Enzymatic hydrolysis of summer and autumn tea and application thereof in improving production performance of laying hens and improving egg quality

Abstract

The present application relates to the technical field of tea deep processing and livestock feed, and particularly relates to an enzymatic summer and autumn tea and application thereof in improving production performance of laying hens and egg quality. The preparation method comprises the following steps: mixing sieved summer and autumn tea powder with water to obtain a summer and autumn tea suspension; adjusting the pH of the summer and autumn tea suspension to 6-6.5, heating to 45-55 DEG C, adding a compound enzyme for enzymolysis for 2.5-4 h, and inactivating the enzyme to obtain a compound enzymatic summer and autumn tea feed additive; wherein the compound enzyme comprises cellulase, xylanase, flavor protease and neutral protease, and the enzyme activity and the mass ratio of the summer and autumn tea E / S are greater than or equal to 100 U / g, greater than or equal to 200 U / g, greater than or equal to 1300 U / g and greater than or equal to 4000 U / g respectively. The present application provides a feed additive for improving egg quality and reducing cholesterol content of eggs, promotes development and utilization of summer and autumn tea resources, reduces feed cost, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tea deep processing and livestock and poultry feed, and in particular to an enzymatic summer and autumn tea and its application in improving the production performance of laying hens and improving egg quality. BACKGROUND

[0002] Tea contains rich nutritional ingredients and is rich in functional ingredients such as tea polyphenols, catechins, and theanine, and has many health effects. China is a major tea producing country, with a high proportion of tea garden area and yield in the world, and a high yield of dry tea. In addition to domestic tea drinking, export trade and part of deep processing, there is a problem of overcapacity of tea production.

[0003] Summer and autumn tea is a tea product picked from June to October. Due to the high temperature and strong sunlight of the growing environment, summer and autumn tea has high fiber content, thin leaf, and coarse and hard leaf quality, and the appearance and color of the dry tea are poor, with heavy bitterness and astringency, but its nutritional and health value is still very high. In recent years, due to the increase in tea picking labor costs, the tea picking rate after June is low. More than 80% of summer and autumn tea resources are directly pruned and discarded or die on the branches every year, causing great waste.

[0004] Summer and autumn tea is rich in tea polyphenols and tea proteins, which is beneficial to improve the health of laying hens and the quality of eggs, and therefore is used as a general additive in chicken feed. However, studies have shown that adding summer and autumn tea to laying hen feed can lead to a decrease in feed palatability and laying hen production performance. SUMMARY

[0005] [TECHNICAL PROBLEM]

[0006] A preparation method of a summer and autumn tea feed additive is provided. The prepared summer and autumn tea feed additive applied in chicken feed can improve the palatability of summer and autumn tea feed, improve the production performance of laying hens (increase egg weight, reduce feed to egg ratio), and improve egg quality (improve egg yolk color, Haugh unit and egg yolk ratio, reduce egg yolk cholesterol, egg yolk triglyceride and saturated fatty acid content, and increase egg yolk unsaturated fatty acid content), opening up a new way for comprehensive utilization of summer and autumn tea resources, saving grain and reducing feed cost.

[0007] [TECHNICAL SCHEME]

[0008] In one aspect, a preparation method of a composite enzymatic summer and autumn tea feed additive is provided, which comprises the following steps:

[0009] (1) mixing sieved summer and autumn tea powder with water to obtain a summer and autumn tea suspension;

[0010] (2) adjusting the pH of the summer and autumn tea suspension to 6-6.5, warming to 45-55℃, adding compound enzymes for enzymolysis for 2.5-4h, and inactivating the enzymes to obtain the compound-enzymolysis summer and autumn tea feed additive; wherein the compound enzymes include cellulase, xylanase, prolaminase, and neutral protease, and the mass ratio of enzyme activity to summer and autumn tea powder E / S is ≥100U / g, ≥200U / g, ≥1300U / g, and ≥4000U / g, respectively.

[0011] In some embodiments, in step (2), the enzymolysis pH is 6, the enzymolysis time is 3.5h, the cellulase, xylanase, prolaminase, and neutral protease have enzyme activity to summer and autumn tea powder E / S of 100U / g, 200U / g, 1366U / g, and 4098U / g, respectively.

[0012] In some embodiments, in step (2), a physical strengthening means is used to uniformly mix the system during enzymolysis.

[0013] In some embodiments, in step (2), stirring is used to uniformly mix the system during enzymolysis.

[0014] In some embodiments, in step (2), the stirring speed during enzymolysis is 100-200rpm.

[0015] In some embodiments, in step (1), the mesh number of the sieve is 60-100 mesh.

[0016] In some embodiments, in step (1), the solid-liquid ratio of summer and autumn tea powder to water is 1:6-1:8 (m / v, g / mL).

[0017] In some embodiments, in step (1), the summer and autumn tea powder is stirred with water at a speed of 100-200rpm for 10-30min to obtain the summer and autumn tea suspension.

[0018] In some embodiments, in step (2), the enzymes are inactivated by warming to 85-90℃ within 10-20min and maintaining for 15-20min.

[0019] In another aspect, the compound-enzymolysis summer and autumn tea feed additive prepared by the method described above is provided. The water extract content of the compound-enzymolysis summer and autumn tea feed additive is not less than 50%, the soluble protein is not less than 9%, the soluble sugar is not less than 9%, the tea polyphenol content is not less than 13%, the crude protein digestibility is not less than 43%, the dry matter digestibility is not less than 62%, the acid detergent fiber digestibility is not less than 37%, and the neutral detergent fiber digestibility is not less than 44%.

[0020] In another aspect, the application provides an application of the aforementioned composite enzymatic hydrolyzed summer and autumn tea feed additive in improving the production performance of laying hens and improving egg quality, characterized in that the improvement of the production performance of laying hens includes increasing egg weight and reducing feed to egg ratio; and the improvement of egg quality includes improving yolk color, Haugh unit and yolk ratio, reducing yolk cholesterol, yolk triglyceride and saturated fatty acid content, and increasing yolk unsaturated fatty acid content.

[0021] In another aspect, the application provides a chicken feed comprising the aforementioned composite enzymatic hydrolyzed summer and autumn tea feed additive, and the addition amount of the composite enzymatic hydrolyzed summer and autumn tea feed additive is 1-10% by dry weight.

[0022] In some embodiments, the chicken feed further comprises 60-67% corn, 24-26% soybean meal, 0-8% laying period premix, and 0-5% stone powder.

