Preparation method and application of ginkgo leaf feed additive

Abstract

The invention provides a preparation method and application of a ginkgo leaf feed additive, and the preparation method comprises the following steps: S1, mixing ginkgo leaf powder with an alkali treatment agent, standing and sealing, adding an acid solution, adjusting the pH value to 4.0-6.0, and filtering to obtain treated ginkgo leaves; s2, adding a compound enzyme preparation into the treated ginkgo leaves, and performing enzymolysis to obtain the ginkgo leaves subjected to enzymolysis; s3, adding a compound fermentation inoculant into the ginkgo leaves subjected to enzymolysis, and performing anaerobic fermentation and drying to obtain the ginkgo leaf feed additive. Active ingredients of the ginkgo leaves can be fully released, the content of ginkgolic acid is reduced, and meanwhile beneficial metabolites are generated; the feed additive is applied to broiler feed, can effectively promote broiler growth, improve broiler intestinal health and enhance immunity, and can replace antibiotics in the feed.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of feed, and particularly relates to a preparation method of a ginkgo leaf feed additive and application thereof. BACKGROUND

[0002] In intensive broiler breeding, antibiotics growth promoters have been widely used for disease prevention and growth promotion, but they have caused serious problems such as bacterial drug resistance and drug residues, which seriously threaten public health. Therefore, it is an urgent need of the industry to find safe and efficient, sustainable antibiotic alternatives.

[0003] Ginkgo leaves are rich in flavonoids, terpene lactones, polysaccharides and other active ingredients, and have antioxidant, anti-inflammatory, circulation improvement and antibacterial effects, and are considered as a potential plant resource for antibiotic alternatives. However, there are problems such as high fiber content, poor palatability, difficulty in direct utilization of flavonoid components, and toxic side effects of ginkgo acid when ginkgo leaves are directly used in broiler feed. Existing feed applications mostly rely on physical extraction, which has the limitations of insufficient release of active ingredients and insufficient detoxification. SUMMARY

[0004] Therefore, the application provides a preparation method of a ginkgo leaf feed additive and application thereof, which can fully release the active ingredients of ginkgo leaves, reduce the content of ginkgo acid, and generate beneficial metabolites at the same time. When applied to broiler feed, it can effectively promote the growth of broilers, improve the intestinal health of broilers, and enhance the immunity.

[0005] To achieve the above-mentioned purpose, the following technical solutions are adopted in the application: In a first aspect, the application provides a preparation method of a ginkgo leaf feed additive, comprising the following steps: S1, mixing ginkgo leaf powder with an alkali treatment agent, standing in a closed state, adding an acid solution, adjusting the pH to 4.0-6.0, filtering, and obtaining treated ginkgo leaves; S2, adding a composite enzyme preparation to the treated ginkgo leaves, and performing enzymatic hydrolysis to obtain enzymatically hydrolyzed ginkgo leaves; S3, adding a composite fermentation agent to the enzymatically hydrolyzed ginkgo leaves, performing anaerobic fermentation, and drying to obtain a ginkgo leaf feed additive.

[0006] Preferably, in step S1, The alkali treatment agent comprises calcium oxide, sodium hydroxide and water, and the mass ratio of calcium oxide, sodium hydroxide and water is 1:1:(50-90); and / or, The mass ratio of the ginkgo leaf powder to the alkali treatment agent is 1:(1-12); and / or, The acid solution comprises phosphoric acid, sulfuric acid and water, and the mass ratio of phosphoric acid, sulfuric acid and water is 1:2:(1-6).

[0007] Preferably, in step S1: The ginkgo leaf powder is obtained by crushing ginkgo leaves to 40-80 mesh; and / or, The temperature of the closed standing is 30-45 ℃, and the time is 5-24 h.

[0008] Preferably, in step S2, the complex enzyme preparation comprises cellulase, xylanase, pectinase, acid protease and beta-glucosidase.

