Bile acid composition for improving utilization rate of grease in feed and application of bile acid composition in animal breeding

Abstract

Hyodeoxycholic acid chenodeoxycholic acid

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a bile acid composition for improving the utilization rate of oil and fat in feed and its application in animal breeding. BACKGROUND

[0002] Oil and fat is one of the most important raw materials in feed, providing energy and essential fatty acids for animals. Increasing the oil and fat content in feed can improve the production performance of animals. Studies have shown that increasing the oil and fat content in feed can increase milk yield and milk protein content in dairy cows, improve egg production rate, egg weight and feed conversion rate in laying hens, and improve fattening performance and carcass quality in mutton sheep. However, high oil and fat levels in feed can also have adverse effects on animals, particularly fish, including reduced growth performance, liver lipid accumulation, and reduced stress resistance. Therefore, improving lipid utilization efficiency and reducing the negative effects of high-fat diets on animals is of great significance to animal health and production performance.

[0003] Emulsifiers are feed additives that can improve oil and fat digestion by emulsifying oil and fat into chylomicrons, thereby improving the degradation efficiency of lipase on oil and fat. Bile acids (BAs) are endogenous emulsifiers synthesized by animals, and their amphiphilic structure naturally has the function of emulsifying oil and fat. Adding bile acids to feed can significantly improve the utilization of oil and fat in animal feed. Supplementing 60 and 80 mg / kg BAs in feed can significantly improve the activity of duodenal lipase in broilers and average daily gain. Supplementing BAs in feed can improve fat digestibility by stimulating the development of the intestinal tract. Adding 0.5%, 1.0% BAs to low fish meal feed can significantly improve the activity of digestive tract lipase in juvenile crabs. Similarly, the results of the same experiment show that BAs (0.3%) significantly improve the body weight and feed conversion rate of grouper, and also significantly increase the activity of intestinal lipase. Adding bile acids to feed can improve the growth performance and nutrient digestibility of weaned piglets.

[0004] There are many types of bile acids, and more than 2000 types of bile acids have been found, and different bile acids have different structures and functions. Currently, the research on bile acids improving oil and fat utilization is limited to the improvement of oil and fat digestion and absorption performance, and the specific mechanism is not clear. At the same time, the specific mechanism of bile acids improving lipase activity is not clear, so it is not possible to determine the optimal bile acid composition for improving oil and fat utilization. SUMMARY

[0005] The application aims to provide a bile acid composition for improving the utilization rate of oil and fat in feed and its application in animal breeding.

[0006] To achieve the above-mentioned application purposes, the application is implemented by the following technical solutions:

[0007] The application provides a bile acid composition for improving the utilization rate of oil and fat in feed, and components of the bile acid composition include: 78-83% of hyodeoxycholic acid and 17-22% of chenodeoxycholic acid by mass percentage.

[0008] The structural formula of the hyodeoxycholic acid is The structural formula of the chenodeoxycholic acid is .

[0009] Further, the bile acid composition is extracted by the high-temperature and high-pressure saponification, oxidation decolorization, acidification crystallization, water washing impurity removal and drying process with pig gall as raw material, and the preparation method of the bile acid composition is specifically as follows:

[0010] (1) Ultra-high temperature and ultra-high pressure saponification: the crushed pig gall is added with 10% of sodium hydroxide and diluted into a liquid with water, and then treated in a reaction kettle at 105-135 DEG C and 1.05-1.10 Mpa for 10-15 h, after the reaction is completed, the liquid is cooled and separated into layers, the supernatant is removed, and pig gall saponification product is obtained;

[0011] (2) Oxidation decolorization: the pig gall saponification product is mixed with water to completely dissolve the pig gall saponification product, and then H2O2 is added for decolorization at 25-30 DEG C for 24 h;

[0012] (3) Acidification crystallization: under the action of stirring, the pH value of the solution is adjusted to 3-3.5 by using hydrochloric acid, and the bile acid crystals are continuously precipitated by continuing stirring;

[0013] (4) Water washing impurity removal and drying: the supernatant is removed by centrifugation, the precipitated bile acid is refined and washed 2-3 times with water, the bile acid is obtained by plate and frame pressure filtration, and then the bile acid composition is obtained by using a spray drying device with the inlet air temperature controlled at 130-180 DEG C and the outlet air temperature controlled at 80-100 DEG C.

[0014] Further, the total bile acid content and the content of hyodeoxycholic acid and chenodeoxycholic acid in the bile acid composition are determined by high performance liquid chromatography; the high performance liquid chromatography conditions are as follows: the chromatographic conditions are determined as follows: a C18 chromatographic column with a length of 150 mm, an inner diameter of 4.6 mm and a particle size of 5; a mobile phase: A liquid: 0.05% trifluoroacetic acid solution, B liquid: acetonitrile; a flow rate: 1.0 mL / min; a detection wavelength: a fluorescence excitation wavelength of 330 nm, an emission wavelength of 410 nm, a column temperature: room temperature, and a sample injection amount: 10 μL.

[0015] Further, the bile acid composition can promote oil emulsification and improve the degradation of lipase on oil.

[0016] Further, the emulsification ability of chenodeoxycholic acid on oil is greater than that of hyodeoxycholic acid.

[0017] Further, the bile acid composition can improve the generation of fatty acids in the oil degradation process.

[0018] Further, the emulsification mode of the bile acid composition on oil is to improve the Zeta potential of oil and reduce the particle size of emulsified oil.