[0023] [Benefits]

[0024] (1) The application provides a preparation method of a summer and autumn tea feed additive. A composite enzyme (enzyme activity to summer and autumn tea mass ratio E / S≥100 U / g, ≥200 U / g, ≥1300 U / g, and ≥4000 U / g, respectively) composed of xylanase, cellulase, flavor protease, and neutral protease is used to hydrolyze summer and autumn tea under specific conditions (pH=6-6.5, temperature 45-55°C) for 2.5-4h. The macromolecular substances such as proteins and crude fibers are decomposed by the enzyme hydrolysis, so as to increase the contents of soluble sugar, soluble protein, polypeptide, and amino acid. The product obtained after the enzyme hydrolysis has a high nutritional value, and the water extract content is not less than 50%, the soluble protein is not less than 9%, the soluble sugar is not less than 9%, the tea polyphenol content is not less than 13%, the crude protein digestibility is not less than 43%, the dry matter digestibility is not less than 62%, the acid detergent fiber digestibility is not less than 37%, and the neutral detergent fiber digestibility is not less than 44%. Compared with the existing fermentation method which usually needs more than 55h, the composite enzyme hydrolysis method of the application has a shorter time consumption, the whole process takes less than 6h, the operation is simple, and the summer and autumn tea feed additive prepared has lower neutral and acid detergent fiber content, which is more helpful to improve the digestibility of the feed, and further improve the production performance of laying hens and the egg quality.

[0025] (2) The summer and autumn tea feed additive prepared by the application can improve the palatability of chicken feed, can be accepted by laying hens, improve the production performance of laying hens (increase egg weight and reduce feed to egg ratio), improve the egg quality (improve yolk color, Haugh unit and yolk ratio, reduce yolk cholesterol, yolk triglyceride and saturated fatty acid content, and increase yolk unsaturated fatty acid content), improve the serum biochemical indexes of laying hens, and improve the body health of laying hens.

[0026] (3) The application can enzymatically hydrolyze the summer and autumn tea which is difficult to utilize into a feed additive meeting the nutritional requirements, not only promotes the development and utilization of summer and autumn tea resources, but also is beneficial to saving grain and reducing feed cost, and improves egg quality and reduces cholesterol content in eggs, and has a wide application prospect; the application has high economic value and social benefits;

[0027] (4) The summer and autumn tea feed additive is added to the basic feed of laying hens at a proportion of 5wt%, which can not only deepen the color of egg yolk and improve the production performance, but also significantly reduce the cholesterol content in eggs, increase the proportion of unsaturated fatty acids, and improve the serum lipid metabolism and antioxidant capacity of laying hens. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The effects of enzymolysis factors A pH, B temperature, C enzymolysis time, D cell wall enzyme addition amount and E compound protease addition amount on the contents of tea polyphenols, soluble sugar and soluble protein in summer and autumn tea in the single-factor experiment. DETAILED DESCRIPTION

[0029] The specific embodiments of the application will be described in detail below in combination with examples, and the described examples are only part of the examples of the application, not all examples, and are intended to explain the application, and cannot be understood as a limitation of the application.

[0030] The determination method involved in the application

[0031] 1. Determination of basic components

[0032] The water extract is determined according to GB / T 8305-2013 “Determination of Tea Water Extract”. The crude fiber content is determined according to GB / T 8310-2013 “Determination of Tea Crude Fiber”. The contents of crude protein and soluble protein are determined by the Kjeldahl method in GB 5009.5-2016 “Determination of Protein in Foods”. The content of tea polyphenols is determined by the Folin phenol method in GB / T 8313-2018 “Determination of Tea Polyphenols and Catechins in Tea”. The content of soluble sugar is determined by the 3,5-dinitrosalicylic acid (DNS) method.

[0033] 2. Determination of acid detergent fiber and neutral detergent fiber

[0034] The contents of NDF and ADF in summer and autumn tea before and after enzymolysis are determined according to GB / T 20806-2022 “Determination of Neutral Detergent Fiber (NDF) in Feed” and NY / T 1459-2022 “Determination of Acid Detergent Fiber (ADF) in Feed”.

[0035] 3. Determination of in vitro digestibility

[0036] Pepsin-trypsin in-vitro digestion simulation: about 2 g of dried and ground sample (accurate to 0.0001 g) was weighed in a 250 mL conical flask. First, 100 mL of 0.1 mol / L phosphate buffer was added to the conical flask, and the pH value was adjusted to 2.00 with 1 mol / L hydrochloric acid solution or 1 mol / L sodium hydroxide solution. 2.8 mL of 30000 U / mL pepsin solution and 2 mL of 2.5 mg / L chloramphenicol solution were added to the conical flask, the rubber plug was tightly covered, and the constant temperature water bath was 39℃ for 2 h. After the pepsin digestion was completed, 20 mL of 0.2 mol / L phosphate buffer and 10 mL of 0.6 mol / L sodium hydroxide solution were added to the conical flask, and the pH was adjusted to 6.80 with 1 mol / L hydrochloric acid solution or 1 mol / L sodium hydroxide solution. 0.5 mL of 8000 U / mL trypsin solution was added to the conical flask, the rubber plug was tightly covered, and the constant temperature water bath was 39℃ for 4 h. After cooling to room temperature, the residue was collected by filtering with 300 mesh filter cloth, and then the residue was washed with acetone, 95% ethanol and deionized water. The washed residue was dried at 60℃ to constant weight, and the in-vitro digestion rates of crude protein, dry matter, neutral detergent fiber and acid detergent fiber of Xiaqiao tea before and after enzymolysis were determined, and the calculation method was as follows:

[0037]

[0038]

[0039]

[0040]

[0041] Example 1

[0042] A preparation method of an enzymatic Xiaqiao tea feed additive, comprising the following steps:

[0043] (1) Xiaqiao tea was crushed and treated, and passed through a 60 mesh sieve; then Xiaqiao tea powder and drinking water were mixed according to a solid-liquid ratio of Xiaqiao tea powder to water of 1:7 (m / v, g / mL), and stirred at a speed of 150 rpm for 10 min to prepare a Xiaqiao tea suspension;

[0044] (2) The Xiaqiao tea suspension was heated to 45℃, and then stirred at a speed of 150 rpm for 10 min, and sodium bicarbonate solution was used to adjust the Xiaqiao tea suspension to a pH value of 6.0 during stirring;

[0045] (3) Add cellulase, xylanase, flavor protease and neutral protease to the summer and autumn tea suspension obtained in step (2) so that the enzyme activity to the mass ratio of summer and autumn tea powder E / S are 100U / g, 200U / g, 1366U / g and 4098U / g respectively. Enzymatic hydrolysis is carried out for 3.5h at pH 6.0 and temperature 50℃ in the mixed system, and the enzymatic hydrolysis is assisted by stirring at a speed of 150rpm throughout the process.

[0046] (4) The temperature of the mixing system is raised to 90℃ within 10-20 min and maintained for 15 min to inactivate the enzyme in the mixing system. The mixture is then dried at 60℃ until the moisture content is ≤10%, and then crushed to obtain the enzymatically hydrolyzed summer and autumn tea feed additive.

[0047] Comparative Example 1

[0048] A method for preparing summer and autumn tea products, referring to Example 1, uses the same batch of summer and autumn tea as raw materials, the only difference being that the steps of adding cellulase, xylanase, flavor protease and neutral protease in step (3) are omitted, and the other operations are the same as in Example 1, to obtain summer and autumn tea products.