[0009] Preferably, the mass ratio of the cellulase, the xylanase, the pectinase, the acid protease and the beta-glucosidase is (10-50):3:15:10:2; and / or, The enzyme activity of the cellulase is 3000-15000 U / g, the enzyme activity of the xylanase is 20000-80000 U / g, the enzyme activity of the pectinase is 10000-30000 U / g, the enzyme activity of the acid protease is 50000-100000 U / g, and the enzyme activity of the beta-glucosidase is 5000-10000 U / g.

[0010] Preferably, in step S2: The mass ratio of the treated ginkgo leaves and the complex enzyme preparation is 100:(0.5-2); and / or, The temperature of the enzymolysis is 40-70 ℃, and the time is 12-48 h; and / or, The enzymolysis is carried out under stirring, wherein the stirring speed is 100-200 rpm.

[0011] Preferably, in step S3, the complex fermentation bacterial agent comprises Bacillus subtilis, Pediococcus acidilactici and Kluyveromyces marxianus.

[0012] Preferably, the mass ratio of the Bacillus subtilis, the Pediococcus acidilactici and the Kluyveromyces marxianus is (2-8):1:2; and / or, The viable bacterial count of the Bacillus subtilis is ≥100 billion CFU / g, the viable bacterial count of the Pediococcus acidilactici is ≥50 billion CFU / g, and the viable bacterial count of the Kluyveromyces marxianus is ≥20 billion CFU / g.

[0013] Preferably, in step S3: The mass ratio of the enzymolyzed ginkgo leaves and the complex fermentation bacterial agent is 100:(0.05-0.5); and / or, The temperature of the anaerobic fermentation is 30-40 ℃, and the time is 24-48 h; and / or, The temperature of the drying is 65 ℃.

[0014] In a second aspect, the present application further provides a broiler feed comprising the ginkgo leaf feed additive prepared by the preparation method of the first aspect.

[0015] Compared with the prior art, the present application has the following beneficial effects: (1) In the preparation method of the present application, the active ingredients in ginkgo leaves can be fully released by soaking the ginkgo leaf powder in an alkali treatment agent, enzymatic hydrolysis by a composite enzyme preparation, and fermentation by a composite fermentation agent, while reducing the content of ginkgo acid and generating beneficial metabolites.

[0016] (2) The ginkgo leaf feed additive prepared by the present application can effectively promote the growth of broilers, improve intestinal health, and enhance immunity when applied to broiler feed, replacing antibiotics in feed. DETAILED DESCRIPTION

[0017] The present application will be further described in detail below with reference to specific examples, so that those skilled in the art can more clearly understand the present application.

[0018] Example 1 The present embodiment provides a preparation method of a ginkgo leaf feed additive, comprising the following steps: (1) The ginkgo leaves are crushed to 40 mesh, and the ginkgo leaves and an alkali treatment agent are uniformly mixed according to a mass ratio of ginkgo leaves: alkali treatment agent = 1:9, and then placed in a closed state at 30°C for 6 hours. Then, an acid solution is added to adjust the pH to 5.0, and then filtered to obtain treated ginkgo leaves. The alkali treatment agent comprises calcium oxide, sodium hydroxide and water in a mass ratio of 1:1:80, and the acid solution comprises phosphoric acid, sulfuric acid and water in a mass ratio of 1:2:4. (2) 0.8% of a composite enzyme preparation is added to the treated ginkgo leaves for enzymatic hydrolysis, and the enzymatic hydrolysis temperature is controlled at 50°C, and the stirring speed is controlled at 180 rpm for 18 hours. The composite enzyme preparation comprises cellulase, xylanase, pectinase, acid protease and β-glucosidase in a mass ratio of 35:3:15:10:2, and the enzyme activity of cellulase is 10000 U / g, the enzyme activity of xylanase is 30000 U / g, the enzyme activity of pectinase is 15000 U / g, the enzyme activity of acid protease is 50000 U / g, and the enzyme activity of β-glucosidase is 6000 U / g. (3) 0.1% of a composite fermentation agent is added to the enzymatically hydrolyzed ginkgo leaves for anaerobic fermentation at 35°C for 36 hours. The composite fermentation agent comprises Bacillus subtilis, Pediococcus acidilactici and Kluyveromyces marxianus in a mass ratio of 3:1:2, and the viable cell count of Bacillus subtilis is 150 billion CFU / g, the viable cell count of Pediococcus acidilactici is 80 billion CFU / g, and the viable cell count of Kluyveromyces marxianus is 30 billion CFU / g. (4) After the fermentation is completed, the product is dried at 65℃ to obtain the ginkgo leaf feed additive.