[0019] Further, the bile acid composition can activate the activity of lipase, improve the content of beta-fold in lipase, and promote the degradation of lipase on oil.

[0020] Further, the activation of lipase activity is achieved by the non-covalent combination of the bile acid composition with the active center of lipase, the improvement of the spatial structure of lipase, and the improvement of the activity of lipase.

[0021] Further, the activation ability of hyodeoxycholic acid on lipase is greater than that of chenodeoxycholic acid.

[0022] Further, the optimized components and contents of the bile acid composition are as follows: hyodeoxycholic acid 78%, chenodeoxycholic acid 17%, and the balance is water.

[0023] The application also provides the use of the bile acid composition in the preparation of a feed additive for improving the oil utilization rate of animal feed.

[0024] The application also provides the use of the bile acid composition in the preparation of a feed additive for improving the growth performance of animals.

[0025] Further, the feed additive contains 0.01%-0.1% of the bile acid composition by mass percentage.

[0026] Furthermore, the animals include aquatic animals and livestock.

[0027] Furthermore, the aquatic animals include perch and Litopenaeus vannamei; the livestock and poultry include broiler chickens and laying hens.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] 1. The bile acid composition provided by this invention is extracted from pig bile paste through high-temperature and high-pressure saponification, oxidative decolorization, acidification crystallization, and water washing to remove impurities and drying processes. The components of the bile acid composition are porcine deoxycholic acid and chenodeoxycholic acid, which are permitted for use in my country's "List of Feed Additive Varieties". The bile acid composition is evaluated for its ability to improve the utilization rate of oil and fat and its mechanism of action, providing theoretical support and practical guidance for improving the utilization of oil and fat in feed.

[0030] 2. The bile acid composition of the present invention enhances the degradation of oils by lipase by activating its activity, thereby increasing the formation of fatty acids during the degradation process. The bile acid composition binds to the active site of lipase in a non-covalent manner, thereby improving lipase activity by altering the spatial structure of the lipase. The bile acid composition can promote the emulsification of oils, and its emulsifying performance is achieved by increasing the zeta potential of the oils and reducing the particle size of the emulsified oils. In the bile acid composition, porcine deoxycholic acid mainly plays the role of activating lipase, while chenodeoxycholic acid mainly plays the role of stimulating the emulsification of oils.

[0031] 3. The bile acid composition of the present invention is added to animal feed at a ratio of 0.01% to 0.1% (by feed weight) to efficiently utilize the oil in the feed. Moreover, the preparation process of the bile acid composition is simple, low-cost, cost-effective, and widely applicable. It has reference value for reducing costs and increasing efficiency in large-scale animal farming, whether for aquatic animals or poultry. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0033] Figure 1 The effect of bile acid combinations 1 to 6 on the zeta potential of lipid emulsion.

[0034] Figure 2 The effect of bile acid combinations 1 to 6 on the particle size of oil emulsions.

[0035] Figure 3 The effect of bile acid combinations 1 to 6 on lipase activity.

[0036] Figure 4 The effect of bile acid combination 1 on the secondary structure of lipase.

[0037] Figure 5 The effect of bile acid combination 2 on the secondary structure of lipase.

[0038] Figure 6 The effect of bile acid combination 1 on the tertiary structure of lipase.

[0039] Figure 7 The effect of bile acid combination 2 on the tertiary structure of lipase.

[0040] Figure 8 The effect of bile acid combination 1 on lipase enzymatic kinetics.

[0041] Figure 9 The effect of bile acid combination 2 on lipase enzymatic kinetics.

[0042] Figure 10 The effects of bile acid combinations 1 to 6 on soybean oil degradation.

[0043] Figure 11 The effects of bile acid combinations 1 to 6 on the degradation of palm oil.

[0044] Figure 12 The effect of bile acid combination 6 on the improvement of watermarked eggs in the later stage of egg production. Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the art or the field of application of such terms. While any substance or material similar to or equivalent to those disclosed herein may be used in practice, preferred substances or materials are described herein.

[0047] Example 1: Preparation of bile acid composition

[0048] This embodiment provides a method for preparing bile acids, including porcine deoxycholic acid and chenodeoxycholic acid, both obtained from the extraction of porcine bile extract.

[0049] 1. Preparation of bile acid composition

[0050] (1) Ultra-high temperature and ultra-high pressure saponification: 10%~15% sodium hydroxide was added to the crushed pig bile paste, and the mixture was diluted with water to form a liquid. The mixture was then treated in a reactor at 105~135℃ and 1.05-1.10 MPa for 10~15 h. After the reaction was completed, the liquid was cooled and separated into layers. The supernatant was removed to obtain the saponified pig bile paste.

[0051] (2) Oxidative decolorization: Mix the pig bile paste saponified with water to completely dissolve the pig bile paste saponified, then add H2O2 and decolorize for 24 h at 25~30℃.

[0052] (3) Acidification crystallization: While stirring, use hydrochloric acid to adjust the pH of the solution to 3.0~3.5, and continue stirring until bile acid crystals precipitate out continuously.

[0053] (4) Washing and drying: Centrifuge to remove supernatant, wash the precipitated bile acids with water 2-3 times, filter through plate and frame filter to obtain bile acids, and then use spray drying equipment with the inlet air temperature controlled at 130-180℃ and the outlet air temperature controlled at 80-100℃ to obtain bile acid composition.

