Traditional Chinese medicine fermented feed additive for improving laying hen performance and preparation method thereof

Abstract

The present invention discloses a traditional Chinese medicine fermented feed additive for improving the performance of laying hens. The preparation method comprises the following steps: (1) using Shuanghuanglian slices or crushed and sieved Shuanghuanglian slices as fermentation materials; (2) adding brown sugar to a potato glucose water culture medium to obtain a fermentation liquid, and then adding the fermentation materials to obtain a fermentation medium; (3) inoculating a composite bacterial agent composed of Bacillus amyloliquefaciens and yeast into the fermentation medium for static fermentation or stirring fermentation. The present invention uses a composite bacterial agent composed of Bacillus amyloliquefaciens and yeast that can efficiently degrade cell wall lignin to break down the cell wall of Shuanghuanglian slices and fully extract the medicinal components. Then, by screening the process conditions for breaking down the cell wall of Shuanghuanglian slices, an optimal fermentation system is established to prepare the traditional Chinese medicine fermented feed additive. Feeding tests and related index measurements show that adding the traditional Chinese medicine fermented feed additive prepared by the present invention to drinking water can significantly improve the laying performance and egg quality of laying hens.

Description

Technical Field

[0001] The invention relates to a feed additive, in particular to a traditional Chinese medicine fermented feed additive for improving the laying performance of laying hens or the quality of eggs and a preparation method thereof, belonging to the field of traditional Chinese medicine fermented feed additives for laying hens. Background Art

[0002] Long-term use of antibiotics can leave residues in animals, reducing the quality of meat and eggs and posing a threat to human health through the spread of drug-resistant bacteria. Relevant regulations mandate the avoidance of antibiotic-resistant fungicides as feed additives, prompting researchers to search for suitable antibiotic alternatives. Fermentation is a key processing technique for traditional Chinese medicine (TCM). During the fermentation process, TCM produces a variety of metabolites that can alter its properties, enhance therapeutic efficacy, and reduce side effects. Studies have shown that fermented TCM, when added to daily animal feed, can provide nutrition, boost immunity, and improve production performance. Furthermore, fermented TCM has minimal toxic side effects and lacks the potential for drug resistance, making it a viable alternative to antibiotics in livestock and poultry farming. TCM wall-breaking technology also allows microorganisms to break down Shuanghuanglian into small molecules, allowing for the full extraction of active ingredients and enhancing the TCM's absorption rate by livestock and poultry.

[0003] Shuanghuanglian (Shuanghuanglian) is a classic Chinese medicine formula, a compound herbal formula made from honeysuckle, scutellaria, and forsythia (Fructus Forsythiae) in a 1:1:2 ratio. It possesses antiviral, bactericidal, anti-inflammatory, and antipyretic properties, and has been widely used to treat respiratory infections. In veterinary practice, it is used to treat colds and fevers in livestock and poultry, as well as respiratory diseases caused by bacteria and viruses. However, cell wall obstruction prevents the full release of active ingredients such as baicalin, forsythin, and chlorogenic acid in Shuanghuanglian slices, and there are no reports of Shuanghuanglian slices being used to improve the production performance of laying hens or egg quality. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a Chinese medicinal fermented feed additive that improves the production performance of laying hens or the quality of eggs;

[0005] The second purpose of the present invention is to apply the traditional Chinese medicine fermented feed additive to improve the laying performance of laying hens or the quality of eggs;

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0007] The present invention provides a traditional Chinese medicine fermented feed additive for improving the laying performance of laying hens or the quality of eggs. The traditional Chinese medicine fermented feed additive is prepared by the following preparation method:

[0008] (1) using Shuanghuanglian decoction pieces composed of honeysuckle, scutellaria baicalensis and forsythia as fermentation materials, or crushing and sieving the Shuanghuanglian decoction pieces to obtain fermentation materials;

[0009] (2) adding brown sugar to a potato dextrose (PD) medium to obtain a fermentation broth, and adding a fermentation material to the fermentation broth to obtain a fermentation medium;

[0010] (3) A composite bacterial agent consisting of Bacillus amyloliquefaciens and yeast is added to the fermentation medium for static fermentation or stirred fermentation to obtain a feed additive.

[0011] As a preferred embodiment of the present invention, in step (1), honeysuckle, scutellaria baicalensis and forsythia suspensa are mixed in a mass ratio of 1:1:2.

[0012] The present invention investigates the effect of materials obtained by pulverizing Shuanghuanglian decoction pieces and passing them through sieves of different mesh sizes (20-80 mesh) on the content of medicinal ingredients. The results show that as the Shuanghuanglian particle size becomes finer, the contents of three medicinal ingredients show a trend of first increasing and then decreasing. Among them, at 60 mesh, the contents of the three ingredients reach the highest, which is significantly different from those at 20 mesh and 80 mesh. However, if the particles are too fine, the intracellular components will be destroyed. Taking the contents of the three components into comprehensive consideration, it is determined that the cell wall-breaking effect is best when the particle size is 60 mesh. Therefore, as a preferred embodiment of the present invention, the sieving described in step (1) is through a 20-80 mesh sieve; more preferably, the sieving described in step (1) is through a 60 mesh sieve.

[0013] As a preferred embodiment of the present invention, in step (2), 2-10% brown sugar is added to the PD culture medium; more preferably, 5% brown sugar is added to the PD culture medium.

[0014] As a preferred embodiment of the present invention, the volume ratio of the fermentation material to the fermentation liquid in step (2) is preferably 1:(1-9), more preferably 1:(7-9), and most preferably 1:7.