[0049] Comparative Example 2

[0050] A method for preparing summer and autumn tea products, referring to Example 1, uses the same batch of summer and autumn tea as raw materials and adopts the following steps: the summer and autumn tea is pulverized and passed through a 60-mesh sieve to obtain summer and autumn tea products.

[0051] Example 2: Process optimization of enzymatic hydrolysis of summer and autumn tea

[0052] Process optimization for enzymatic hydrolysis of summer and autumn teas includes:

[0053] 2.1 Enzyme screening:

[0054] Experimental steps: Similar to the method in Example 1, summer and autumn tea → pulverize (60 mesh) → add water, solid-liquid ratio (m / v, g / mL) is 1:7, adjust pH → add enzyme → extract with stirring in constant temperature water bath → inactivate enzyme with boiling water (10 min) → centrifuge (5000 r / min, 10 min) → take supernatant.

[0055] 2.1.1 Screening of cell wall-breaking enzymes

[0056] Four cell wall-breaking enzymes—cellulase, pectinase, xylanase, and hemicellulase—were selected to enzymatically hydrolyze the same batch of summer and autumn tea. The optimal operating conditions for each enzyme are shown in Table 1. Using the soluble sugar content in the supernatant as an indicator, suitable cell wall-breaking enzymes were screened for compounding. The results of the soluble sugar content in the supernatant in this experiment are shown in Table 2.

[0057] Table 1 Optimal operating conditions for different cell wall-breaking enzymes

[0058]

[0059] Table 2. Effects of single cell wall-breaking enzymes on the soluble sugar and soluble protein content in summer and autumn tea.

[0060]

[0061] Table 2 shows that the type of cell wall-breaking enzyme has a significant impact on the soluble sugar content in enzymatically hydrolyzed summer and autumn tea. The soluble sugar content in enzymatically hydrolyzed summer and autumn tea, from highest to lowest, is: cellulase > xylanase > pectinase > hemicellulase. Compared with unhydrolyzed summer and autumn tea, cellulase hydrolysis increased the soluble sugar content by 70.53%, the highest increase. Compared with unhydrolyzed summer and autumn tea, xylanase hydrolysis increased the soluble sugar content by 39.71%, the second highest increase. Therefore, to obtain a higher soluble sugar content, a combination of cellulase and xylanase will be considered in future studies. Because summer and autumn tea has a high fiber content and coarse leaf texture, cellulase and xylanase can effectively break down plant cell walls, decomposing macromolecules such as cellulose into soluble sugars and promoting the release of effective components within the cells.

[0062] 2.1.2 Screening of proteases

[0063] Four proteases—neutral protease, acidic protease, flavor protease, and papain—were selected to enzymatically hydrolyze the same batch of summer and autumn tea. The optimal operating conditions for each enzyme are shown in Table 3. Using the soluble protein content in the supernatant as an indicator, suitable proteases were screened for compounding. The results of the soluble protein content in the supernatant in this experiment are shown in Table 4.

[0064] Table 3 Optimal activation conditions for different proteases

[0065]

[0066] Table 4. Effects of protease monoenzymes on soluble sugar and soluble protein content in summer and autumn tea.

[0067]

[0068] Table 4 shows that the type of protease has a significant impact on the soluble protein content in enzymatically hydrolyzed summer and autumn teas. The soluble protein content in enzymatically hydrolyzed summer and autumn teas, from highest to lowest, is: neutral protease > flavor protease > papain > acidic protease. Compared to unhydrolyzed summer and autumn teas, the soluble protein content increased by 48.66% after hydrolysis with neutral protease, showing the highest increase. The soluble protein content increased by 44.31% after hydrolysis with flavor protease, showing the second highest increase. Therefore, to obtain even higher soluble protein content, a combination of neutral and flavor proteases will be considered in future studies. Neutral and flavor proteases can cleave peptide bonds, altering protein structure and hydrolyzing large proteins into polypeptides, small peptides, and amino acids. Simultaneously, proteases can also hydrolyze other substances linked to proteins, increasing the protein dissolution rate.

[0069] 2.1.3 Optimization of the ratio of cellulase and xylanase

[0070] Based on the screening of cell wall-breaking enzymes in 2.1.1, and under the premise of keeping the total amount of cell wall-breaking enzymes added constant, the addition ratio of cellulase and xylanase was optimized. The results are shown in Table 5.

[0071] Table 5 shows that, under the premise of keeping the amount of cell wall-breaking enzyme constant, the ratio of xylanase to cellulase has a significant impact on the soluble sugar content in summer and autumn tea after enzymatic hydrolysis. When the ratio of xylanase to cellulase is 2:1, the soluble sugar content in the summer and autumn tea after enzymatic hydrolysis is the highest (reaching 43.26 mg / g), which is 42.68% higher than that of cellulase alone (reaching 30.32 mg / g) and 74.15% higher than that of xylanase alone (reaching 24.84 mg / g). This indicates that the combined enzymatic hydrolysis of xylanase and cellulase at a 2:1 ratio has a significant synergistic effect on increasing the soluble sugar content of summer and autumn tea. When xylanase and cellulase act together on summer and autumn tea, they can promote the degradation of the cell wall and significantly increase the soluble sugar content. Therefore, a xylanase to cellulase ratio of 2:1 was selected for subsequent experiments.

[0072] Table 5. Effects of cell wall-breaking enzyme complex on soluble sugar and soluble protein content in summer and autumn tea.

[0073] Xylanase: cellulase ratio Soluble sugar (mg / g) Soluble protein (mg / g) 1：3 35.61 ± 0.68 e ]] 29.68 ± 0.81 a ]] 1：2 38.78 ± 0.14 d ]] 28.31 ± 0.42 b ]] 1：1 40.73 ± 1.38 c ]] 28.46 ± 0.50 ab ]] 2：1 43.26 ± 0.16 a ]] 28.00 ± 0.10 b ]] 3：1 42.09 ± 0.34 b ]] 28.54 ± 0.08 ab ]]

[0074] 2.1.4 Optimization of the ratio of neutral protease and flavor protease

[0075] Based on the screening of proteases in 2.1.2, and under the premise of keeping the total amount of proteases added constant, the addition ratio of neutral protease and flavor protease was optimized. The results are shown in Table 6.

[0076] Table 6. Effects of protease complex enzymes on soluble sugar and soluble protein content in summer and autumn tea.

[0077] Flavour protease: neutral protease ratio Soluble sugar (mg / g) Soluble protein (mg / g) 1：3 19.21±0.16 55.70 ± 1.07 a ]] 1：2 19.30±0.08 49.07 ± 1.52 b ]] 1：1 19.27±0.29 46.99 ± 0.72 c ]] 2：1 18.97±0.24 46.24 ± 0.20 c ]] 3：1 19.30±0.19 40.70 ± 1.74 d ]]

[0078] As shown in Table 6, under the premise of keeping the total amount of protease added constant, the ratio of neutral protease and flavor protease has a significant impact on the soluble protein content in summer and autumn tea after enzymatic hydrolysis.

