Feed additive for improving fat absorption of poultry and preparation method thereof

Abstract

The invention belongs to the technical field of feed additives, and relates to a preparation method of a feed additive for improving poultry fat absorption. According to the invention, mixed grease of algae oil and perilla oil or mixed grease of fish oil and linseed oil and MCT are subjected to ester exchange under the synergistic catalysis of polysulfobetaine modified lipase and auxiliary enzyme to obtain MLCT structure grease, the MLCT structure grease and a natural antioxidant form a composite system, and water-soluble dietary fibers are adopted for embedding, so that the composite antioxidant is obtained. The compound vitamins, the compound amino acid, the mildew preventive and the adhesive are used as auxiliary materials, so that the feed additive capable of improving the fat absorption of poultry is prepared.

Description

Technical Field

[0001] This invention relates to the field of feed additive technology, and in particular to a feed additive that improves the absorption of fat in poultry and its preparation method. Background Technology

[0002] As people's demands for the quality and quantity of poultry products continue to rise, the animal feed industry is also constantly innovating and developing. Traditional feed mainly provides basic nutrition, but it is insufficient in promoting livestock and poultry growth rate, improving feed utilization, and enhancing immunity. Under intensive farming, livestock and poultry are prone to slow growth and frequent diseases due to stress and nutritional imbalances. Feed additives can specifically address these problems. Feed additives can optimize the nutritional structure of traditional feed, promote the digestion and absorption of nutrients by livestock and poultry, thereby achieving rapid and healthy growth, improving breeding efficiency, meeting the large market demand for livestock and poultry products, and reducing costs.

[0003] Oils and fats are an important energy source for poultry, playing a crucial role in their growth, development, and production performance. However, poultry's own bile and digestive enzymes have limited capacity to digest and absorb large amounts of exogenous feed fats. Therefore, finding a fat source that is more easily digested and absorbed by poultry and developing technologies and products that can improve the digestibility and utilization of fats in poultry has become one of the research hotspots in the feed industry.

[0004] The emergence of MLCT has provided new ideas and directions for the feed industry, and its application in animal feed is receiving increasing attention. MLCT is a special type of triglyceride whose glycerol molecule is simultaneously bound to medium-chain fatty acids and long-chain fatty acids. This structure allows MLCT to combine the advantages of both medium-chain and long-chain fatty acids, providing both rapid energy supply and essential fatty acids for the human body. Chinese patent application CN 117617365A discloses a feed composition and feed additive and its application in laying hen farming. This composition includes medium- and short-chain fatty acids, medium-chain fatty acids, and long-chain fatty acids, as well as saturated and unsaturated fatty acids, ω-3 fatty acids, and ω-6 fatty acids. Compared to traditional additives such as soybean oil or palm oil, it is more beneficial for improving nutrient absorption efficiency, improving the health of laying hens, effectively enhancing laying hen production performance, and increasing feed utilization. The composition contains components that are sensitive to temperature, humidity, and pH. Improper storage conditions may lead to the deterioration and oxidation of fatty acids, which in turn affects the quality and effectiveness of the feed additive. Furthermore, the mechanism of action is complex, and the synergistic mechanism of action of multiple fatty acids has not yet been clarified, which is not conducive to further technical improvement and optimization. Summary of the Invention

[0005] This invention aims to provide a feed additive that improves poultry fat absorption and its preparation method. The feed additive uses algal oil, fish oil, and other substances rich in long-chain fatty acids, which are combined with MCT (methyl sulfonate betaine) through transesterification with a modified lipase and coenzyme to obtain MLCT (methyl sulfonate betaine lipase). This MLCT is then compounded with antioxidants such as tea polyphenols and curcumin to form a stable complex through intermolecular hydrogen bonds. Finally, it is encapsulated with water-soluble dietary fiber to prepare a modified MLCT that improves poultry fat absorption. With the addition of compound vitamins, compound amino acids, and other adjuvants, a feed additive that enhances poultry fat absorption is obtained.

[0006] To achieve the above objectives, the present invention provides a method for preparing a feed additive that improves the absorption of fat in poultry, comprising:

[0007] Step S1: Initially, 1 / 2 of MCT and oil are mixed, modified lipase and coenzyme are added, and after the first stage reaction, the remaining 1 / 2 of MCT is added, and after the second stage reaction, the reaction is carried out at room temperature to obtain crude MLCT product, which is then purified to obtain MLCT.