[0015] The present invention found that different dosage ratios of fermentation materials and fermentation liquid (i.e., material-liquid ratio) have a significant effect on the fermentation effect. As the material-liquid ratio increases, the contents of the three components, chlorogenic acid, baicalin and forsythin, first increase and then decrease. The contents of chlorogenic acid, baicalin and forsythin all reach the highest at 1:7. Moreover, at a material-liquid ratio of 1:7, chlorogenic acid, baicalin and forsythin are significantly different from those at 1:1 and 1:3, and baicalin and forsythin are significantly different from those at 1:9. Taking into account practical applications and costs, the present invention finally determines that the material-liquid ratio of 1:7 is optimal.

[0016] As a preferred embodiment of the present invention, in step (2), the initial pH value of the fermentation medium is controlled to be 5.0-9.0, more preferably, the initial pH value of the fermentation medium is controlled to be 7.0-8.0.

[0017] The present invention investigates the effects of different initial pH values ​​(5.0, 6.0, 7.0, 8.0, and 9.0) of the fermentation medium on the active ingredients. The results show that with the increase of the initial pH, the contents of chlorogenic acid, baicalin, and forsythin first increase and then decrease. Among them, when the initial pH value of the fermentation medium is 5.0-7.0, the contents of the three components gradually increase, and when the initial pH value of the fermentation medium is 7.0-9.0, the contents gradually decrease. When the initial pH value of the fermentation medium is 7.0, the contents of baicalin and forsythin are the highest, and the contents of baicalin and forsythin are significantly different from those at pH 5.0 and 9.0; when the initial pH of the fermentation medium is 8.0, the content of chlorogenic acid is the highest and is significantly different from those at pH 5.0 and 9.0. Taking into account the results of the contents of the three components, the present invention determined that the initial fermentation starting pH of the fermentation medium was 6.0-8.0 for response surface experiment, and finally determined through response surface experiment that the initial pH value of the fermentation medium was 7.0, which was the best.

[0018] As a preferred embodiment of the present invention, in step (3), 1-9% of a composite bacterial agent consisting of Bacillus amyloliquefaciens and yeast is introduced into the fermentation medium; more preferably, 3% of a composite bacterial agent consisting of Bacillus amyloliquefaciens and yeast is introduced into the fermentation medium; most preferably, 3% of a composite bacterial agent consisting of Bacillus amyloliquefaciens and yeast in a mass ratio of 1:1 is introduced into the fermentation medium.

[0019] In order to effectively break the cell walls of Shuanghuanglian slices and facilitate the release of effective ingredients, the present invention screens the compatibility combination of EM bacteria, yeast and Bacillus amyloliquefaciens, hoping to obtain the best composite bacterial agent. According to the screening test results, the composite bacterial agent composed of Bacillus amyloliquefaciens and yeast has the largest number of bacteria, and the medicinal ingredients are significantly higher than other treatments. It can effectively break the cell walls of the slices and is more conducive to the release of effective ingredients.

[0020] The microbial preservation number of the Bacillus amyloliquefaciens described in the present invention is: CGMCC No. 15178; the classification name is: Bacillus amyloliquefaciens; the preservation time is: January 11, 2018; the preservation unit is: General Microbiology Center of China Culture Collection Administration; the preservation address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0021] The present invention further investigated the effects of varying inoculum sizes (1-9%) of the composite inoculum on the active ingredients in fermentation. The results revealed that as the inoculum size of the composite inoculum increased, the contents of chlorogenic acid, baicalin, and forsythin gradually increased and then decreased. Chlorogenic acid and baicalin reached their highest levels at an inoculum size of 3%, while forsythin reached its highest level at an inoculum size of 5%. Although forsythin reached its highest level at an inoculum size of 5%, its content was not significantly different from that at an inoculum size of 3%. Chlorogenic acid and baicalin reached their highest levels at 3%. Therefore, considering the simultaneous extraction of the three components and cost savings, the present invention determined that the optimal inoculum size for the composite inoculum is 3%.

[0022] As a preferred embodiment of the present invention, the fermentation temperature in step (3) is 24-42°C; more preferably, the fermentation temperature in step (3) is 28°C.

[0023] The present invention investigates the effects of different fermentation temperatures (24-42°C) on the active ingredients in fermentation. The results show that as the fermentation temperature increases, the content of chlorogenic acid, baicalin, and forsythin first increases and then decreases. The content of the three ingredients at 28°C is significantly different from that at 42°C; chlorogenic acid and forsythin are significantly different at 28°C and 37°C, and forsythin is significantly different at 28°C and 24°C. Since the content of the three ingredients is the highest at 28°C, the present invention conducts a response surface experiment at a temperature of 24-33°C, and ultimately determines that the fermentation effect is best when the fermentation temperature is 28°C.

[0024] As a preferred embodiment of the present invention, the fermentation time in step (3) is 1-9 days; more preferably, the fermentation time in step (3) is 5 days.

[0025] The present invention investigated the effects of different fermentation times (1-9 days) on the active ingredients in the fermented product. The results showed that with increasing fermentation time, the levels of chlorogenic acid, baicalin, and forsythin initially increased and then decreased. Chlorogenic acid and baicalin levels were highest at 5 days, while forsythin reached its highest level at 7 days. Taking into account factors such as simultaneous extraction of the three components and time savings, the present invention determined that the optimal fermentation time was 5 days.

[0026] As a preferred embodiment of the present invention, the stirring fermentation in step (3) can be carried out at a rotation speed of 120-210 r / min. More preferably, the stirring fermentation is carried out at a rotation speed of 150 r / min.