[0079] When the ratio of flavor protease to neutral protease was 1:3, the soluble protein content in summer and autumn tea after enzymatic hydrolysis was the highest (reaching 55.70 mg / g), an increase of 56.64% compared to neutral protease alone (reaching 35.56 mg / g) and 61.26% compared to flavor protease alone (reaching 34.54 mg / g). This demonstrates that the combined enzymatic hydrolysis of flavor protease and neutral protease at a 1:3 ratio has a significant synergistic effect on increasing the soluble protein content of summer and autumn tea. This confirms that the combined use of neutral protease and flavor protease has a synergistic effect on increasing the soluble protein content of summer and autumn tea. Therefore, a flavor protease to neutral protease ratio of 1:3 was selected for subsequent experiments.

[0080] 2.1.5 Single-factor experiment

[0081] Five factors were selected: treatment temperature (35, 40, 45, 50, 55, 60℃), pH (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0), enzymatic hydrolysis time (1, 1.5, 2, 2.5, 3, 3.5, 4 h), cell wall-breaking enzyme dosage (100, 150, 200, 250, 300, 350 U / g), and complex protease dosage (1000, 2000, 3000, 40000, 5000, 6000, 7000 U / g). Enzymatic hydrolysis was performed under the above experimental conditions, and the contents of soluble sugars and soluble proteins in the extract were determined. The results are as follows. Figure 1 As shown.

[0082] Depend on Figure 1 A shows that the soluble sugar content in summer and autumn tea is highest at pH 6.5, and the soluble protein content is highest at pH 6. However, since increasing the pH will lead to a decrease in the content of tea polyphenols, considering factors such as economy and environmental protection, the optimal pH is selected as 6-6.5 for subsequent experiments.

[0083] Depend on Figure 1 B shows that the soluble sugar content is highest at 45℃, and the soluble protein content is highest at 55℃; the tea polyphenol content does not change significantly with enzymatic hydrolysis temperature (p>0.05), but decreases significantly at 60℃ (p<0.05). Therefore, the optimal enzymatic hydrolysis temperature is 45–55℃ for subsequent experiments.

[0084] Depend on Figure 1 As shown in Figure C, the contents of soluble sugars and soluble proteins in summer and autumn teas increased rapidly with increasing enzymatic hydrolysis time, reaching their maximum at 2.5 h and 3 h, respectively, after which there was no significant difference with increasing time (p>0.05). Therefore, an enzymatic hydrolysis time of 2.5–3.5 h was preferred for subsequent experiments.

[0085] Depend on Figure 1 As shown in Figure D, the soluble sugar content increased with increasing cell wall-breaking enzyme dosage, reaching its maximum at 250 U / g. Further increases in the cell wall-breaking enzyme dosage did not significantly alter the soluble sugar content (p>0.05). Therefore, a cell wall-breaking enzyme dosage of 200–300 U / g was selected for subsequent experiments.

[0086] like Figure 1 As shown in Figure E, when the amount of compound protease added is less than 6000 U / g, the content of soluble protein in summer and autumn tea increases with the increase of enzyme addition. When the amount of compound protease added exceeds 6000 U / g, the increase in soluble protein content slows down. Therefore, a compound protease addition amount of 5000–7000 U / g was selected for subsequent experiments.

[0087] 2.1.6 Plackett-Burman Experiment: The Plackett-Burman design was used to screen out the key factors affecting the enzymatic hydrolysis of summer and autumn tea. Based on the single-factor experiments, tea polyphenols (Y1), soluble sugar content (Y2), and soluble protein content (Y3) were used as response values ​​to evaluate the five influencing factors and screen out the main effect factors. Two levels were selected for each factor, resulting in a total of 12 experimental groups. The experimental factors and level values ​​are shown in Table 7.

[0088] Table 7. Factors and Levels in the Plackett-Burman Experimental Design

[0089]

[0090] The Plackett-Burman assay can quickly identify key influencing factors from multiple factors using a minimal number of trials. This experiment used the soluble sugar, soluble protein, and tea polyphenol content of summer and autumn tea after enzymatic hydrolysis as evaluation indicators. The Plackett-Burman assay was used to screen out important factors affecting the enzymatic hydrolysis process of summer and autumn tea, with the response values ​​being tea polyphenol yield (Y1), soluble sugar yield (Y2), and soluble protein yield (Y3). The experimental results are shown in Tables 8 and 9.

[0091] Table 8 Results of the Plackett-Burman Experimental Design

[0092] No. X1 X2 X3 X4 X5 [Y1] [Y2] [Y3] 1 1 -1 -1 -1 1 78.58 83.88 59.86 2 1 -1 1 1 1 77.26 91.59 83.94 3 1 1 -1 -1 -1 78.05 77.01 58.36 4 1 1 1 -1 -1 75.98 78.56 69.11 5 -1 -1 -1 -1 -1 81.40 73.12 52.87 6 1 -1 1 1 -1 75.04 82.83 80.16 7 -1 1 1 1 -1 78.81 78.40 76.10 8 -1 1 1 -1 1 80.76 89.79 63.14 9 -1 -1 -1 1 -1 81.03 75.00 63.95 10 -1 -1 1 -1 1 79.14 88.53 62.52 11 -1 1 -1 1 1 80.99 79.86 70.73 12 1 1 -1 1 1 77.79 83.09 75.91

[0093] Table 9. Analysis of Variance of the Plackett-Burman Trial

[0094]

[0095] Table 9 shows that the experimental model has a high degree of fit and reliability, and can describe the influence of each factor on the response value. Based on the p-value, the significance of each factor can be analyzed. Finally, time, pH, cell wall-breaking enzyme addition amount, and protease addition amount were selected as the main influencing factors, and the optimal level range will be further explored in the next experiment.

[0096] 2.1.7 Box-Behnken Response Surface Method Experiment:

[0097] Based on the Plackett-Burman experiment, pH (A), enzymatic hydrolysis time (B), amount of complex protease added (C), and amount of cell wall-breaking enzyme added (D) were selected as factors for further optimization. Following the Box-Behnken design principle, a 4-factor, 3-level experimental design was established. The experimental factors and level values ​​are shown in Table 10.

[0098] Table 10. Factors and levels in the Box-Behnken response surface methodology experiment.