[0019] Example 2 The embodiment provides a preparation method of a ginkgo leaf feed additive, and the method comprises the following steps: (1) The ginkgo leaves are crushed to 40 meshes, and the ginkgo leaves and an alkali treatment agent are uniformly mixed according to a mass ratio of ginkgo leaves: alkali treatment agent = 1:1, and then are placed at 30℃ for 5 hours, and then an acid solution is added to adjust the pH to 4.0, and then filtration is performed to obtain treated ginkgo leaves; wherein the alkali treatment agent comprises calcium oxide, sodium hydroxide and water in a mass ratio of 1:1:50, and the acid solution comprises phosphoric acid, sulfuric acid and water in a mass ratio of 1:2:1; (2) 0.5% of a compound enzyme preparation is added to the treated ginkgo leaves for enzymolysis, the enzymolysis temperature is controlled to be 40℃, and stirring is performed at a rotating speed of 100 rpm for 12 hours; wherein the compound enzyme preparation comprises cellulase, xylanase, pectinase, acid protease and β-glucosidase in a mass ratio of 10:3:15:10:2, the enzyme activity of the cellulase is 3000 U / g, the enzyme activity of the xylanase is 20000 U / g, the enzyme activity of the pectinase is 10000 U / g, the enzyme activity of the acid protease is 50000 U / g, and the enzyme activity of the β-glucosidase is 5000 U / g; (3) 0.05% of a compound fermentation bacteria agent is added to the enzymolyzed ginkgo leaves for anaerobic fermentation at 30℃ for 24 hours; wherein the compound fermentation bacteria agent comprises bacillus subtilis, pediococcus acidilactici and maxichlorella in a mass ratio of 2:1:2, the viable bacterial count of the bacillus subtilis is 100 billion CFU / g, the viable bacterial count of the pediococcus acidilactici is 50 billion CFU / g, and the viable bacterial count of the maxichlorella is 20 billion CFU / g; (4) After the fermentation is completed, the product is dried at 65℃ to obtain the ginkgo leaf feed additive.

[0020] Example 3 The embodiment provides a preparation method of a ginkgo leaf feed additive, and the method comprises the following steps: (1) The ginkgo leaves are crushed to 80 meshes, and the ginkgo leaves and an alkali treatment agent are uniformly mixed according to a mass ratio of ginkgo leaves: alkali treatment agent = 1:12, and then are placed at 45℃ for 24 hours, and then an acid solution is added to adjust the pH to 6.0, and then filtration is performed to obtain treated ginkgo leaves; wherein the alkali treatment agent comprises calcium oxide, sodium hydroxide and water in a mass ratio of 1:1:90, and the acid solution comprises phosphoric acid, sulfuric acid and water in a mass ratio of 1:2:6; (2) 2% of a complex enzyme preparation is added to the treated ginkgo leaves for enzymolysis, the enzymolysis temperature is controlled at 70 ℃, and stirring is performed at a rotating speed of 200 rpm for 48 h; the complex enzyme preparation comprises cellulase, xylanase, pectinase, acid protease and β-glucosidase at a mass ratio of 50:3:15:10:2, the enzyme activity of the cellulase is 15000 U / g, the enzyme activity of the xylanase is 80000 U / g, the enzyme activity of the pectinase is 30000 U / g, the enzyme activity of the acid protease is 100000 U / g, and the enzyme activity of the β-glucosidase is 10000 U / g; (3) 0.5% of a complex fermentation bacterial agent is added to the enzymolyzed ginkgo leaves, and anaerobic fermentation is performed at 40 ℃ for 48 h; the complex fermentation bacterial agent comprises Bacillus subtilis, Pediococcus acidilactici and Kluyveromyces marxianus at a mass ratio of 8:1:2, the viable bacterial count of the Bacillus subtilis is 150 billion CFU / g, the viable bacterial count of the Pediococcus acidilactici is 800 billion CFU / g, and the viable bacterial count of the Kluyveromyces marxianus is 300 billion CFU / g; (4) After the fermentation is completed, the product is subjected to drying treatment at 65 ℃, and the ginkgo leaf feed additive is obtained.