[0027] The present invention investigated the effects of different stirring speeds (0 r / min, 120 r / min, 150 r / min, 180 r / min, and 210 r / min) on the active ingredients. The results showed that with the increase in speed, the content trends of chlorogenic acid, baicalin, and forsythin all first increased and then decreased. Among them, at a speed of 150 r / min, the content of baicalin and chlorogenic acid reached the highest level and was significantly different from that at 210 r / min; forsythin reached the highest level at 180 r / min, with significant differences from static and 210 r / min. There was no significant difference between forsythin at 180 r / min and 150 r / min. Taking into account the simultaneous extraction of the three components, a speed of 120-180 r / min was determined for response surface experiments. Based on the response surface test results, 150 r / min was finally determined to be the optimal stirring speed.

[0028] A 30-day feeding trial and related indicator measurements showed that adding the feed additive of the present invention to drinking water effectively improved the laying performance and egg quality of laying hens, with feeding effects significantly superior to those of the control group. Therefore, the feed additive provided by the present invention can be used to improve the laying performance or egg quality of laying hens; preferably, the improvement in laying performance of laying hens involves increasing the average daily feed intake, egg production rate, and Haugh value of eggs, while reducing the feed-to-egg ratio.

[0029] The present invention further provides a method for improving the production performance of laying hens or the quality of eggs, comprising: adding 0.3-0.7% wt of a feed additive to the feed or drinking water of the laying hens; preferably, adding 0.5% wt of the feed additive to the feed or drinking water of the laying hens.

[0030] The present invention adopts a composite bacterial agent composed of Bacillus amyloliquefaciens and yeast for efficiently degrading cell wall lignin, breaks the cell wall of Shuanghuanglian slices for degradation and fully extracts the medicinal ingredients, and establishes an optimal fermentation system and prepares a feed additive by screening the process conditions for breaking the cell wall of Shuanghuanglian slices. A 30-day feeding test and relevant index determination show that adding 0.5% of the feed additive to drinking water can effectively promote the laying performance and egg quality of laying hens, and the feeding effect is significantly better than that of the control group. The present invention can be used as a new feed additive in the livestock and poultry breeding industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The effect of material-liquid ratio on the active ingredients.

[0032] Figure 2 The effect of inoculation amount of composite bacterial agent for lignin degradation on fermentation.

[0033] Figure 3 The effect of fermentation temperature on fermentation.

[0034] Figure 4The effect of fermentation time on fermentation.

[0035] Figure 5 The effect of fermentation speed on fermentation.

[0036] Figure 6 This is the effect of the initial pH of fermentation on fermentation.

[0037] Figure 7 The effect of Chinese medicine particle size on fermentation.

[0038] Figure 8 These are the contour lines and response surfaces of the interaction between fermentation initial pH and fermentation temperature on the active ingredients.

[0039] Figure 9 These are the contour lines and response surfaces of the interaction between the initial pH of fermentation and the rotation speed during fermentation on the active ingredients.

[0040] Figure 10 These are the contour lines and response surfaces of the interaction between fermentation temperature and fermentation speed on the active ingredients. DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0042] Example 1 Preparation of Traditional Chinese Medicine Fermented Feed Additive

[0043] (1) crushing the Shuanghuanglian decoction pieces composed of honeysuckle, scutellaria baicalensis and forsythia suspensa in a mass ratio of 1:1:2, and passing the Shuanghuanglian decoction pieces through a 60-mesh sieve to obtain a fermentation material;

[0044] (2) adding 5% brown sugar to the PD medium to obtain a fermentation broth, adding a fermentation material to the fermentation broth to obtain a fermentation medium, wherein the volume ratio of the fermentation material to the fermentation broth is 1:7, and the initial pH value of the fermentation medium is controlled to be 7.0;

[0045] (3) 3% of a composite bacterial agent consisting of Bacillus amyloliquefaciens and yeast in a mass ratio of 1:1 was added to the fermentation medium for stirring and fermentation at a temperature of 28° C. and a stirring speed of 150 r / min. The fermentation was carried out for 5 days to obtain a traditional Chinese medicine fermented feed additive.

[0046] Example 2 Preparation of Traditional Chinese Medicine Fermented Feed Additive

[0047] (1) crushing the Shuanghuanglian decoction pieces composed of honeysuckle, scutellaria baicalensis and forsythia suspensa in a mass ratio of 1:1:2, and passing the Shuanghuanglian decoction pieces through a 20-mesh sieve to obtain a fermentation material;

[0048] (2) adding 3% brown sugar to the PD medium to obtain a fermentation broth, adding a fermentation material to the fermentation broth to obtain a fermentation medium, wherein the volume ratio of the fermentation material to the fermentation broth is 1:2, and the initial pH value of the fermentation medium is controlled to be 5.0;

[0049] (3) 9% of a composite bacterial agent consisting of Bacillus amyloliquefaciens and yeast in a mass ratio of 1:2 was added to the fermentation medium for stirring and fermentation at a temperature of 42° C. and a stirring speed of 120 r / min. The fermentation was carried out for 9 days to obtain a traditional Chinese medicine fermented feed additive.

[0050] Example 3 Preparation of Chinese medicinal fermented feed additive

[0051] (1) Pulverizing Shuanghuanglian decoction pieces composed of honeysuckle, scutellaria baicalensis, and forsythia suspensa in a mass ratio of 1:1:2 and passing through an 80-mesh sieve to obtain a fermentation material;

[0052] (2) adding 6% brown sugar to the PD medium to obtain a fermentation broth, adding a fermentation material to the fermentation broth to obtain a fermentation medium, wherein the volume ratio of the fermentation material to the fermentation broth is 1:9, and the initial pH value of the fermentation medium is controlled to be 9.0;

[0053] (3) 2% of a composite bacterial agent consisting of Bacillus amyloliquefaciens and yeast in a mass ratio of 2:1 was added to the fermentation medium for stirring and fermentation at a temperature of 32° C. and a stirring speed of 180 r / min. The fermentation was carried out for 4 days to obtain a traditional Chinese medicine fermented feed additive.