[0099]

[0100] Table 11 Results of Box-Behnken response surface experimental design

[0101] No. A B C D Y1 Y2 Y3 1 1 1 0 0 72.87 89.62 82.34 2 1 0 0 -1 74.75 85.00 77.67 3 0 1 0 -1 78.80 81.74 84.17 4 0 1 0 1 78.73 89.48 86.86 5 0 -1 0 1 81.87 81.13 78.79 6 0 0 0 0 80.97 83.63 85.01 7 0 0 1 0 73.55 86.56 78.93 8 -1 0 1 0 82.34 75.85 76.09 9 0 -1 0 -1 82.20 76.89 78.62 10 -1 -1 0 0 84.30 71.18 67.96 11 -1 0 0 1 82.78 78.73 71.39 12 1 0 0 1 74.89 88.82 78.17 13 0 0 1 1 78.33 85.71 86.39 14 0 0 0 0 81.51 83.25 83.78 15 0 0 -1 1 81.58 83.96 81.23 16 -1 0 -1 0 82.96 73.73 62.95 17 0 0 0 0 81.15 83.02 82.95 18 0 0 0 0 80.68 83.44 83.13 19 1 0 -1 0 76.09 86.23 73.13 20 0 -1 1 0 81.69 79.15 85.71 21 -1 1 0 0 82.31 78.91 73.16 22 -1 0 0 -1 82.45 71.84 69.37 23 0 1 -1 0 79.09 85.00 83.80 24 1 -1 0 0 77.17 84.53 69.20 25 0 3 1 -1 80.25 78.63 86.16 26 0 1 1 0 77.64 85.19 87.95 27 0 -1 -1 0 82.27 76.04 68.35 28 0 0 0 0 79.05 82.59 82.48 29 0 0 -1 -1 81.15 77.97 77.67

[0102] The response values ​​were tea polyphenol yield (Y1), soluble sugar yield (Y2), and soluble protein yield (Y3). The results were analyzed using Design-Expert software, and the experimental results are shown in Table 11. The regression equation with tea polyphenol yield (Y1) as the dependent variable and pH (A), time (B), amount of compound protease added (C), and amount of cell wall-breaking enzyme added (D) as independent variables was obtained.

[0103] Y1=80.67-3.99A-1.67B-0.7779C-0.1176D-0.5789AB-0.4794AC-0.0452AD-0.2171BC+0.0633BD-0.5879CD-1.62A2-0.0552B2-0.3039C2-0.1999D2

[0104] The regression equation was obtained with soluble sugar yield (Y2) as the dependent variable and pH (A), time (B), amount of complex protease added (C), and amount of cell wall-breaking enzyme added (D) as independent variables:

[0105] Y2=83.19+5.88A+3.42B+0.6800C+2.98D-0.6604AB-0.4481AC-0.7665AD-0.7311BC+0.8726BD+0.2712CD-1.55A2-0.5645B2-1.17C2-0.4347D2

[0106] The regression equation was obtained with soluble protein yield (Y3) as the dependent variable and pH (A), time (B), amount of complex protease added (C), and amount of cell wall-breaking enzyme added (D) as independent variables:

[0107] Y3=83.47+3.21A+4.14B+4.51C+0.7649D+1.98AB-1.83AC-0.3799AD-3.30BC+0.6293BD-0.8324CD-9.44A2-1.12B2-0.9421C2+0.0732D2.

[0108] Analysis of variance was performed on the regression model, and the results are shown in Tables 12, 13, and 14. As can be seen from the tables, the p-values ​​in the regression equations are all less than 0.001, indicating a highly significant model fit. The p-values ​​for the lack-of-fit terms are all greater than 0.05, indicating no significant lack of fit. Therefore, the model is suitable for this experiment and has a high degree of fit. R 2 The degree of agreement between the reaction model and the actual results. The correlation coefficient R of the model (Y1). 2 =0.9794, R 2 adj =0.9588; the correlation coefficient R of model (Y2) 2 =0.9965, R 2 adj =0.9929; the correlation coefficient R of model (Y3) 2 =0.9849, R 2 adj =0.9697. This indicates that the model fits the actual experimental results well and can be used to analyze and predict the content of tea polyphenols, soluble sugars, and soluble proteins in enzymatically hydrolyzed summer and autumn tea in real experiments.

[0109] Table 12 Response Surface Analysis of Variance (Tea Polyphenols)

[0110]

[0111] Note: ** indicates a significant difference (p<0.01); * indicates a significant difference (p<0.05).

[0112] Table 13 Response Surface Analysis of Variance (Soluble Sugars)

[0113]

[0114]

[0115] Note: ** indicates a significant difference (p<0.01); * indicates a significant difference (p<0.05).

[0116] Table 14 Response Surface Analysis of Variance (Soluble Proteins)

[0117]

[0118] Note: ** indicates a significant difference (p<0.01); * indicates a significant difference (p<0.05).

[0119] The optimal processing conditions for enzymatic hydrolysis of summer and autumn tea were determined using Design-Expert 13.0.1.0 software: hydrolysis pH 6.017, hydrolysis time 3.5 h, compound protease addition 5464.221 U / g, and cell wall-breaking enzyme addition 300 U / g. Under these conditions, the predicted content of tea polyphenols in the enzymatically hydrolyzed summer and autumn tea was 79.305 mg / g, soluble sugar content 89.160 mg / g, and soluble protein content 87.661 mg / g. Considering the feasibility of practical operation, the conditions were set as follows: hydrolysis pH 6.0, hydrolysis time 3.5 h, compound protease addition 5464 U / g, and cell wall-breaking enzyme addition 300 U / g (see Example 1). Three parallel experiments were conducted, and the obtained tea polyphenol content was 80.93 mg / g, soluble sugar content was 90.71 mg / g, and soluble protein content was 92.08 mg / g. The relative errors with the predicted values ​​were 2.01%, 1.70%, and 4.80%, respectively. The small difference between the experimental and predicted values ​​indicates that the enzyme treatment process for summer and autumn tea obtained through response surface methodology is effective and reliable, and has practical significance.

[0120] Test Example 1

[0121] The nutritional components of the summer and autumn tea products prepared in Example 1 and Comparative Example 1 were compared, and the test results are shown in Table 15. Compared to Comparative Example 1, the crude fiber, acid detergent fiber, and neutral detergent fiber content in the compound enzymatically hydrolyzed summer and autumn tea feed additive of Example 1 were significantly reduced, while the water extract, soluble protein, and soluble sugar content were significantly increased: The water extract content in the compound enzymatically hydrolyzed summer and autumn tea feed additive of Example 1 increased by 20.39% compared to Comparative Example 1; the acid detergent fiber and neutral detergent fiber content in the compound enzymatically hydrolyzed summer and autumn tea feed additive of Example 1 were 12.40% and 14.87%, respectively, which were reduced by 28.49% and 42.43% compared to Comparative Example 1; the soluble protein content in the compound enzymatically hydrolyzed summer and autumn tea feed additive of Example 1 increased by 2.85 times, and the soluble sugar content increased by 4.10 times; the crude fiber content in the compound enzymatically hydrolyzed summer and autumn tea feed additive of Example 1 was 10.98%, which was reduced by 24.22% compared to Comparative Example 1.