[0054] 2. Measurement of the components of the bile acid composition

[0055] The total bile acid content and the contents of porcine deoxycholic acid and chenodeoxycholic acid in the bile acid composition obtained above were determined by high performance liquid chromatography (HPLC) using the following experiments:

[0056] (1) Before detection by high performance liquid chromatography, the bile acid composition was derivatized in acetonitrile solution using 4-bromomethyl-7-methoxycoumarin as a derivatizing reagent to obtain bile acid fluorescent derivatives.

[0057] (2) High-performance liquid chromatography (HPLC) conditions were as follows: A C18 column, 150 mm long, 4.6 mm inner diameter, and 5 μm particle size. Mobile phase: Solution A: 0.05% trifluoroacetic acid solution; Solution B: acetonitrile. Flow rate: 1.0 mL / min. Detection wavelength: fluorescence excitation wavelength 330 nm, emission wavelength 410 nm. Column temperature: room temperature. Injection volume: 10 μL. Elution gradient: 0–2 min 40% Solution A + 60% Solution B, 3–18 min 20% Solution A + 80% Solution B, 19–26 min 40% Solution A + 60% Solution B. The retention time of porcine deoxycholic acid was 9.2 min, and the retention time of chenodeoxycholic acid was 14.8 min.

[0058] 3. The bile acid composition is made by mixing the components porcine deoxycholic acid and chenodeoxycholic acid in the following mass ratio:

[0059] Combination 1 (100% porcine deoxycholic acid, 0% chenodeoxycholic acid);

[0060] Combination 2 (0% porcine deoxycholic acid, 100% chenodeoxycholic acid);

[0061] Combination 3 (20% porcine deoxycholic acid, 80% chenodeoxycholic acid);

[0062] Combination 4 (50% porcine deoxycholic acid, 50% chenodeoxycholic acid);

[0063] Combination 5 (80% porcine deoxycholic acid, 20% chenodeoxycholic acid);

[0064] Combination 6 (78% porcine deoxycholic acid, 17% chenodeoxycholic acid, balance water).

[0065] Based on different combinations of porcine deoxycholic acid and chenodeoxycholic acid, this study explores the role of bile acids in animal husbandry and conducts experiments.

[0066] Example 2: Investigating the emulsifying properties of bile acid compositions on oils and fats.

[0067] 10 mL of phosphate buffer (pH 7.2) was added to 1 g of soybean oil. Samples from combinations 1 to 6, each with a final concentration of 10 mg / mL bile acids, were added separately, homogenized for 2 min, and incubated at room temperature for 1 h. The zeta potential and particle size of the emulsified oils were measured using a nanoparticle size and zeta potential analyzer. Oil samples without bile acid combinations served as a control group, with three replicates per group. The effect of samples from combinations 1 to 6 on the emulsifying properties of palm oil was determined using the same method. In oil-forming emulsion systems, zeta potential and particle size are frequently used to characterize emulsion stability. A higher absolute value of the zeta potential indicates stronger electrostatic repulsion between oil droplets, resulting in a more stable emulsion system. Conversely, smaller droplet sizes in the emulsion also indicate a more stable emulsion system.

[0068] The zeta potentials of emulsions formed after emulsifying fats with different combinations of bile acids are shown in the figure. Figure 1 In soybean oil emulsions, the absolute values ​​of the Zeta potentials of samples with bile acid combinations 1 through 6 were significantly higher than those of the control group (P < 0.05), while there were no significant differences between the groups of combinations 1 through 6 (P > 0.05). The absolute value of the Zeta potential of combination 2 with added bile acids reached 50 mV, and the absolute values ​​of the Zeta potentials of the other combinations with added bile acids were significantly higher than those of the control group (P < 0.05). Similarly, in palm oil emulsions, the absolute values ​​of the Zeta potentials of samples with bile acid combinations 1 through 6 were also extremely significantly higher than those of the control group (P < 0.05), while there were no significant differences between the groups of combinations 1 through 6 (P > 0.05). The absolute value of the Zeta potential of combination 1 with added bile acids reached 42 mV, and the absolute value of the Zeta potential of combination 2 with added bile acids reached 44 mV. In the study of the effects of different bile acids on the emulsifying properties of oils, the addition of bile acids consistently increased the absolute value of the emulsion's Zeta potential. Chedeoxycholic acid (CHEC) showed superior results compared to porcine deoxycholic acid (PDCA), indicating that the emulsion formed with CHEC was more stable. This is attributed to CHEC's higher hydrophobicity, allowing it to adhere more tightly to the oil droplet surface, forming a more stable molecular film. This effectively reduces interfacial tension and thus improves the stability of the emulsion system.

[0069] Oil droplet size in oil emulsions is another key parameter for evaluating the stability of the emulsion system. Smaller droplet sizes result in larger specific surface areas, stronger Brownian motion, and lower frequencies of collision and aggregation, leading to more uniform dispersion in the aqueous phase. The droplet sizes of the bile acid combinations 1 through 6 after emulsifying soybean oil are shown in [the table / reference needed]. Figure 2In the soybean oil emulsification experiment, bile acid combination 1 significantly reduced the particle size of the soybean oil emulsion (P<0.05), decreasing from 3.70 µm to 1.64 µm. Compared to soybean oil, palm oil is more difficult for animals to utilize. In the palm oil emulsification experiments using samples from bile acid combinations 1 to 6, chenodeoxycholic acid in bile acid combination 1 and porcine deoxycholic acid in bile acid combination 2 both significantly reduced the particle size of the soybean oil emulsion (P<0.01), while the particle size of the palm oil emulsion decreased from 3.62 µm to 2.16 µm and 2.55 µm, respectively. These results indicate that chenodeoxycholic acid has a greater impact on the particle size of oil emulsions than porcine deoxycholic acid, resulting in a more stable emulsion system. Therefore, chenodeoxycholic acid is more effective than porcine deoxycholic acid in emulsifying oils.