[0054] Example 4 Preparation of Chinese medicinal fermented feed additive

[0055] (1) Shuanghuanglian decoction pieces consisting of honeysuckle, scutellaria baicalensis, and forsythia suspensa in a mass ratio of 1:1:2 were prepared;

[0056] (2) adding 5% brown sugar to the PD culture medium to obtain a fermentation broth, and adding Shuanghuanglian decoction pieces to the fermentation broth to obtain a fermentation medium, wherein the volume ratio of Shuanghuanglian decoction pieces to the fermentation broth is 1:7, and the initial pH value of the fermentation medium is controlled to be 7.0;

[0057] (3) 3% of a composite bacterial agent consisting of Bacillus amyloliquefaciens and yeast in a mass ratio of 1:1 was added to the fermentation medium for stirring and fermentation at a temperature of 28° C. and a stirring speed of 150 r / min. The fermentation was carried out for 5 days to obtain a traditional Chinese medicine fermented feed additive.

[0058] Test Example 1 Screening test of composite bacterial agent for lignin degradation by wall-breaking fermentation of Shuanghuanglian slices

[0059] 1. Test methods

[0060] In this experiment, three strains were used to screen fermentation agents, mainly to analyze the growth adaptability of the bacteria and the effective components of the fermentation products. The bacterial agents used in this experiment were EM bacterial powder (purchased from a commercial biological company), yeast powder (purchased from a supermarket), and Bacillus amyloliquefaciens (preserved in the laboratory of the inventor, the strain deposit number is CGMCC No. 15178, the classification name is: Bacillus amyloliquefaciens, the strain deposit time is January 11, 2018). The combination of various strains is shown in Table 1.

[0061] Shuanghuanglian fermentation broth: Shuanghuanglian slices were prepared by mixing honeysuckle, scutellaria, and forsythia in a ratio of 1:1:2. The mixture was ground and passed through a 60-mesh sieve. The inoculum from Table 1 was added at a 3% inoculum level, and brown sugar was added at a 5% fermentation level. The Shuanghuanglian slices were then added to a sterilized 100mL fermentation bottle at a material-liquid ratio of 1:7. Fermentation was performed at an initial pH of 7.0, 28°C, and 150 rpm for 5 days.

[0062] Table 1 Combination of bacterial strains

[0063] Treatment group EM bacteria powder Yeast powder Bacillus amyloliquefaciens CK 0 0 0 Process 1 1 1 0 Process 2 1 0 1 Process 3 0 1 1 Process 4 1 1 1

[0064] The adaptability of different bacterial agents in Shuanghuanglian decoction pieces was tested.

[0065] 2. Test results

[0066] The test results are shown in Table 2. When the fermentation reached 15 days, the bacterial count in the fermentation broth was measured. The results showed that all three bacterial combinations could grow in the fermentation broth, with treatments 3 and 4 having the largest bacterial counts. Furthermore, the active ingredients were significantly higher than those in the other treatments, effectively breaking the cell walls of the slices and facilitating the release of active ingredients. Considering cost, treatment 3, a combination of yeast powder and Bacillus amyloliquefaciens, was selected as the bacterial combination for the wall-breaking fermentation of the Shuanghuanglian slices. The two bacterial species were mixed and cultured in a 1:1 ratio to form the composite bacterial agent for the Shuanghuanglian slice wall-breaking fermentation and lignin degradation of the present invention.

[0067] Table 2 Analysis of the fermentation effect of different bacterial strain combinations on Shuanghuanglian decoction pieces

[0068]

[0069] Experimental Example 2 Optimization of Fermentation Process Conditions of Traditional Chinese Medicine Fermented Feed Additives

[0070] 1 Materials and Methods

[0071] 1.1 Materials and Reagents

[0072] Honeysuckle, Scutellaria baicalensis and Forsythia suspensa were provided by Fuyang Normal University; the lignin-degrading composite bacterial agent consisted of Bacillus amyloliquefaciens and yeast in a mass ratio of 1:1; fermented brown sugar was purchased from the market.

[0073] 1.2 Instruments and Equipment

[0074] Electronic balance: Mettler-Toledo Instrument Co., Ltd.; grinder: Zhejiang Wenling Dade Traditional Chinese Medicine Machinery Co., Ltd.; high-pressure sterilizer: Shandong Xinhua Pharmaceutical Instrument Co., Ltd.; constant temperature incubator: Shanghai Senxin Laboratory Instrument Co., Ltd.; pH meter: Shanghai Gaozhi Precision Instrument Co., Ltd.; constant temperature shaker: Shanghai Shidan Pharmaceutical Technology Co., Ltd.; SHZ-D(III) circulating water vacuum pump: Gongyi Yuhua Instrument Co., Ltd.; N-1100D-WD rotary evaporator: Gongyi Yuhua Instrument Co., Ltd.; Agilent 7890A gas chromatograph-mass spectrometer: Agilent Technologies, Inc., USA; UV spectrophotometer: Beijing Puxi General Instrument Co., Ltd.; high-performance liquid chromatograph: Agilent Technologies, Inc., USA; multifunctional egg quality tester: Tianxiang Feiyu International Co., Ltd., USA.