[0122] Table 15 Nutritional Information (on a dry basis)

[0123] Ingredient (%) Comparative Example 1 Example 1 Crude fiber 14.49±0.44 10.98±0.35 Crude protein 16.99±0.29 16.25±0.80 Tea polyphenol 14.95±0.20 13.61±0.07 Water extract 45.36±0.94 54.61±0.86 Acid detergent fiber 17.34±0.10 12.40±0.19 Neutral detergent fiber 25.83±0.51 14.87±0.62 Soluble protein 2.39±0.03 9.21±0.20 Soluble sugar 1.78±0.12 9.07±0.14

[0124] Test Example 2

[0125] The digestibility of nutrients in the summer and autumn tea products prepared in Example 1 and Comparative Examples 1-2 was compared, and the results are shown in Table 16. Table 16 shows that, compared to Comparative Example 2, the digestibility of crude protein, dry matter, acid detergent fiber, and neutral detergent fiber in the compound enzymatically hydrolyzed summer and autumn tea feed additive of Example 1 was significantly improved: compared to Comparative Example 2, the crude protein digestibility of the compound enzymatically hydrolyzed summer and autumn tea feed additive of Example 1 increased by 2.22 times, the dry matter digestibility increased by 19.29%, the acid detergent fiber digestibility increased by 12.47%, and the neutral detergent fiber digestibility increased by 8.74%. This may be because enzymatic hydrolysis degrades some cellulose and hydrolyzes some peptide bonds in proteins, reducing the molecular weight of proteins and forming polypeptides and small peptides, which facilitate contact and reaction with proteases during in vitro digestion. Therefore, the digestibility and utilization rate of summer and autumn tea is improved after enzymatic hydrolysis. Enzymatic hydrolysis can also reduce the particle diameter of summer and autumn tea powder to a certain extent and destroy plant cell walls, thus making it more easily digested by pepsin and pancreatic enzymes.

[0126] Table 16 Changes in the digestibility of nutrients (on a dry basis)

[0127] Comparative Example 2 Example 1 Crude protein digestibility, % 13.65±0.53 44.00±0.98 Dry matter digestibility, % 54.10±0.64 64.54±1.67 Acid detergent fiber digestibility, % 34.15±0.34 38.41±0.49 Neutral detergent fiber digestibility, % 41.53±0.93 45.16±0.82

[0128] Example 3 - Laying hen feed containing 1% compound enzymatic hydrolysed summer and autumn tea feed additive.

[0129] This embodiment provides a laying hen feed that improves egg quality and reduces egg cholesterol. The feed contains the compound enzymatic hydrolyzed summer and autumn tea feed additive prepared in Example 1. The feed formula, by weight percentage, is: corn 65.73%, soybean meal 25.27%, limestone powder 3.00%, premix 5.00%, and the compound enzymatic hydrolyzed summer and autumn tea feed additive prepared in Example 1 (by dry weight) 1.00%. The composition of the premix is ​​described below in Table 18.

[0130] The laying hen feed is prepared by mixing all the ingredients according to the above formula.

[0131] Example 4 - Layer hen feed containing 3% compound enzymatic hydrolysed summer and autumn tea feed additive

[0132] This embodiment provides a laying hen feed that improves egg quality and reduces egg cholesterol. The feed contains the compound enzymatic hydrolyzed summer and autumn tea feed additive prepared in Example 1. The feed formula, by weight percentage, is: corn 64.28%, soybean meal 24.72%, limestone powder 3.00%, premix 5.00%, and the compound enzymatic hydrolyzed summer and autumn tea feed additive prepared in Example 1 (by dry weight) 3.00%. The composition of the premix is ​​described in Table 18 below.

[0133] The laying hen feed is prepared by mixing all the ingredients according to the above formula.

[0134] Example 5 - Laying hen feed containing 5% compound enzymatic hydrolysed summer and autumn tea feed additive

[0135] This embodiment provides a laying hen feed that improves egg quality and reduces egg cholesterol. The feed contains the compound enzymatic hydrolyzed summer and autumn tea feed additive prepared in Example 1. The feed formula, by weight percentage, is: corn 62.84%, soybean meal 24.16%, limestone powder 3.00%, premix 5.00%, and the compound enzymatic hydrolyzed summer and autumn tea feed additive prepared in Example 1 (by dry weight) 5.00%. The composition of the premix is ​​described in Table 18 below.

[0136] The laying hen feed is prepared by mixing all the ingredients according to the above formula.

[0137] Comparative Example 3 - Laying hen feed containing 1% summer and autumn tea feed additive.

[0138] A type of laying hen feed, referring to Example 3, except that the compound enzymatic hydrolyzed summer and autumn tea feed additive prepared in Example 1 is replaced with the summer and autumn tea product (untreated summer and autumn tea powder) of Comparative Example 2.

[0139] Comparative Example 4 - Laying hen feed containing 3% summer and autumn tea feed additive.

[0140] A type of laying hen feed, referring to Example 4, except that the compound enzymatic hydrolyzed summer and autumn tea feed additive prepared in Example 1 is replaced with the summer and autumn tea product (untreated summer and autumn tea powder) of Comparative Example 2.

[0141] Comparative Example 5 - Laying hen feed containing 5% summer and autumn tea feed additive.

[0142] A type of laying hen feed, referring to Example 5, except that the compound enzymatic hydrolyzed summer and autumn tea feed additive prepared in Example 1 is replaced with the summer and autumn tea product (untreated summer and autumn tea powder) of Comparative Example 2.

[0143] Example 6 - Application of laying hen feed containing summer and autumn tea feed additives in improving egg quality and reducing egg cholesterol

[0144] 1. Feeding and Management

[0145] Two hundred and twenty 34-week-old Xianju laying hens of similar physical condition were randomly divided into seven groups, with three replicates per group and ten hens per replicate. The specific grouping details are shown in Table 17. The hens were cage-housed with free access to feed and water, and their manure was cleaned and disinfected regularly. The experiment lasted for 42 days. The composition and nutrient levels of the basal diet required for free access are shown in Table 4.

[0146] Table 17 Grouping of Experimental Animals

[0147]

[0148]

[0149] Table 18 Composition and Nutritional Levels of Basal Diets

[0150] Diet composition Content (%) Nutritional level Content (%) Corn 66.45 Metabolizable energy (MJ·kg -1 )]]> 11.41 Soybean meal 25.55 Crude protein 18.83 Limestone 3.00 Crude fiber 5.01 Premix 5.00 Calcium 3.14 Total 100.00 Total phosphorus 0.41

[0151] Note: 1. The premix provides the following per kilogram of feed: VA 7000 IU, VB1 1.6 mg, VB2 6.1 mg, VB6 4 mg, VB 12 0.10mg, VD3 2000IU, VE 20IU, VK3 2mg, Biotin 0.21mg, Folic Acid 1.1mg, D-Pantothenic Acid 11mg, Niacin 15.8mg, Cu 10.5mg, Fe 110.5mg, Mn 105mg, Zn 94mg, I 0.53mg, Se 0.11mg.

[0152] 2. Nutritional levels are calculated values.