[0008] Step S2: Mix MLCT with antioxidants, add lecithin and deionized water, and homogenize to obtain MLCT emulsion;

[0009] Step S3: Disperse water-soluble dietary fiber in deionized water, add MLCT emulsion, adjust pH to 2.5-5, add cross-linking agent, stir to gel the emulsion, and freeze-dry to obtain modified MLCT;

[0010] Step S4: Mix the modified MLCT, compound vitamins, compound amino acids, antifungal agent and binder to obtain the feed additive.

[0011] Preferably, in step S1, the oil is any one or more of a mixture of algal oil and perilla oil or a mixture of fish oil and flaxseed oil, wherein the mass ratio of algal oil to perilla oil is (4-7):1, and the mass ratio of fish oil to flaxseed oil is (3-5):1.

[0012] Preferably, in step S1, the coenzyme is any one or more of lipoxygenase or phospholipase A.

[0013] Preferably, in step S1, the mass ratio of MCT, oil, modified lipase and coenzyme is (1-3):1:(0.05-0.1):(0.01-0.03).

[0014] Preferably, in step S1, the temperature of the first stage reaction is 30-40°C, and the reaction time is 0.5-1 hour.

[0015] Preferably, in step S1, the temperature of the second stage reaction is 25–35°C, and the reaction time is 1–3 hours.

[0016] Preferably, in step S1, the reaction time at room temperature is 3-5 hours.

[0017] Preferably, in step S2, the antioxidant is any one or more of tea polyphenols or curcumin.

[0018] Preferably, in step S2, the mass ratio of MLCT, antioxidant, lecithin and deionized water is 1:(0.055-0.15):(0.12-0.25):(2-3).

[0019] Preferably, in step S3, the water-soluble dietary fiber is any one or more of pectin, sodium alginate, and guar gum.

[0020] Preferably, in step S3, the crosslinking agent is a calcium chloride solution.

[0021] Preferably, in step S3, the concentration of the calcium chloride solution is 0.5% to 1%.

[0022] Preferably, in step S3, the stirring temperature is 30–45°C and the stirring time is 1–3 hours.

[0023] Preferably, in step S3, the freeze-drying operation is as follows: -40℃ to -20℃, holding for 2 to 4 hours; -30℃ to -10℃, holding for 2 to 4 hours; 20℃ to 30℃, holding for 6 to 8 hours.

[0024] Preferably, in step S3, the mass ratio of the water-soluble dietary fiber, deionized water, MLCT emulsion and crosslinking agent is 1:(5-10):(2-4):(0.12-0.2).

[0025] Preferably, in step S4, the complex vitamins include vitamin A, vitamin D, vitamin E, and vitamin K.

[0026] Preferably, in step S4, the mass ratio of vitamin A, vitamin D, vitamin E and vitamin K in the complex vitamin is 1:(1-1.5):(0.75-1.2):(0.5-0.75).

[0027] Preferably, in step S4, the composite amino acid includes cysteine, arginine and leucine; the mass ratio of cysteine, arginine and leucine in the composite amino acid is 1:(1-1.2):(1-1.1).

[0028] Preferably, in step S4, the antifungal agent is any one or more of potassium sorbate, sodium sorbate, and sodium benzoate.

[0029] Preferably, in step S4, the binder is sodium tripolyphosphate; the mass ratio of the modified MLCT, compound vitamins, compound amino acids, antifungal agent and binder is 1:(0.1-0.3):(0.05-0.12):(0.05-0.075):(0.005-0.02).

[0030] Preferably, in step S1, the method for preparing the modified lipase includes:

[0031] Step A1: Disperse polysulfonate betaine in MES buffer solution, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS), and stir to obtain activated polysulfonate betaine;

[0032] Step A2: Mix activated polysulfonate betaine and lipase, disperse in phosphate buffer at pH 7.0, and stir to obtain modified lipase.

[0033] Preferably, in step A1, the mass ratio of polysulfonate betaine, MES buffer solution, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:(5-10):(1-1.2):(1-1.2).

[0034] Preferably, in step A1, the stirring time is 1 to 2 hours.

[0035] Preferably, in step A2, the lipase is Lipozyme 435.

[0036] Preferably, in step A2, the mass ratio of activated polysulfonate betaine, phosphate buffer, and lipase is 1:(5-10):(0.05-0.2).