[0075] 1.3 Test methods

[0076] 1.3.1 Preparation of Shuanghuanglian slices

[0077] Shuanghuanglian decoction pieces were prepared by mixing honeysuckle, scutellaria baicalensis and forsythia in a ratio of 1:1:2, and the mixed pieces were crushed and passed through a 60-mesh sieve.

[0078] 1.3.2 Preparation of culture medium and seed solution

[0079] Prepare PD culture medium (PD culture medium formula: 200 g of potatoes (peeled), 12 g of sucrose, and 1000 mL of water) and sterilize under high pressure; the inoculation amount of the lignin-degrading composite bacterial agent is 3%, the amount of fermented brown sugar added is 5%, and Shuanghuanglian decoction pieces are added to a sterilized 100 mL fermentation bottle at a material-liquid ratio of 1:7 for fermentation. The initial pH is 7.0, and the seed liquid is obtained by fermentation.

[0080] 1.3.3 Fermentation

[0081] The seed solution obtained in 1.3.2 was fermented at 28°C and 150 rpm for 5 days.

[0082] 1.4 Screening of factors affecting the fermentation process of Shuanghuanglian slices

[0083] 1.4.1 Effect of material-liquid ratio on active ingredients

[0084] The inoculation amount of the lignin-degrading composite fungus agent was 5%, the fermentation brown sugar addition amount was 5%, the fermentation temperature was 28°C, the fermentation speed was 180r / min, and the fermentation time was 7d. The effects of different material-liquid ratios (1:1, 1:3, 1:5, 1:7, 1:9) on the active ingredients in the fermentation were studied.

[0085] 1.4.2 Effect of inoculation amount of lignin-degrading composite fungus on its active ingredients

[0086] The addition amount of fermented brown sugar was 5%, the material-liquid ratio was 1:5, the fermentation temperature was 28℃, the fermentation speed was 180r / min, and the fermentation time was 7d. The effects of different inoculation amounts of lignin-degrading composite fungi (1%, 3%, 5%, 7%, 9%) on the active ingredients were studied.

[0087] 1.4.3 Effect of fermentation temperature on active ingredients

[0088] The inoculation amount of the lignin-degrading composite fungus agent was 5%, the fermentation brown sugar addition amount was 5%, the material-liquid ratio was 1:5, the fermentation speed was 180r / min, and the fermentation time was 7d. The effects of different fermentation temperatures (24℃, 28℃, 33℃, 37℃, 42℃) on the active ingredients were studied.

[0089] 1.4.4 Effect of fermentation time on active ingredients

[0090] The inoculation amount of the lignin-degrading composite fungus agent was 5%, the fermentation brown sugar addition amount was 5%, the material-liquid ratio was 1:5, the fermentation temperature was 28°C, and the fermentation speed was 180r / min. The effects of different fermentation times (1d, 3d, 5d, 7d, 9d) on the active ingredients were studied.

[0091] 1.4.5 Effect of fermentation speed on active ingredients

[0092] The inoculation amount of the lignin-degrading composite fungus agent was 5%, the fermentation brown sugar addition amount was 5%, the material-liquid ratio was 1:5, the fermentation temperature was 28°C, and the fermentation time was 7 days. The effects of different fermentation speeds (0r / min, 120r / min, 150r / min, 180r / min, 210r / min) on the active ingredients were studied.

[0093] 1.4.6 Effect of fermentation initial pH on active ingredients

[0094] The inoculation amount of the lignin-degrading composite fungus agent was 5%, the fermentation brown sugar addition amount was 5%, the material-liquid ratio was 1:5, the fermentation temperature was 28°C, the fermentation speed was 180r / min, and the fermentation time was 7d. The effects of different pH values ​​(5.0, 6.0, 7.0, 8.0, 9.0) on the active ingredients were studied.

[0095] 1.4.7 Effect of Shuanghuanglian Pieces Particle Size on Medicinal Components

[0096] The inoculation amount of the lignin-degrading composite fungus agent was 5%, the fermentation brown sugar addition amount was 5%, the material-liquid ratio was 1:5, the fermentation temperature was 28°C, the fermentation speed was 180r / min, and the fermentation time was 7d. The effects of different particle sizes (decoction pieces, 20 mesh, 40 mesh, 60 mesh, 80 mesh) on the active ingredients were studied.

[0097] 1.5 Response surface optimization experimental design

[0098] On the basis of the single-factor experiment, the initial pH, temperature and rotation speed were selected to optimize the fermentation conditions. The experimental results were subjected to regression analysis using Design-Expert software. The quadratic polynomial regression equations of chlorogenic acid, baicalin and forsythin for the coding variables A, B and C were obtained as follows. The response surface optimization experiment is shown in Table 3.

[0099] Table 3 Response surface design factors and levels

[0100]

[0101] 2 Test results

[0102] 2.1 Single-factor experiment

[0103] Depend on Figure 1 As can be seen, as the solid-liquid ratio increases, the contents of chlorogenic acid, baicalin, and forsythin first increase and then decrease. The contents of chlorogenic acid, baicalin, and forsythin all reach their highest levels at a solid-liquid ratio of 1:7. Moreover, at a solid-liquid ratio of 1:7, chlorogenic acid, baicalin, and forsythin are significantly different from those at 1:1 and 1:3, and baicalin and forsythin are significantly different from those at 1:9. Although there are no significant differences in the contents of the three components at solid-liquid ratios of 1:5 and 1:7, a solid-liquid ratio of 1:7 is selected for practical applications.