[0153] 2. Indicator Measurement and Methods

[0154] 2.1 Determination of production performance

[0155] During the experiment, eggs were collected at 16:00 every day. Egg weight, number of eggs laid, number of soft-shelled eggs, and feed intake were recorded in repeated units. Leftover feed was cleaned up and weighed every week. Average egg weight, average daily feed intake, feed conversion ratio (the ratio of feed consumed by the hen during the laying period to the total number of eggs laid), and laying rate were calculated.

[0156] 2.2 Determination of egg quality

[0157] On day 42 of the experiment, six eggs were randomly selected from each replicate for egg quality testing. Egg weight, yolk color (the darker the yolk, the higher the value), and Haugh units (Haugh units are an indicator of egg freshness and protein quality; the better the egg quality, the higher the Haugh unit value) were measured using an egg quality analyzer; the weight of the yolks was measured using an electronic balance.

[0158] 2.3 Determination of egg yolk cholesterol and triglyceride content

[0159] Egg yolks and egg whites were separated. The yolks were weighed using an electronic analytical balance, and the weight was recorded. The yolks were then mixed thoroughly and freeze-dried in a freeze dryer for 72 hours. The weight was recorded afterward, and the yolks were stored at -20°C. Egg yolk cholesterol was determined using a total cholesterol kit from Nanjing Jiancheng Laboratory; egg yolk triglycerides were determined using a triglyceride kit from Nanjing Jiancheng Laboratory.

[0160] 2.4 Determination of egg yolk fatty acid composition

[0161] The fatty acid composition of egg yolk was determined by gas chromatography-mass spectrometry (GC-MS). 1.0 g of sample was weighed into a 50 mL centrifuge tube, and 20 mL of chloroform-methanol (2 / 1, v / v) was added and mixed thoroughly. After standing for 20 min, the mixture was centrifuged at 5000 rpm, and the filtrate was collected. 0.88% NaCl solution was added, and the chloroform layer was collected and dried under nitrogen. 20 mg of oil sample was weighed and added to 3 mL of 0.1 mol / L NaOH methanol solution. Saponification was carried out at 60 °C for 30 min until the oil droplets disappeared. After cooling, 2 mL of 14% BF3-MeOH solution was added, and the mixture was heated at 60 °C for 15 min. After cooling, 2 mL of n-hexane and anhydrous sodium sulfate were added, and the mixture was filtered through a 0.22 μm organic filter membrane and analyzed by GC-MS. GC-MS parameter settings: Column: HP-5MS capillary column (30m×0.25mm×0.25μm), injection port temperature: 250℃, MS detector temperature: 280℃, split ratio: 10:1. Temperature program: 150℃ for 20 min, then ramp to 230℃ at 10℃ / min and hold for 15 min.

[0162] 2.5 Statistical analysis

[0163] SPSS 27.0 software was used to perform statistical analysis on the experimental data.

[0164] 3. Results and Analysis

[0165] 3.1 Effect of the compound enzymatic summer and autumn tea feed additive of the present application on the production performance of laying hens

[0166] The effects of the compound enzymatically hydrolyzed summer and autumn tea feed additive of this invention on the egg production performance of laying hens are shown in Table 19. As shown in Table 19, compared to the control group, after replacing part of the basal diet with the compound enzymatically hydrolyzed summer and autumn tea feed additive of this invention, the average daily feed intake decreased, while the average egg weight increased by 1.33–3.11%, and the feed conversion ratio decreased by 2.26–12.81%, significantly improving the egg production performance of laying hens. Compared to the unenzymatically hydrolyzed summer and autumn tea additives of Comparative Examples 3–5, the compound enzymatically hydrolyzed summer and autumn tea feed additive of this invention improved palatability, increased the average daily feed intake to a certain extent, further increased the egg production rate, further reduced the feed conversion ratio, and improved feed conversion ratio and egg production performance of laying hens. Among them, the layer hen feed containing 5% compound enzymatic hydrolyzed summer and autumn tea feed additive in Example 5 showed the best performance in improving the production performance of layer hens. Compared with using an equal amount of unhydrolyzed summer and autumn tea as feed additive, palatability was improved: the average daily feed intake increased by 3.70%; production performance was significantly improved: the average egg weight increased by 3.86%, the laying rate increased by 18.95%, and the feed conversion ratio decreased by 15.97%.

[0167] Table 19 Effects of each group on laying hen production performance

[0168] Group Average egg weight / g Average daily feed intake / g Egg laying rate / % Feed to egg ratio Blank group 48.85 ± 3.05 b ]] 96.38 ± 0.41 a ]] 37.40 ± 7.06 ab ]] 5.31 ± 0.42 b ]] Comparative Example 3 50.05 ± 2.8 ab ]] 95.55 ± 0.39 ab ]] 36.98 ± 7.71 ab ]] 5.21 ± 0.44 b ]] Comparative Example 4 49.57 ± 3.64 ab ]] 92.69 ± 1.64 c ]] 36.11 ± 7.61 bc ]] 5.20 ± 0.38 b ]] Comparative Example 5 49.22 ± 2.38 b ]] 90.38 ± 2.73 d ]] 33.41 ± 6.95 c ]] 5.51 ± 0.28 a ]] Example 3 50.37 ± 2.95 ab ]] 96.09 ± 0.41 a ]] 37.01 ± 7.43 ab ]] 5.19 ± 0.47 b ]] Example 4 49.50 ± 2.87 ab ]] 93.80 ± 1.95 bc ]] 37.75 ± 7.34 ab ]] 5.04 ± 0.35 c ]] Example 5 51.12 ± 3.05 a ]] 93.72 ± 2.25 bc ]] 39.74 ± 8.07 a ]] 4.63 ± 0.28 c ]]

[0169] Note: Different lowercase letters indicate significant differences between different groups for the same item (P<0.05).

[0170] 3.2 Effect of the compound enzymatic summer and autumn tea feed additive of the present application on the egg quality of each group

[0171] The effects of the compound enzymatic hydrolyzed summer and autumn tea feed additive of this invention on egg quality are shown in Table 20. As shown in Table 20, compared to the control group, after replacing part of the basal diet with the compound enzymatic hydrolyzed summer and autumn tea feed additive of this invention, the yolk color deepened, the Haugh unit increased by 6.04–8.79%, and the yolk ratio increased by 5.44–10.50%. Compared to the unenzymatic summer and autumn tea additives in Comparative Examples 3–5, the compound enzymatic hydrolyzed summer and autumn tea feed additive of this invention further deepened the yolk color and increased the Haugh unit. Among them, the laying hen feed containing 5% compound enzymatic hydrolyzed summer and autumn tea feed additive in Example 5 showed the best effect in improving egg quality, with the deepest yolk color, highest yolk ratio, highest Haugh unit, and best freshness. The compound enzymatic hydrolyzed summer and autumn tea feed additive of this invention has a significant improvement effect on yolk color, Haugh unit, and yolk ratio, and can significantly improve egg quality.