[0037] Preferably, in step A2, the stirring temperature is 25-30°C and the stirring time is 1-2 hours.

[0038] On the other hand, the present invention provides a feed additive that improves the absorption of fat in poultry.

[0039] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0040] (1) This application uses a mixture of algal oil and perilla oil or fish oil and flaxseed oil with MCT, and prepares MLCT structured lipids that can promote the absorption of poultry fat through transesterification under the catalysis of polysulfonate betaine-modified lipase and coenzyme. Among them, the MLCT structured lipids prepared by using a mixture of algal oil and perilla oil with MCT have the best effect on promoting the absorption of poultry fat. The long-chain fatty acids provided by algal oil, such as DHA and EPA, and α-linolenic acid in perilla oil are all essential fatty acids required for the growth and development of poultry, and have a variety of physiological functions, but they need to form micelles with bile acids before they can be absorbed; while the medium-chain fatty acids provided by MCT have small molecules and high water solubility, and can be directly absorbed by intestinal epithelial cells without the need for bile acid emulsification, and quickly enter the liver via the portal vein for oxidation and energy supply, providing immediate energy for poultry; the glycerol backbone of MLCT is simultaneously linked to medium-chain fatty acids and long-chain fatty acids, and can release both types of fatty acids simultaneously after hydrolysis, which not only ensures rapid energy supply, but also meets the requirements of essential fatty acids, avoiding the limitations of single fatty acid absorption. In transesterification, the lipase modified with polysulfonate betaine possesses zwitterionic groups on its surface, enhancing its affinity for the oil-water interface and improving its catalytic efficiency for mixed oils. Its catalysis causes medium-chain fatty acids to tend to attach to the sn-1 and sn-3 positions of glycerol, while long-chain fatty acids tend to attach to the sn-2 position. This structure highly matches the hydrolytic preference of avian pancreatic lipase, making it easier for pancreatic lipase to recognize ester bonds at the sn-1 and sn-3 positions. The sn-2 monoglyceride released after hydrolysis can be directly absorbed by intestinal epithelial cells, reducing the retention of long-chain fatty acids in the intestine and minimizing oxidative loss. A dual-enzyme system is constructed by adding a coenzyme and the modified lipase. The modified lipase is responsible for transesterification, while the coenzyme scavenges reactive oxygen species generated during the reaction, reducing the oxidative degradation of DHA / α-linolenic acid without affecting the catalytic activity of the lipase. Simultaneously, by selectively hydrolyzing phospholipid impurities in the oil, it avoids competition with the lipase for active sites, thus improving the yield of MLCT (Multi-Layer Transesterification).

[0041] (2) This application adds natural antioxidants, such as curcumin or tea polyphenols. The phenolic hydroxyl groups in their structure interact with the ester oxygen atoms on the MLCT backbone or the hydroxyl groups in the fatty acid chains through intermolecular hydrogen bonds to form a stable complex system. The phenolic hydroxyl groups of natural antioxidants can neutralize the reactive oxygen free radicals generated during the oxidation of oils and block the oxidation chain reaction. The hydrogen bond makes it easier for antioxidants to be distributed in the MLCT oil phase, improving their dispersibility and antioxidant efficiency in oils and extending the shelf life and activity of MLCT. The complex system of antioxidants and MLCT can promote the intestinal absorption of antioxidants through the digestive and absorption pathway of MLCT, improving their bioavailability in animals. Due to the presence of antioxidants, the oxidative damage of MLCT oxidation products to the intestines, liver and other tissues of laying hens can be reduced, protecting the integrity of the intestinal mucosa and liver function, and indirectly promoting the absorption of nutrients. Curcumin and tea polyphenols also have anti-inflammatory and immunomodulatory effects, which, in synergy with the nutritional function of MLCT, can improve the disease resistance of laying hens, reduce the impact of diseases on production performance, reduce the deposition of oxidation products in laying hens, and help improve egg quality and muscle quality.