[0104] Depend on Figure 2 As the inoculum size of the lignin-degrading composite inoculum increased, the contents of chlorogenic acid, baicalin, and forsythin gradually increased and then decreased. Chlorogenic acid and baicalin reached their highest levels at an inoculum size of 3%, while forsythin reached its highest level at an inoculum size of 5%. Although forsythin reached its highest level at an inoculum size of 5%, its content was not significantly different from that at an inoculum size of 3%. Chlorogenic acid and baicalin reached their highest levels at an inoculum size of 3%. Therefore, considering the simultaneous extraction of all three components and cost savings, an inoculum size of 3% was selected.

[0105] Depend on Figure 3As shown, the contents of chlorogenic acid, baicalin, and forsythin initially increased and then decreased with increasing fermentation temperature. The contents of all three components at 28°C were significantly different from those at 42°C. Chlorogenic acid and forsythin were significantly different at 28°C compared to 37°C, and forsythin was significantly different at 28°C compared to 24°C. Given that all three components reached their highest levels at 28°C, the response surface experiment was conducted at a temperature between 24°C and 33°C.

[0106] Depend on Figure 4 The results show that the contents of chlorogenic acid, baicalin, and forsythin first increased and then decreased, with the highest contents of chlorogenic acid and baicalin on day 5 and forsythin on day 7. Considering the simultaneous extraction of the three components and saving time, the fermentation time was selected as 5 days.

[0107] Depend on Figure 5 As shown, the contents of chlorogenic acid, baicalin, and forsythin all increased first and then decreased with increasing rotational speed. At a rotational speed of 150 r / min, the contents of baicalin and chlorogenic acid reached their highest levels, significantly different from those at 210 r / min. Forsythin reached its highest level at 180 r / min, significantly different from both static and 210 r / min. There was no significant difference between 180 r / min and 150 r / min forsythin. Considering the simultaneous extraction of the three components, a rotational speed of 120-180 r / min was determined for the response surface experiment.

[0108] Depend on Figure 6 It can be seen that with the increase of initial pH, the content of chlorogenic acid, baicalin, and forsythin showed a trend of first increasing and then decreasing. The content of the three components gradually increased when the pH value was 5.0-7.0, and gradually decreased when the pH value was 7.0-9.0. At pH 7.0, the content of baicalin and forsythin was the highest, and the content of baicalin and forsythin was significantly different from that at pH 5.0 and 9.0. When the pH was 8.0, the content of chlorogenic acid was the highest and significantly different from that at pH 5.0 and 9.0. Since there was no significant difference in the content of chlorogenic acid at pH 7.0 and 8.0, considering the results of the content of the three components, the fermentation starting pH was determined to be 6.0-8.0 for the response surface experiment.

[0109] Depend on Figure 7 As shown, as Shuanghuanglian particle size decreases, the content of the three active ingredients increases and then decreases. At 60 mesh, the content of the three ingredients reaches its highest level, showing significant differences from the decoction pieces, 20 mesh, and 80 mesh sizes. However, too low a particle size can damage intracellular components. Taking into account the content of the three ingredients, a particle size of 60 mesh was determined to be the most effective for cell wall destruction.

[0110] 2.2 Response surface methodology optimization results

[0111] Table 4 Response surface experimental design and result analysis

[0112]

[0113]

[0114] Table 5 Variance analysis of chlorogenic acid response surface

[0115]

[0116] Table 6 Analysis of variance results of baicalin response surface

[0117]

[0118] Table 7 Variance analysis of forsythiaside response surface

[0119]

[0120] Note: * indicates significant difference (*P<0.05); ** indicates extremely significant difference (**P<0.01).

[0121] The response surface analysis results of chlorogenic acid were subjected to second-order response surface regression analysis, and the analysis results are shown in Table 3.

[0122] The regression model for chlorogenic acid was Y = 8.33 - 0.14A - 0.25B + 0.053C + 0.023AB - 0.14AC + 0.16BC - 1.42A² - 0.88B² - 0.84C², where Y represents chlorogenic acid, and A, B, and C correspond to the pH, temperature, and rotational speed codes, respectively. Analysis of variance confirmed that the chlorogenic acid regression model was highly significant (P < 0.0001), while the lack-of-fit term was not significant (P = 0.9837). The linear terms A, B, and C; the interaction terms AC and BC; and the quadratic terms A², B², and C² were all significant, indicating that they significantly influence chlorogenic acid content. The R² of the chlorogenic acid model was 0.9991, and the Radj was 0.9978. This indicates that 99.91% of the chlorogenic acid content is caused by the experimental factor, and CV% = 0.70 and Adep Precision = 72.391 prove that the model has high accuracy and credibility.

[0123] The response surface results of baicalin were subjected to second-order response surface regression analysis, and the analysis results are shown in Table 6.

[0124] The regression model of baicalin was Y=17.20-0.19A+1.20B+0.35C-0.33AB-0.96AC+0.19BC-2.50A 2 -2.14B 2 -2.35C 2, where Y is baicalin, and A, B, and C correspond to the codes of pH, temperature, and speed, respectively. The baicalin regression model was found to be significant according to the variance analysis. The regression of the model was extremely significant (P<0.0001), and the lack of fit term was not significant (P=0.9585). The linear terms A, B, and C, the interaction terms AB, AC, and the quadratic term A 2 、B 2 、C 2 were all significant, indicating that they had a significant effect on the content of baicalin. 2 =0.9964, R adj =0.9918, indicating that 99.64% of the baicalin content was caused by the experimental factor, CV% =1.58, Adep Precision =35.447, proving that the model has high accuracy and credibility.

[0125] The response surface results of forsythin were subjected to second-order response surface regression analysis, and the analysis results are shown in Table 7.