[0021] Comparative Example 1 The comparative example provides a ginkgo leaf feed additive, and the ginkgo leaves are crushed to 40 mesh.

[0022] Comparative Example 2 The comparative example provides a preparation method of a ginkgo leaf feed additive, which is different from example 1 in that: (1) the ginkgo leaves are crushed to 40 mesh, the ginkgo leaves and water are uniformly mixed according to a mass ratio of ginkgo leaves:water = 1:9, and the mixture is statically placed in a closed container at 30 ℃ for 6 h, then an acid solution is added, the pH is adjusted to 5.0, and filtration is performed to obtain treated ginkgo leaves; the acid solution comprises phosphoric acid, sulfuric acid and water at a mass ratio of 1:1:4.

[0023] Comparative Example 3 The comparative example provides a preparation method of a ginkgo leaf feed additive, which is different from example 1 in that: step (2) is not performed.

[0024] Comparative Example 4 The comparative example provides a preparation method of a ginkgo leaf feed additive, which is different from example 1 in that: step (3) is not performed.

[0025] The nutritional indexes of the ginkgo leaf feed additives obtained in examples 1-3 and comparative examples 1-4 are detected respectively, and the results are shown in Table 1.

[0026] Table 1 Nutritional indexes of ginkgo leaf feed additives in examples and comparative examples

[0027] As shown in Table 1, the preparation method of the application can improve the release of total flavonoids and terpene lactones in ginkgo leaves, improve the biological activity of ginkgo leaf feed additive; can improve the content of crude protein, reducing sugar and lactic acid in ginkgo leaf feed additive, improve the flavor of ginkgo leaf feed additive, improve the nutritional value of feed; can also significantly reduce the content of ginkgo acid in ginkgo leaf feed additive, and detoxification is sufficient.

[0028] Application example Select 1-day-old healthy good health of 1200 broilers, randomly divided into 4 groups, 20 replicates per group, 15 chickens per replicate, feeding experiment, see Table 2 for details.

[0029] Table 2: Broiler feeding scheme

[0030] During the test, the broilers were free to eat and drink water, 24 h light, the temperature of the chicken house was controlled at 32~34 ℃ in the first week, then the temperature was reduced by 3~4 ℃ per week, until the temperature was reduced to about 25 ℃ and maintained until the end of the test; The relative humidity of the chicken house during the test period was 50~60 %; natural ventilation, according to the routine immunization procedure, the health status, feeding and feces of the chicken group were observed daily, and the chicken house was cleaned and disinfected regularly; The test period was 42 days, and the feed intake and body weight of the broilers were recorded; the results are shown in Table 3, and the same row data in the table indicates that the difference is significant (P≤0.05), and the same indicates that the difference is not significant (P>0.05).

[0031] Table 3: Growth of broilers in each group

[0032] As shown in Table 3, compared with the basal diet, 5% untreated ginkgo leaf replacing the basal diet significantly reduces the final weight and survival rate of broilers, and significantly increases the feed to weight ratio, which has a negative impact on the growth of broilers; 5% ginkgo leaf feed additive replacing the basal diet can significantly increase the final weight and average daily gain of broilers, and increase the survival rate; compared with the antibiotic group, 5% ginkgo leaf feed additive replacing the basal diet can increase the final weight, average daily gain and survival rate of broilers, and the effect is better. It is shown that the ginkgo leaf feed additive prepared by the application is applied to the broiler feed, which not only can reduce the use of antibiotics, but also can promote the growth of broilers, and bring economic benefits to the breeders.

[0033] In the application, the specific raw materials are not specified and are already existing substances that can be directly purchased from the market.