[0172] Table 20 Effects of each group on egg quality

[0173] Group Egg yolk color Hazen unit Egg yolk ratio / % Blank group 7.20 ± 0.45 b ]] 74.88 ± 4.97 b ]] 31.43 ± 1.35 b ]] Comparative Example 3 7.80 ± 0.84 ab ]] 77.90 ± 5.04 ab ]] 31.24 ± 0.90 b ]] Comparative Example 4 8.00 ± 0.71 ab ]] 79.70 ± 3.57 ab ]] 33.40 ± 1.39 a ]] Comparative Example 5 8.20 ± 0.84 ab ]] 78.54 ± 6.84 ab ]] 31.36 ± 1.01 b ]] Example 3 8.20 ± 0.84 ab ]] 79.40 ± 4.91 ab ]] 34.73 ± 2.20 a ]]> Example 4 8.20 ± 0.84 ab ]] 81.10 ± 4.19 a ]] 33.14 ± 1.55 ab ]]> Example 5 8.80 ± 0.84 a ]] 81.46 ± 5.20 a ]] 33.54 ± 0.94 ab ]]

[0174] Note: Different lowercase letters indicate significant differences between different groups for the same item (P<0.05).

[0175] 3.3 Effect of the compound enzymatic summer and autumn tea feed additive of the present application on the egg yolk cholesterol and triglyceride content of each group

[0176] The effects of the compound enzymatic hydrolyzed summer and autumn tea feed additive of the present invention on the cholesterol and triglyceride content of egg yolk are shown in Table 21. As shown in Table 21, compared with the control group, after replacing part of the basal diet with the compound enzymatic hydrolyzed summer and autumn tea feed additive of the present invention, the cholesterol content of egg yolk decreased by more than 6.15%, and the triglyceride content of egg yolk decreased by more than 3.00%. Compared with the unhydrolyzed summer and autumn tea additives of Comparative Examples 3 to 5, the compound enzymatic hydrolyzed summer and autumn tea feed additive of the present invention further reduced the cholesterol and triglyceride content of egg yolk. Compared with the control group, Example 5 showed the largest reduction in cholesterol and triglyceride content of egg yolk, decreasing by 16.65% and 13.22%, respectively. The compound enzymatic hydrolyzed summer and autumn tea feed additive of the present invention can significantly reduce egg cholesterol.

[0177] Table 21. Cholesterol and triglyceride content of egg yolks in each group

[0178]

[0179]

[0180] Note: Different lowercase letters indicate significant differences between different groups for the same item (P<0.05).

[0181] 3.4 Effect of the compound enzymatic summer and autumn tea feed additive of the present application on the fatty acid content in the egg yolk of each group

[0182] The effect of the compound enzymatic hydrolyzed summer and autumn tea feed additive of the present invention on the fatty acid composition ratio of egg yolk is shown in Table 22. As shown in Table 22, compared with the control group, the proportion of SFA (saturated fatty acids) decreased and the proportion of UFA (unsaturated fatty acids) increased in Examples 3 to 5. Specifically, the proportions of egg yolk C18:2 and total PUFA significantly increased in Examples 3 and 4 (P < 0.05); and the content of egg yolk C18:2 and total PUFA significantly increased in Example 5 (P < 0.05). Compared with the unenzymatically hydrolyzed summer and autumn tea additives of Comparative Examples 3 to 5, the compound enzymatic hydrolyzed summer and autumn tea feed additive of the present invention further increased the proportion of UFA (unsaturated fatty acids) and decreased the proportion of SFA (saturated fatty acids). These results indicate that adding the compound enzymatic hydrolyzed summer and autumn tea feed additive of the present invention to laying hen feed increases the proportion of UFA (unsaturated fatty acids) and decreases the proportion of SFA (saturated fatty acids), which can effectively optimize the fatty acid composition of eggs and enhance their nutritional value and health benefits.

[0183] Table 22 Fatty acid composition of egg yolks in each group

[0184]

[0185] Note: Different lowercase letters indicate significant differences between different groups for the same item (P<0.05).

[0186] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing a compound enzymatic hydrolysed summer and autumn tea feed additive, characterized in that, Includes the following steps: (1) Mix the sieved summer and autumn tea powder with water to obtain a summer and autumn tea suspension; (2) Adjust the pH of the summer and autumn tea suspension to 6-6.5, raise the temperature to 45-55℃, add a compound enzyme for enzymatic hydrolysis for 2.5-4 hours, and inactivate the enzyme to obtain a compound enzymatic hydrolyzed summer and autumn tea feed additive; wherein the compound enzyme includes cellulase, xylanase, flavor protease and neutral protease, and the enzyme activity to the mass ratio of summer and autumn tea powder E / S is ≥100U / g, ≥200U / g, ≥1300U / g and ≥4000U / g, respectively.

2. The method according to claim 1, characterized in that, In step (2), the enzymatic hydrolysis pH is 6, the enzymatic hydrolysis time is 3.5h, and the cellulase, xylanase, flavor protease and neutral protease make the enzyme activity to the mass ratio E / S of summer and autumn tea powder 100U / g, 200U / g, 1366U / g and 4098U / g, respectively.

3. The method according to claim 1, characterized in that, In step (2), stirring is used during enzymatic hydrolysis to ensure uniform mixing of the system. The stirring speed during enzymatic hydrolysis is 100-200 rpm.

4. The method according to claim 1, characterized in that, The mesh size for sieving in step (1) is 60-100 mesh.

5. The method according to claim 1, characterized in that, In step (1), the solid-liquid ratio of summer and autumn tea powder to water is 1:6 to 1:8 (m / v, g / mL).

6. The method according to claim 1, characterized in that, In step (2), the enzyme is inactivated by heating to 85-90℃ within 10-20 minutes and maintaining the temperature for 15-20 minutes.

7. The compound enzymatic hydrolyzed summer and autumn tea feed additive prepared by any one of claims 1-6, characterized in that, The compound enzymatically hydrolyzed summer and autumn tea feed additive has a water extract content of no less than 50%, a soluble protein content of no less than 9%, a soluble sugar content of no less than 9%, a tea polyphenol content of no less than 13%, a crude protein digestibility of no less than 43%, a dry matter digestibility of no less than 62%, an acid detergent fiber digestibility of no less than 37%, and a neutral detergent fiber digestibility of no less than 44%.

8. The application of the compound enzymatic hydrolyzed summer and autumn tea feed additive according to claim 7 in improving the laying hen's production performance and egg quality, characterized in that, The improvement of laying hen production performance includes increasing egg weight and reducing feed conversion ratio; the improvement of egg quality includes improving yolk color, Haugh unit and yolk ratio, reducing yolk cholesterol, yolk triglyceride and saturated fatty acid content, and increasing yolk unsaturated fatty acid content.

9. A type of chicken feed, characterized in that, The compound enzymatic hydrolyzed summer and autumn tea feed additive according to claim 7 is added at a rate of 1-10% on a dry weight basis.

10. The chicken feed according to claim 9, characterized in that, Also includes: 60-67% corn, 24-26% soybean meal, 0-8% laying period premix and 0-5% limestone powder.

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