[0126] The regression model of forsythin is Y=17.10+0.31A-0.84B-0.41C-0.15AB-0.78AC+0.19BC-2.65A 2 -1.45B 2 -1.67C 2 , where Y is forsythiaside, and A, B, and C correspond to the codes of pH, temperature, and speed, respectively. The variance analysis showed that the forsythiaside regression model was significant. The regression model was extremely significant (P<0.0001), and the lack of fit term was not significant (P=0.6889). The linear terms A, B, and C, the interaction terms AB, AC, and the quadratic term A 2 、B 2 、C 2 The results were significant, indicating that they had a great influence on the content of forsythiaside. 2 =0.9866, R adj =0.9694, indicating that 99.64% of the forsythin content is caused by this experimental factor, CV% =2.49, Adep Precision =18.978, proving that the model has high accuracy and credibility.

[0127] 2.3 Determination and verification of optimal conditions for the fermentation of Shuanghuanglian slices

[0128] Design-Expert software analysis revealed the optimal fermentation conditions for Shuanghuanglian slices: a fermentation starting pH of 6.99, a fermentation temperature of 27.89°C, and a fermentation speed of 149.99 rpm. Considering practical applications, the fermentation conditions were set to a fermentation starting pH of 7.0, a fermentation temperature of 28°C, and a fermentation speed of 150 rpm. Under these conditions, the measured chlorogenic acid, baicalin, and forsythin contents were 9.36 mg / mL, 18.52 mg / mL, and 17.63 mg / mL, respectively. These values ​​were not significantly different from the model-predicted values ​​(chlorogenic acid 8.33 mg / mL, baicalin 17.17 mg / mL, and forsythin 17.12 mg / mL). Compared to the pre-optimized fermentation conditions, chlorogenic acid, baicalin, and forsythin contents increased by 75.6%, 43.23%, and 47.4%, respectively. This shows that the optimal process parameters are accurate and reliable, indicating that the model can better predict the content of the extracted active ingredients of Shuanghuanglian ( Figures 8-10 ).

[0129] The optimal fermentation process parameters for the wall-breaking fermentation of Shuanghuanglian slices determined by experimental screening in the present invention are as follows: a material-liquid ratio of 1:7, an inoculation amount of a lignin-degrading composite bacterial agent of 3%, a fermentation temperature of 28°C, a fermentation time of 5 days, a fermentation speed of 150 r / min, a fermentation initial pH of 7.0, and a Chinese medicine particle size of 60 meshes.

[0130] Experimental Example 3 Effect of Chinese herbal fermented feed additives on laying performance and egg quality of laying hens

[0131] 1 Materials and Methods

[0132] The trial was completed from November to December 2021.

[0133] The basal feed was purchased from Shandong Liuhe Feed Co., Ltd. Honeysuckle, Scutellaria baicalensis, and Forsythia suspensa were provided by Fuyang Normal University, and the lignin-degrading composite microbial agent was provided by the Biological Microbial Agent Research and Development Center of Northeast Agricultural University.

[0134] The experiment used a single-factor design. 288 healthy laying hens of uniform weight and egg production rate of <80% at 50 weeks of age were randomly divided into four treatments, with eight replicates per treatment and nine hens per replicate. The control group was fed a basal diet and had normal drinking water. All experimental groups were fed a basal diet, with 0.3% of the fermented Chinese medicinal feed additive (prepared in Example 1) added to the drinking water of Group A, 0.5% of the fermented Chinese medicinal feed additive (prepared in Example 1) added to the drinking water of Group B, and 0.7% of the fermented Chinese medicinal feed additive (prepared in Example 1) added to the drinking water of Group C.

[0135] 1.2 Experimental diet and feeding management

[0136] The experimental diet used a corn-soybean meal-based basal diet, formulated according to the nutrient requirements of the Chinese Chicken Husbandry Standard (2004). Seven laying hens per cage were housed in flat cages. All laying hens were housed under the same conditions throughout the experiment, with the ambient temperature gradually decreasing from 36°C to 22°C. The hens had free access to food and water and 24-hour light until the end of the experiment. The cages were disinfected weekly and immunized according to standard procedures.

[0137] 1.3 Measurement indicators and methods

[0138] 1.3.1 Egg production performance

[0139] Eggs were collected daily from each replicate, and the egg production rate was calculated. Eggs were weighed using an electronic balance and the average egg weight was calculated to the nearest 0.01 g. Egg production rate = number of eggs per day / number of hens tested × 100%; average egg weight = total egg weight / total number of eggs.

[0140] 1.3.2 Production performance

[0141] Record the average feed intake of each replicate daily and calculate the feed-to-egg ratio. Average daily feed intake = (feed amount - remaining amount) / number of experimental chickens; feed-to-egg ratio = weekly feed intake / weekly egg weight.

[0142] 1.3.3 Egg quality

[0143] One egg was randomly selected from each replicate weekly. The longitudinal and transverse diameters of the egg were measured with a vernier caliper to calculate the egg shape index (EGI) to the nearest 0.01 mm. The ends and middle of the egg were measured with a micrometer screw, and the average was used to calculate the eggshell thickness to the nearest 0.01 mm. Yolk color, yolk height, and Haugh units were measured using a multifunctional egg quality meter. The egg shape index = EGI divided by the transverse diameter.

[0144] 1.4 Data Analysis

[0145] The experimental data were organized using Excel 2010, and statistical analysis and significance tests (P < 0.05) were performed using SPSS 22 statistical software. Graphs were prepared and variance analysis was performed using GraphPad Prism 5. Response surface design and data analysis were performed using Design-Expert software.