[0034] The above merely preferred embodiments of the present application and are not intended to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a ginkgo biloba leaf feed additive, characterized by, The method comprises the following steps: S1, mixing ginkgo leaf powder with an alkali treatment agent, standing in a closed state, adding an acid solution, adjusting the pH to 4.0-6.0, filtering, and obtaining treated ginkgo leaves; S2, adding a composite enzyme preparation to the treated ginkgo leaves, and performing enzymatic hydrolysis to obtain enzymatically hydrolyzed ginkgo leaves; S3, adding a composite fermentation agent to the enzymatically hydrolyzed ginkgo leaves, performing anaerobic fermentation, and drying to obtain ginkgo leaf feed additives.

2. The method of claim 1, wherein the ginkgo biloba leaf feed additive is prepared by the steps of: In step S1, ​ the alkali treatment agent comprises calcium oxide, sodium hydroxide, and water, wherein the mass ratio of calcium oxide, sodium hydroxide, and water is 1:1:(50-90); and / or the mass ratio of the ginkgo leaf powder to the alkali treatment agent is 1:(1-12); and / or the acid solution comprises phosphoric acid, sulfuric acid, and water, wherein the mass ratio of phosphoric acid, sulfuric acid, and water is 1:2:(1-6).

3. The method of claim 1, wherein the ginkgo biloba leaf feed additive is prepared by the steps of: In step S1, ​ the ginkgo leaf powder is obtained by crushing ginkgo leaves to 40-80 mesh; and / or the temperature for standing in a closed state is 30-45 ℃, and the time is 5-24 h.

4. The method for preparing the ginkgo leaf feed additive according to claim 1, characterized in that, In step S2, the composite enzyme preparation comprises cellulase, xylanase, pectinase, acid protease, and β-glucosidase.

5. The method of claim 4, wherein the ginkgo biloba leaf feed additive is prepared by the steps of: a) mixing the ginkgo biloba leaf extract with the feed additive carrier; b) drying the mixture; and c) pulverizing the dried mixture. The mass ratio of the cellulase, the xylanase, the pectinase, the acid protease, and the β-glucosidase is (10-50):3:15:10:2; and / or the enzyme activity of the cellulase is 3000-15000 U / g, the enzyme activity of the xylanase is 20000-80000 U / g, the enzyme activity of the pectinase is 10000-30000 U / g, the enzyme activity of the acid protease is 50000-100000 U / g, and the enzyme activity of the β-glucosidase is 5000-10000 U / g.

6. The method for preparing the ginkgo leaf feed additive according to claim 1, characterized in that, In step S2, the mass ratio of the treated ginkgo leaves to the composite enzyme preparation is 100:(0.5-2); and / or the temperature for enzymatic hydrolysis is 40-70 ℃, and the time is 12-48 h; and / or the enzymatic hydrolysis is performed under stirring, wherein the stirring speed is 100-200 rpm.

7. The method for preparing the Ginkgo leaf feed additive according to claim 1, characterized in that, In step S3, the composite fermentation agent comprises Bacillus subtilis, Pediococcus acidilactici, and Kluyveromyces marxianus.

8. The method of claim 7, wherein the ginkgo biloba leaf feed additive is prepared by the steps of: a) mixing the ginkgo biloba leaf extract with the feed additive carrier; b) drying the mixture; and c) pulverizing the dried mixture. The mass ratio of the Bacillus subtilis, the Pediococcus acidilactici, and the Kluyveromyces marxianus is (2-8):1:2; and / or the viable bacterial count of the Bacillus subtilis is ≥100 billion CFU / g, the viable bacterial count of the Pediococcus acidilactici is ≥50 billion CFU / g, and the viable bacterial count of the Kluyveromyces marxianus is ≥20 billion CFU / g.

9. The method of claim 1, wherein the ginkgo biloba leaf feed additive is prepared by the steps of: In step S3, ​ the mass ratio of the enzymatically hydrolyzed ginkgo leaves to the composite fermentation agent is 100:(0.05-0.5); and / or the temperature for anaerobic fermentation is 30-40 ℃, and the time is 24-48 h; and / or the drying temperature is 65 ℃.

10. A broiler feed, characterized in that, The ginkgo leaf feed additives are prepared by the preparation method of any one of claims 1-9.

Images