[0042] (3) This application uses water-soluble dietary fibers such as pectin, sodium alginate, and guar gum to encapsulate MLCT, thereby improving the stability, processability, and absorption efficiency of MLCT. Dietary fiber has a high molecular weight, multiple hydroxyl groups, and multi-branched structure, with a large number of hydrogen bonds and pores between molecules. The three-dimensional network structure or porosity of dietary fiber can physically adsorb small MLCT droplets, and encapsulate the oil particles inside the fiber matrix through van der Waals forces and hydrophobic interactions. The outer shell is formed by dietary fiber, which has a good physical barrier effect, can isolate MLCT from direct contact with the external environment, and prolong the shelf life. The outer shell of dietary fiber is relatively stable in the acidic environment of the stomach, which can protect MLCT from premature decomposition. After entering the intestine, under the action of pancreatic juice and cellulase secreted by intestinal flora, the outer shell of dietary fiber gradually degrades, and MLCT is slowly released, prolonging its interaction time with intestinal lipase and improving digestion and absorption efficiency. At the same time, dietary fiber itself can be fermented and utilized by intestinal flora to produce short-chain fatty acids, regulate intestinal pH and flora balance, provide a more suitable intestinal environment for the digestion and absorption of MLCT, and indirectly enhance its nutritional efficacy. Attached Figure Description

[0043] Figure 1 This is a flowchart illustrating the preparation process of a feed additive that enhances fat absorption in poultry.

[0044] Figure 2 This is a schematic diagram showing the free fatty acid content measured in the in vitro simulated digestion experiment of Experiment Example 1.

[0045] Figure 3 This is a schematic diagram of the apparent fat digestibility measured in Experiment Example 2.

[0046] Figure 4 This is a schematic diagram of the fat content in different parts of a chick during the tissue fat deposition determination experiment in Example 5. Detailed Implementation

[0047] The following embodiments are only used to illustrate the technical solutions of the present invention more clearly, and should not be used to limit the scope of protection of the present invention.

[0048] The main compounds used in the examples and comparative examples were all commercially available products and were not subjected to any further purification treatment.

[0049] Example 1

[0050] A modified lipase, the preparation method of which includes:

[0051] Step A1: Disperse 10g of polysulfonate betaine in 50g of MES buffer solution, add 10g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10g of N-hydroxysuccinimide, stir for 1h to obtain activated polysulfonate betaine.

[0052] Step A2: Mix 10g of activated polysulfonated betaine and 0.5g of Lipozyme 435, disperse in 50g of pH 7.0 phosphate buffer, stir at 25°C for 2h to obtain modified lipase.

[0053] Example 2

[0054] A modified lipase, the preparation method of which includes:

[0055] Step A1: Disperse 10g of polysulfonate betaine in 75g of MES buffer solution, add 11g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 11g of N-hydroxysuccinimide, stir for 2h to obtain activated polysulfonate betaine.

[0056] Step A2: Mix 10g of activated polysulfonated betaine and 1g of Lipozyme 435, disperse in 75g of pH 7.0 phosphate buffer, stir at 30℃ for 1h to obtain modified lipase.

[0057] Example 3

[0058] A modified lipase, the preparation method of which includes:

[0059] Step A1: Disperse 10g of polysulfonate betaine in 100g of MES buffer solution, add 12g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 12g of N-hydroxysuccinimide, stir for 1h to obtain activated polysulfonate betaine.

[0060] Step A2: Mix 10g of activated polysulfonated betaine and 2g of Lipozyme 435, disperse in 100g of pH 7.0 phosphate buffer, stir at 25°C for 1h to obtain modified lipase.

[0061] Example 4

[0062] A feed additive for improving fat absorption in poultry, the preparation method of which includes:

[0063] Step S1: Initially, 50g of MCT, 80g of algal oil and 20g of perilla oil were mixed, and 5g of the modified lipase prepared in Example 1 and 1g of lipoxygenase were added. The mixture was reacted at 40°C for 1 hour, and the remaining 50g of MCT was added. The mixture was reacted at 35°C for 3 hours, and finally reacted at room temperature for 5 hours to obtain crude MLCT product. After purification, MLCT was obtained.

[0064] In step S2, 100g of MLCT was mixed with 5.5g of tea polyphenols, 12g of lecithin and 200g of deionized water were added, and homogenized to obtain an MLCT emulsion.

[0065] Step S3: Disperse 50g of guar gum in 250g of deionized water, add 100g of MLCT emulsion, adjust the pH to 2.5-5, add 6g of calcium chloride solution (0.5g of calcium chloride dissolved in 99.5g of deionized water to prepare 100g of 0.5% calcium chloride solution), stir at 30℃ for 3h to gel, keep warm at -20℃ for 4h, keep warm at -10℃ for 4h, keep warm at 20℃ for 8h to obtain modified MLCT.