[0146] 2 Test results

[0147] 2.1 Effects of adding Chinese herbal fermented feed additives to drinking water on laying performance of laying hens

[0148] The test results are shown in Table 8.

[0149] Table 8 Effects of Chinese herbal fermented feed additives on laying performance of laying hens

[0150]

[0151] Note: Data are mean + standard deviation. Data in the same column are marked with different letters, indicating significant differences among treatments (P < 0.05).

[0152] As shown in Table 8, the average daily feed intake of laying hens in the groups supplemented with Chinese medicinal fermented feed additives gradually increased compared with the control group; among them, the average egg weight, egg production rate and feed-to-egg ratio when 0.5% and 0.7% of Chinese medicinal fermented feed additives were added were significantly different from those in the control group; the egg production rate was the highest when 0.5% was added and increased by 11.70% compared with the control group; the feed-to-egg ratio was the lowest when 0.5% was added and decreased by 13.18% compared with the control group; this shows that adding 0.5% of Chinese medicinal fermented feed additives has a better effect on the growth of laying hens.

[0153] 2.2 Effects of adding Chinese herbal fermented feed additives to drinking water on egg quality

[0154] Table 9 Effects of Chinese herbal fermented feed additives on egg quality

[0155]

[0156] As shown in Table 9, compared with the control group, there were no significant differences in eggshell thickness, egg shape index and albumen height in the group with added Chinese herbal fermented feed additives; although there was no significant difference in the yolk color of the eggs with added Chinese herbal fermented feed additives in drinking water, it was darker; there were significant differences in the Haugh value between the groups with added Chinese herbal fermented feed additives, and the Haugh value increased by 6.9% when 0.5% was added. Considering factors such as egg quality, egg production performance and application cost, the addition of 0.5% Chinese herbal fermented feed additives was selected.

Claims

1. A Chinese medicinal fermented feed additive for improving laying performance of laying hens or egg quality, characterized in that: The preparation method of the traditional Chinese medicine fermented feed additive comprises: (1) using Shuanghuanglian decoction pieces composed of honeysuckle, scutellaria baicalensis and forsythia suspensa as fermentation materials, or crushing and sieving Shuanghuanglian decoction pieces to obtain fermentation materials; (2) adding brown sugar to a potato glucose water culture medium to obtain a fermentation broth, and adding the fermentation material to the fermentation broth to obtain a fermentation medium; (3) A composite bacterial agent consisting of Bacillus amyloliquefaciens and yeast in a mass ratio of 1:1 is added to the fermentation medium for static fermentation or stirring fermentation to obtain a Chinese medicinal fermented feed additive; the microbial preservation number of the Bacillus amyloliquefaciens is: CGMCC No. 15178.

2. The Chinese medicinal fermented feed additive according to claim 1, characterized in that In step (1), honeysuckle, scutellaria baicalensis and forsythia suspensa are mixed in a mass ratio of 1:1:2; the sieving in step (1) is through a 20-80 mesh sieve.

3. The Chinese medicinal fermented feed additive according to claim 1, characterized in that In step (2), 2-10% brown sugar is added to the potato glucose water culture medium.

4. The Chinese medicinal fermented feed additive according to claim 3, characterized in that In step (2), 5% brown sugar is added to the potato glucose water culture medium.

5. The Chinese medicinal fermented feed additive according to claim 1, characterized in that In step (2), the volume ratio of the fermentation material to the fermentation liquid is 1:(1-9).

6. The Chinese medicinal fermented feed additive according to claim 5, characterized in that: In step (2), the volume ratio of the fermentation material to the fermentation liquid is 1:(7-9).

7. The Chinese medicinal fermented feed additive according to claim 1, characterized in that In step (2), the initial pH value of the fermentation medium is controlled to be 5.0-9.

0.

8. The Chinese medicinal fermented feed additive according to claim 7, characterized in that: In step (2), the initial pH value of the fermentation medium is controlled to be 7.0-8.

0.

9. The Chinese medicinal fermented feed additive according to claim 1, characterized in that: In step (3), 1-9% of a composite bacterial agent consisting of Bacillus amyloliquefaciens and yeast is introduced into the fermentation medium.

10. The Chinese medicinal fermented feed additive according to claim 9, characterized in that: In step (3), 3% of a composite bacterial agent consisting of Bacillus amyloliquefaciens and yeast in a mass ratio of 1:1 is added to the fermentation medium.

11. The Chinese medicinal fermented feed additive according to claim 1, characterized in that: The fermentation temperature in step (3) is 24-42°C.

12. The Chinese medicinal fermented feed additive according to claim 11, characterized in that: The fermentation temperature in step (3) is 28°C.

13. The Chinese medicinal fermented feed additive according to claim 1, characterized in that: The fermentation time in step (3) is 1-9 days; the stirring fermentation in step (3) is carried out at a rotation speed of 120-210 r / min.

14. The Chinese medicinal fermented feed additive according to claim 13, characterized in that: The fermentation time in step (3) is 5 days; the stirring fermentation in step (3) is carried out at a rotation speed of 150 r / min.

15. Use of the fermented Chinese medicinal feed additive according to any one of claims 1 to 14 for non-therapeutic purposes in improving the laying performance of laying hens or the quality of eggs.

16. A method for improving the production performance of laying hens or the quality of eggs for non-therapeutic purposes, characterized in that: Add 0.3-0.7%wt of the feed additive according to any one of claims 1-14 to the feed or drinking water of laying hens.

17. The method according to claim 16, characterized in that Add 0.5%wt of the feed additive according to any one of claims 1 to 14 to the feed or drinking water of laying hens.

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