[0070] Example 3: Activation performance of bile acid composition on lipase

[0071] Methanol solutions of bile acid combinations 1 through 6 were prepared at a concentration of 50 mg / mL and serially diluted to concentrations of 0, 2, 4, 6, and 8 mg / mL. A pancreatic lipase solution with a final concentration of 5 mg / mL was prepared using Tris-HCl (pH 8.2) buffer. 20 µL of each bile acid combination (1 through 6) was added to 50 µL of pancreatic lipase solution and 100 µL of Tris-HCl buffer, mixed, and incubated at 37°C for 15 min. 30 µL of 4-nitrobenzene laurate substrate (10 mmol / L) was added, mixed, and incubated at 37°C for 20 min. The absorbance was measured at 405 nm. The treatment without pancreatic lipase served as the control group, the treatment without bile acids served as the experimental control group, and the treatment without both bile acids and pancreatic lipase served as the blank control group. Each treatment was repeated three times. The activation rate was calculated using the following formula:

[0072] Activation rate (%) = {〔(A 样品试验 - A 样品对照 ) / (A 对照试验 - A对照空白 )〕- 1} × 100%;

[0073] In the formula: A 样品试验 A represents the absorbance of the sample group. 样品对照 A is the absorbance value of the sample control group; 对照试验 A represents the absorbance of the control group. 对照空白 The absorbance values ​​are for the control group.

[0074] Depend on Figure 3It was found that samples from bile acid combinations 1 to 6 all enhanced pancreatic lipase activity at concentrations of 2-8 mg / mL. Among them, bile acid combination 1 showed the greatest increase in lipase activity at 4 mg / mL, reaching 43%; bile acid combination 2 showed the greatest increase at 4 mg / mL, reaching 19%. Subsequently, the effect of bile acids on lipase activity decreased with increasing concentration. Overall, the activation capacity of bile acid combination 1 for lipase was 17%-45%, stronger than that of bile acid combination 2 (3%-21%). Therefore, in terms of lipase activation, porcine deoxycholic acid is stronger than chenodeoxycholic acid.

[0075] Example 4: Changes in the structure of lipase caused by different bile acids

[0076] Solutions of bile acid combination 1 and bile acid combination 2 (4 mg / mL) were prepared. Pancreatic lipase solution (0.25 mg / mL) was added at bile acid to pancreatic lipase molar ratios of 2:1 and 4:1, respectively, and the solutions were mixed thoroughly. The mixtures were incubated at 37℃ for 30 min. Changes in the secondary structure of pancreatic lipase were detected by circular dichroism spectroscopy. The control group (without bile acid) was used. The circular dichroism spectroscopy conditions were: quartz cell aperture 0.1 mm, temperature 25℃, scanning range 190–250 nm, scanning speed 50 nm / min, repeated three times, and the average value was taken. The obtained circular dichroism spectral data were used to calculate the content of each secondary structure of lipase on the Dichroweb website (http: / / dichroweb.cryst.bbk.ac.uk / html / apply.shtml). The results of the circular dichroism spectroscopy are shown below. Figure 4 and Figure 5 The results showed that bile acid combination 1 ( Figure 4 ) and bile acid combination 2 ( Figure 5 This significantly affects the secondary structure of lipase, showing a distinct negative absorption peak at 216 nm, indicating an increased β-sheet content. Furthermore, as the ratio of bile acid combination 1 and bile acid combination 2 to lipase increases from 2:1 to 4:1, the β-sheet content in lipase further increases. Simultaneously, it binds to… Figure 4 and Figure 5 It is evident that, compared to chenodeoxycholic acid in bile acid combination 2, porcine deoxycholic acid in bile acid combination 1 significantly increases the β-sheet content. This structural change is closely related to the effect of bile acids on lipase activity. The increased catalytic activity of lipase is due to the structural alteration, leading to the exposure of the lipase active site or the stabilization of the active conformation.

[0077] Bile acids and lipases were labeled as ligands and receptors, respectively. The molecular structures of the ligands, porcine deoxycholic acid and chenodeoxycholic acid, were obtained from the PubChem database (https: / / testpubchem.ncbi.nlm.nih.gov / ), and the molecular structure of the receptor, pancreatic lipase (accession number: 1ETH), was obtained from the PDB database (https: / / www.rcsb.org / ). After dehydration and hydrogenation of the ligands and receptors using Autodocktools 1.5.7 software, docking simulations of bile acids and pancreatic lipases were performed using Autodock Vina 1.1.2 software, and the docking results were displayed using Pymol 3.0.4 software. The active site of the lipase is a catalytic triplet structure composed of α-helices and β-sheets. The molecular docking results of the ligands, porcine deoxycholic acid and chenodeoxycholic acid, with the receptor, porcine trypsin are shown below. Figure 6 and Figure 7 As shown, the porcine deoxycholic acid in bile acid combination sample 1 ( Figure 6 ) and bile acids combined in 2 samples chenodeoxycholic acid ( Figure 7 All of these can bind to the interior of the active site. Normally, the catalytic triplet of the lipase active site is embedded under a flexible loop structure "lid." When the lipase is at the oil-water interface, the loop structure opens, allowing the lipid to enter the active site for degradation. The binding of bile acids in this region stabilizes the open state of the loop structure. Simultaneously, molecular docking results show that the bile acid combination 1 sample contains porcine deoxycholic acid (…). Figure 6 It can form hydrogen bonds with lipase Trp107 with a bond length of 3.0 Å, while the formation of hydrogen bonds was not predicted in the docking results of chenodeoxycholic acid. Figure 7 The formation of hydrogen bonds makes the loop structure near the active site of lipase more stable, thus exhibiting a stronger activating effect on lipase.

