lignin, reducing the inhibitory effect of lignin on enzymes in subsequent processes.

[0081] Based on the mass of the straw, the addition amount of baking soda is 1%, the addition amount of glycerol is 0.5%, the addition amount of Tween 20 is 0.1%, and the addition amount of fermented soybean meal is 1%.

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

[0083] Based on the mass of the pretreated raw material, 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 were sequentially added to the pretreated raw material. The moisture was controlled at 40%, and enzymatic hydrolysis was carried out at 30°C for 24 hours to allow the enzyme preparation to fully react and maintain the pH at 4.

[0084] (3) Fermentation

[0085] 20 billion cfu / g of Kluyveromyces marxianus (CICC 1765) bacterial powder was added to the enzymatically hydrolyzed raw material and mixed with the raw material to keep the viable bacteria count in the raw material at 10 6 cfu / g. The moisture was controlled at 50%, and fermentation was carried out at 30°C for 5 days. The final sample was dried at 50°C, and no 3-hydroxybutyric acid content was detected by high-performance liquid chromatography.

[0086] The above examples are only illustrative of the present invention and do not constitute a limitation on the protection scope of the present invention. Any design identical or similar to the present invention falls within the protection scope of the present invention.

Claims

1. A preparation method of a fermented feed containing 3-hydroxybutyric acid, characterized in that, It includes the following steps: (1) Destroy the structure of lignocellulose in straw to obtain a pretreated raw material; (2) Enzymatically hydrolyze the pretreated raw material; (3) Add Kluyveromyces marxianus to the enzymatically hydrolyzed raw material for mixed fermentation to obtain a fermented feed containing 3-hydroxybutyric acid.

2. The preparation method of the fermented feed containing 3-hydroxybutyric acid according to claim 1, characterized in that: The specific operation of step (1) is that after the straw is crushed, baking soda, glycerol, Tween 20 and fermented soybean meal are added and mixed, the moisture is controlled at 25-40%, and it is placed at 25-30 °C for 1-4 days to obtain a pretreated raw material.

3. The preparation method of the fermented feed containing 3-hydroxybutyric acid according to claim 2, characterized in that: Calculated by the mass of straw, the addition amount of baking soda is 1-3%, the addition amount of glycerol is 0.5-1%, the addition amount of Tween 20 is 0.1-0.5%, and the addition amount of fermented soybean meal is 1-5%.

4. The preparation method of the fermented feed containing 3-hydroxybutyric acid according to claim 1, characterized in that: The specific operation of step (2) is that calculated by the mass of the pretreated raw material, 0.5-1% sulfuric acid, 100-500 U / g phytase, 200-1000 U / g cellulase, and 100-500 U / g xylanase are added to the pretreated raw material for enzymatic hydrolysis.

5. The preparation method of the fermented feed containing 3-hydroxybutyric acid according to claim 4, wherein: Step (2) also includes adding 0.01-0.05% calcium pantothenate, 0.01-0.05% nicotinic acid, and 0.5-1% ammonium sulfate calculated by the mass of the pretreated raw material.

6. The preparation method of the fermented feed containing 3-hydroxybutyric acid according to claim 4, characterized in that: In step (2), the enzymatic hydrolysis temperature is 30-50 °C, the enzymatic hydrolysis time is 24-48 h, and the pH is maintained between 3 and 6.

7. The preparation method of the fermented feed containing 3-hydroxybutyric acid according to claim 1, characterized in that: In step (3), based on the mass of the raw material after enzymatic hydrolysis, the viable cell count of Kluyveromyces marxianus is 10 6 -10 7 cfu / g.

8. The preparation method of the fermented feed containing 3-hydroxybutyric acid according to claim 1, characterized in that: In step (3), the fermentation temperature is 25-35 °C, and the fermentation time is 5-10 days.

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

10. Use of the fermented feed containing 3-hydroxybutyric acid according to claim 9 in the preparation of a functional feed.