[0078] Example 5: Effects of different bile acids on the kinetics of lipase enzymatic reactions

[0079] Pancreatic lipase (5 mg / mL) was mixed with different concentrations of porcine deoxycholic acid and chenodeoxycholic acid (0, 4, 8, 12, 16 mg / mL). Then, 0.05, 0.075, 0.1, 0.2, and 0.4 mg / mL of the substrate 4-nitrobenzene laurate were added, and the mixture was reacted for 30 min. The absorbance was measured at 405 nm. Each treatment was repeated three times, and double reciprocal kinetic curves were plotted. The analytical equations are as follows:

[0080] 1 / V = ​​K m / V max (1 + [I] / K) i )×1 / 〔S〕+ 1 / Vmax

[0081] Where: V is the enzyme reaction rate, Δabsorbance / min; Vmax is the maximum enzyme reaction rate, Δabsorbance / min; [S] is the substrate concentration, mg / mL; [I] is the concentration of porcine deoxycholic acid and chenodeoxycholic acid, mg / mL; K m K is the Michaelis constant; i The activation constant is the value of the free enzyme.

[0082] porcine deoxycholic acid in bile acid combination 1 ( Figure 8 ) and bile acid combination 2 chenodeoxycholic acid ( Figure 9 Kinetic analysis of the effect of bile acid on lipase showed that the straight lines of the Lineweaver-Burk double reciprocal curves intersected in the second and first quadrants, respectively. Furthermore, with increasing bile acid concentration, Km gradually decreased, while Vmax gradually increased. This indicates that bile acids can affect the catalytic efficiency of lipase by altering its kinetic parameters. Overall, the porcine deoxycholic acid (POC) in bile acid combination 1... Figure 8 The effect of chenodeoxycholic acid on promoting lipase enzymatic reactions was higher than that of bile acid combination 2. Figure 9 The results were consistent with those of the lipase activation rate test.

[0083] Example 6: Effect of bile acid composition on the degradation properties of fats and oils

[0084] 5 g of soybean oil was mixed with 25 mL of phosphate buffer. Samples from combinations 1 to 6 of bile acids, with final concentrations of 0, 5, 10, 15, 20, 25, 30, 35, 40, and 45 mg / mL, were then added. 300 mg of pancreatic lipase was added, and the mixture was incubated at 40℃ and 180 rpm for 3.5 h. The reaction was then terminated by boiling in a water bath for 10 min, cooled to room temperature, and 2-3 drops of phenolphthalein were added. The mixture was titrated with 0.05 mol / L potassium hydroxide standard titrant until a red color appeared that did not fade within 30 seconds. The treatment without bile acids served as the control group, with three replicates per group. The effect of samples from combinations 1 to 6 of bile acids on fatty acid production from palm oil was determined using the same method.

[0085] The effects of bile acid combinations 1-6 on the degradation of soybean oil by lipase to produce fatty acids are as follows: Figure 10As shown in the figure, in combination 1, the acid value reached its maximum at a concentration of 5 mg / mL of porcine deoxycholic acid (PDA), reaching 34.88 mg KOH / g, which was 56% higher than the control group without added PDA (22.33 mg KOH / g). In combination 2, the acid value reached its maximum at a concentration of 15 mg / mL of chenodeoxycholic acid (CEA), reaching 30.75 mg KOH / g, which was 38% higher than the control group without added PDA (22.23 mg KOH / g). Subsequently, with the increase of the added amounts of porcine and chenodeoxycholic acid, the acid value tended to decrease, but remained higher than the control group.

[0086] The effects of bile acid combinations 1-6 on the degradation of palm oil by lipase to produce fatty acids are as follows: Figure 11 As shown, samples from bile acid combinations 1 to 6 all improved the efficiency of lipase degradation of palm oil to produce fatty acids. Among them, combination 1, with a porcine deoxycholic acid concentration of 15 mg / mL, achieved the highest acid value of 26.53 mg KOH / g, a 71% increase compared to the control group (15.50 mg KOH / g) without added bile acid. Combination 2, with a chenodeoxycholic acid concentration of 45 mg / mL, achieved the highest acid value of 25.22 mg KOH / g, a 65% increase compared to the control group (15.31 mg KOH / g) without added bile acid. Combination 6 achieved the highest acid value. Subsequently, with increasing bile acid concentration, the acid value tended to decrease, but remained higher than the control group.

[0087] Bile acids influence the efficiency of lipid degradation into fatty acids in two ways. Firstly, bile acids promote lipid emulsification, resulting in emulsions that are more conducive to lipase degradation. Secondly, bile acids can alter the structure of lipases, increasing their activity and further promoting lipid degradation. In the first aspect, chenodeoxycholic acid plays a dominant role, while in the second aspect, porcine deoxycholic acid plays a dominant role. Therefore, by using different proportions of porcine and chenodeoxycholic acid, the efficiency of lipid degradation can be maximized.

[0088] Example 7: Application of bile acid composition in promoting oil utilization in sea bass farming

[0089] Eight hundred healthy largemouth bass, weighing 150±5 g, were randomly divided into seven groups for the experiment. Before the experiment officially began, the bass were temporarily held in a recirculating aquaculture system for two weeks to allow them to adapt to the experimental diet and rearing conditions. During the temporary holding period, they were fed a basic diet free of exogenous bile acids (commercially available extruded feed specifically for bass: crude protein ≥48.0%, crude fiber ≤4.0%, crude fat ≥9.0%, crude ash ≤12.0%).

[0090] After the experiment began, the control group was fed a basal diet, while the experimental groups were fed a basal diet supplemented with bile acids (samples 1-6) at 200 mg / kg. Feeding was done twice daily, at 09:00 and 16:00 during the initial holding and experimental periods, respectively. Water conditions, fish feeding, and mortality were recorded daily during the feeding period. Water temperature and dissolved oxygen were maintained at 24-29℃ and 4.5-6.5 mg / L, respectively, while nitrite and ammonia nitrogen levels were controlled below 0.02 mg / L and 0.1 mg / L, respectively. The experimental period was 70 days.

[0091] After the rearing period, all treatment groups were starved for 24 hours. Five fish from each group were randomly selected, anesthetized with 80 mg / L chlorobutanol, and their body length and weight were measured. Then, blood was collected by tail amputation into centrifuge tubes containing an anticoagulant (EDTA-K2), mixed, and centrifuged at 4°C and 4000 rpm for 10 min. The serum was separated and stored at -80°C. Finally, the fish were dissected, and the total weight of the viscera and liver were measured. The intestinal tract (2 cm from the middle section) was collected, flash-frozen in liquid nitrogen, and stored at -80°C.

[0092] Each treatment group was weighed, and the feed conversion ratio, weight gain rate, feed intake rate, and specific growth rate were statistically analyzed. Based on the body length, body weight, visceral weight, liver weight, and spleen weight of each fish, the condition factor, liver-to-body ratio, and visceral-to-body ratio were calculated. The following indicators were calculated using the following formulas:

[0093] Feed conversion ratio = Wf / (Wt--Wo);

[0094] Weight gain rate / % = (Wt-Wo) / Wo × 100;

[0095] Feed intake rate (% / d) = 100 × Wf / [70 × (Wt + Wo) / 2];

[0096] Specific growth rate / % = (lnWt - lnWo) / 70 × 100;

[0097] Body condition / (g·cm-3) = (W / L3)×100;

[0098] Liver-to-body weight ratio = Wg / Wt × 100%;

[0099] Visceral ratio = Wn / Wt × 100%;

[0100] In the formula: Wf is the total mass of feed (g), Wt is the final mass (g), Wo is the initial mass (g), 70 is the number of days of feeding (d), W is the fish body mass (g), L is the fish body length (cm), Wg is the liver mass (g), and Wn is the visceral mass mass (g).

[0101] The effects of different bile acids on the growth performance indicators of largemouth bass are shown in Table 1. Compared with the control group, the addition of bile acids to the diet in combinations 1, 2, and 6 significantly increased the body length, weight, weight gain rate, and specific growth rate of bass in each experimental group; at the same time, it reduced the feed intake rate and feed conversion ratio of bass (P < 0.05). Among them, although the bile acid combination 6 showed no significant difference from the single bile acid combination 1 and bile acid combination 2 in all indicators (P > 0.05), its performance was slightly better than that of the single experimental groups, especially in terms of visceral-to-body ratio (P < 0.05).

[0102] Table 1. Effects of different bile acid combinations on growth performance indicators of sea bass

[0103]

[0104] Note: Different lowercase letters in the superscript of data from the same row indicate significant differences (P < 0.05).

[0105] Accurately weigh 1 g of intestinal tissue, add 9 mL of physiological saline, mechanically homogenize under ice bath conditions, centrifuge at 3000 rpm for 10 min, and collect the supernatant for testing (the protein content of the homogenate supernatant should be determined simultaneously). Lipase activity was determined by colorimetric enzymatic method using a commercial kit, and the assay method was performed according to the kit instructions.

[0106] The effects of different bile acids on the intestinal enzyme activities of largemouth bass are shown in Table 2. Compared with the control group, the addition of bile acids to the feed of samples 1 to 6 significantly increased the activities of amylase (unit: U / mg) and lipase (unit: U / gprot) in the intestinal tissue of bass (P<0.05), while the activity of protease (unit: U / mgprot) showed an increasing trend, but did not reach the level of significant difference (P>0.05).

[0107] Table 2. Effects of different bile acid combinations on the activity of digestive enzymes in the intestine of sea bass.

[0108]

[0109] Data from each group of experiments were processed using Excel software. One-way ANOVA was performed using the general linear model in SPSS software, and Dencan's method was used for multiple comparisons. Results are expressed as mean ± standard deviation, and P < 0.05 was considered statistically significant. Bar chart analysis was used to analyze the changes in various parameters of the sea bass.

[0110] Example 8: Bile acid composition promotes oil utilization in shrimp farming

[0111] One hundred and eighteen hundred healthy Litopenaeus vannamei shrimp with an initial weight of 0.16 ± 0.01 g and consistent genetic background were selected as experimental shrimp and randomly divided into 36 tanks, with 50 shrimp per tank, for a total of 6 groups and 6 replicates per group. The control group was fed a diet without added bile acids, while the other groups were fed experimental diets containing bile acids (combinations 1 to 6) at a concentration of 200 mg / kg. After a 14-day pre-feeding period, a formal feeding and rearing trial was conducted for eight weeks. The results in Table 3 show that the addition of bile acids to the diet tended to improve the survival rate, condition factor, hepatosome index, feed intake, feed conversion ratio, protein efficiency, and protein deposition rate of Litopenaeus vannamei (P>0.05). Shrimp growth performance increased with increasing bile acid content in the diet, with combination 6 showing the best growth performance. The compositional analysis in Table 4 shows that the addition of bile acids to the diet significantly reduced the crude fat content of the whole shrimp, with the crude fat content of the whole shrimp in combinations 1 to 6 being significantly lower than that in the control group (P<0.05).

[0112] The analysis of digestive enzyme activities in Table 5 revealed that the addition of bile acids to the feed of samples 1-6 significantly increased the activity of hepatopancreatic protease (U / mgprot) (P<0.05), hepatopancreatic lipase (U / gprot) (P<0.05), and midgut lipase (P<0.05), with sample 6 showing the greatest increase in enzyme activity. Conclusion: Under the experimental conditions, 200 mg / kg of bile acids (combinations 1-6) is an effective feed additive for Litopenaeus vannamei, with sample 6 showing the best efficacy and the most significant improvement in growth and lipase activity.

[0113] Table 3. Effects of different bile acid combinations in feed on growth performance and feed utilization of Litopenaeus vannamei.

[0114]

[0115] Table 4. Effects of different bile acid combinations added to the feed on the nutritional composition of whole Litopenaeus vannamei.

[0116]

[0117] Table 5. Effects of different bile acid combinations added to feed on the digestive enzyme activity of Litopenaeus vannamei.

[0118]

[0119] Example 9: Bile acid composition promotes oil utilization in broiler farming

[0120] 12,000 healthy, one-day-old "AA" white-feathered broilers of similar weight were selected and divided into a control group and an experimental group, with 6,000 birds in each group and 3 replicates in each group, with 2,000 birds in each replicate, and half males and half females.

[0121] The experiment used a corn-soybean meal basal diet. The control group's diet was the basal diet (without any other bile acid products). The experimental group's diet consisted of the basal diet supplemented with 200 mg / kg of bile acid combination 6 sample. The basal diet was formulated in accordance with the People's Republic of China National Standard "Compound Feed for Laying Hens and Broilers" (GB / T 5916—2020).

[0122] Throughout the experiment, chickens were cage-raised with free access to feed and water, and were vaccinated according to the normal immunization schedule. The chicken coop was kept well-ventilated and clean during the experiment, and regular cleaning and disinfection were carried out. Daily inspections were conducted to observe the chickens' feed intake, droppings, and health status. If any dead or sick chickens were found and culled during the experiment, the number of dead or culled chickens and their feed intake were recorded.

[0123] Methods for measuring growth performance indicators: Before the start of the experiment and at 42 days of age (feeding was stopped 12 hours in advance), the weight of each group of broilers was measured. At the end of the experiment, the feed consumption was recorded, and the average daily feed intake (ADFI), average daily weight gain (ADG), and feed conversion ratio (F / G) of each group of broilers were calculated. The mortality rate was also recorded, and the breeding benefits were calculated.

[0124] The calculation formula is as follows:

[0125] Average daily weight gain (ADG) = (average final weight - average initial weight) / number of days in the trial;

[0126] Average daily feed intake (ADFI) = (Total feed consumption - Feed consumption of dead and culled chickens) / (Number of days in the trial × Number of chickens);

[0127] Feed conversion ratio (F / G) = Average daily feed intake / Average daily weight gain × 100%;

[0128] Mortality rate = (Number of dead chickens / Total number of chickens) × 100%.

[0129] Experimental data (growth performance indicators) were initially statistically analyzed using Excel, and then analyzed for variance using SPSS 17.0 software. Multiple comparisons were performed using Duncan's method. P < 0.05 was used as the criterion for statistical significance, and results are expressed as mean ± standard deviation.

[0130] Table 6. Effects of bile acid combination 6 on growth performance of 1-36 day old broiler chickens

[0131]

[0132] Note: No letters above the top of the data in the same column indicate no significant difference (P>0.05), while different lowercase letters indicate significant difference (P<0.05).

[0133] Based on the experimental results in Table 6, it can be seen that adding 200g / ton of bile acid combination 6 to the diet of white-feathered broilers significantly increased the final body weight of the broilers in the experimental group by 5.72% compared with the control group; the F / G and mortality rate of the experimental group were significantly lower than those of the control group, decreasing by 2.65% and 25.32% respectively; and the ADG and ADFI were higher than those of the control group, but the differences were not significant.

[0134] Bile acid compositions, as bioactive emulsifiers, can promote the absorption of fats and fat-soluble vitamins, while also protecting the liver and gallbladder and promoting detoxification. Adding bile acid compositions to broiler diets reduces the need for added oils, saving feed costs. This not only increases feed intake and daily weight gain, and lowers the feed conversion ratio, but also improves intestinal and overall health, significantly reducing mortality and increasing profitability. In this experiment, overall feed costs decreased by 20-25 yuan / ton, while sales revenue increased, with an average increase of 0.90 yuan in gross profit per broiler.

[0135] Example 10: Application of bile acid composition in promoting egg production in laying hens

[0136] Watermarked eggs, also known as dark-spotted eggs, show transparent marks resembling rainwater runoff or raindrops when observed under directional light in a dark place. These eggs pose risks in the distribution process (short shelf life and high spoilage), in sales (short production period), in commercial egg production (more cracked eggs), and in branded egg supply (significantly reduced premium egg rate). These are pressing issues that egg farms urgently need to address.

[0137] The experiment used six laying hen farms, each with approximately 32,000 hens, for a total of 240,000 hens. Two farms had particularly high rates of watermarked eggs; one farm housed 221-day-old Hy-Line White hens, and the other housed 260-day-old Nongda No. 3 hens. The Hy-Line White farm had 32,000 hens, consuming 115 grams per hen / day, for a total of 4.6 tons of feed per day. The Nongda No. 3 farm had 32,000 hens, consuming 90 grams per hen / day, for a total of 3.6 tons of feed per day. Six different bile acid combinations were continuously supplemented at 500 mg / kg. The experiment lasted 15 days.

[0138] The experimental results showed that the sample with added bile acid combination 6 effectively solved the problem of watermarked eggs in the later stage of egg production. Figure 12In other production performance indicators, feed intake decreased by 3 grams per bird per day for Nongda No. 3, totaling 192 jin / day for 32,000 birds. With a feed cost of approximately 1.65 yuan per jin, this translates to a daily feed cost saving of 316.8 yuan. For Hy-Line White, feed intake decreased by 7 grams per bird per day for 32,000 birds, totaling 448 jin / day for 1.65 yuan per jin, resulting in a daily feed cost saving of 448 yuan. Egg production rate increased by 0.5 percentage points, producing approximately 160 more eggs per day (about 8 eggs per jin), or about 20 jin more eggs per day. For Hy-Line White, egg production rate increased by 1.7 percentage points, producing approximately 544 more eggs per day (about 8 eggs per jin), or about 68 jin more eggs per day. Premium egg rate increased by 5 percentage points, producing approximately 1600 eggs per day (about 200 jin of premium eggs). Hy-Line White eggs increased by 7 percentage points, producing approximately 2,240 eggs per day, or about 280 jin (140 catties) of premium eggs.

[0139] In summary, feed intake, egg production rate, and premium egg rate can generate a value of 884,176 yuan for the customer annually. The product requires investment every two months, with each investment costing approximately 19,723 yuan, totaling about 118,338 yuan annually. The return on investment is approximately 1:7.4.

[0140] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A bile acid composition for improving the utilization rate of fats in feed, characterized in that, The bile acid composition comprises, by mass percentage, 78%–83% porcine deoxycholic acid and 17%–22% chenodeoxycholic acid; The structural formula of the porcine deoxycholic acid is: The structural formula of the chenodeoxycholic acid is as follows: .

2. The bile acid composition according to claim 1, characterized in that, The bile acid composition is extracted from pig bile paste through high-temperature, high-pressure saponification, oxidative decolorization, acidification crystallization, water washing to remove impurities, and drying processes. The specific preparation method of the bile acid composition is as follows: (1) Ultra-high temperature and ultra-high pressure saponification: After crushing the pig bile paste, add 10%~15% sodium hydroxide, dilute with water to form a liquid, and treat in a reaction vessel at 105~135℃ and 1.05-1.10 MPa for 10-15 h. After the reaction is completed, let the liquid cool and separate into layers, remove the supernatant, and obtain the pig bile paste saponified product. (2) Oxidative decolorization: Mix the saponified pig bile paste with water to completely dissolve the saponified pig bile paste, then add H2O2 and decolorize for 24 h at 25-30℃; (3) Acidification crystallization: While stirring, use hydrochloric acid to adjust the pH of the solution to 3.0~3.5, and continue stirring until bile acid crystals precipitate out continuously; (4) Washing and drying: Centrifuge to remove supernatant, wash the precipitated bile acids with water 2-3 times, filter through plate and frame filter to obtain bile acids, and then use spray drying equipment with the inlet air temperature controlled at 130-180℃ and the outlet air temperature controlled at 80-100℃ to obtain bile acid composition.

3. The bile acid composition according to claim 1, characterized in that, The bile acid composition can promote oil emulsification and enhance the degradation of oils by lipases. The bile acid composition emulsifies oils by increasing the zeta potential of the oils and reducing the particle size of the emulsified oils.

4. The bile acid composition according to claim 1, characterized in that, The bile acid composition described above can activate the activity of lipase, increase the β-sheet content in lipase, and promote the degradation of lipids by lipase.

5. The bile acid composition according to claim 1, characterized in that, The optimized composition and content of the bile acid composition are as follows: by mass percentage, 78% porcine deoxycholic acid, 17% chenodeoxycholic acid, and the balance being water.

6. The use of the bile acid composition of claim 1 in the preparation of feed additives that improve the utilization rate of fats in animal feed.

7. The use of the bile acid composition of claim 1 in the preparation of feed additives that improve animal growth performance.

8. The application according to claim 6 or 7, characterized in that, The feed additive contains 0.01% to 0.1% bile acid composition by weight percentage.

9. The application according to claim 6 or 7, characterized in that, The animals mentioned include aquatic animals and livestock.

10. The application according to claim 9, characterized in that, The aquatic animals include perch and Litopenaeus vannamei; the livestock and poultry include broiler chickens and laying hens.

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