[0066] Step S4: Mix 100g of modified MLCT, 10g of compound vitamins (15g of vitamin A, 15g of vitamin D, 12g of vitamin E and 8g of vitamin K to prepare 50g of compound vitamins), 5g of compound amino acids (16g of cysteine, 17g of arginine and 17g of leucine to prepare 50g of compound amino acids), 5g of potassium sorbate and 0.5g of sodium tripolyphosphate to obtain a feed additive.

[0067] Example 5

[0068] A feed additive for improving fat absorption in poultry, the preparation method of which includes:

[0069] Step S1: Initially, 100g of MCT, 80g of fish oil and 20g of flaxseed oil were mixed, and 7.5g of the modified lipase prepared in Example 2 and 2g of phospholipase A were added. The mixture was reacted at 30°C for 0.5h, and the remaining 100g of MCT was added. The mixture was reacted at 25°C for 2h, and finally reacted at room temperature for 4h to obtain crude MLCT product. After purification, MLCT was obtained.

[0070] In step S2, 100g of MLCT is mixed with 10g of curcumin, 20g of lecithin and 250g of deionized water are added, and homogenized to obtain an MLCT emulsion.

[0071] Step S3: Disperse 50g of sodium alginate in 350g of deionized water, add 150g of MLCT emulsion, adjust the pH to 2.5-5, add 9g of calcium chloride solution (1g of calcium chloride is dissolved in 99g of deionized water to prepare 100g of 1% calcium chloride solution), stir at 40℃ for 2h to gel, keep warm at -30℃ for 3h, keep warm at -20℃ for 3h, and keep warm at 25℃ for 7h to obtain modified MLCT.

[0072] Step S4: Mix 100g of modified MLCT, 20g of compound vitamins (12g of vitamin A, 18g of vitamin D, 12g of vitamin E and 8g of vitamin K to prepare 50g of compound vitamins), 10g of compound amino acids (16.1g of cysteine, 17.7g of arginine and 16.2g of leucine to prepare 50g of compound amino acids), 6g of sodium benzoate and 1g of sodium tripolyphosphate to obtain a feed additive.

[0073] Example 6

[0074] A feed additive for improving fat absorption in poultry, the preparation method of which includes:

[0075] Step S1: Initially, 150g of MCT, 87g of algal oil and 13g of perilla oil were mixed, and 10g of the modified lipase prepared in Example 3 and 3g of lipoxygenase were added. The mixture was reacted at 35°C for 0.5h, and the remaining 150g of MCT was added. The mixture was reacted at 30°C for 1h, and finally reacted at room temperature for 3h to obtain crude MLCT product. After purification, MLCT was obtained.

[0076] In step S2, 100g of MLCT is mixed with 15g of tea polyphenols, 25g of lecithin and 300g of deionized water are added, and homogenized to obtain an MLCT emulsion.

[0077] Step S3: Disperse 50g of pectin in 500g of deionized water, add 200g of MLCT emulsion, adjust the pH to 2.5-5, add 10g of calcium chloride solution (0.5g of calcium chloride dissolved in 99.5g of deionized water to prepare 100g of 0.5% calcium chloride solution), stir at 45℃ for 1h to gel, keep warm at -40℃ for 2h, keep warm at -30℃ for 2h, keep warm at 30℃ for 6h to obtain modified MLCT.

[0078] Step S4: Mix 100g of modified MLCT, 30g of compound vitamins (11.2g of vitamin A, 16.8g of vitamin D, 13.4g of vitamin E and 8.6g of vitamin K to prepare 50g of compound vitamins), 12g of compound amino acids (15.2g of cysteine, 18.24g of arginine and 16.56g of leucine to prepare 50g of compound amino acids), 7.5g of sodium sorbate and 2g of sodium tripolyphosphate to obtain a feed additive.

[0079] Comparative Example 1

[0080] A feed additive that improves the absorption of fat in poultry is prepared differently from that in Example 6 in step S1, only algal oil is used, that is, 100g of algal oil is added.

[0081] Comparative Example 2

[0082] A feed additive that improves the absorption of fat in poultry is prepared differently from that in Example 6 in step S1, only perilla oil is used, that is, 100g of perilla oil is added.

[0083] Comparative Example 3

[0084] A feed additive that improves the absorption of fat in poultry is prepared in a manner that differs from that in Example 6 in that lipoxygenase is not added in step S1.

[0085] Comparative Example 4

[0086] A feed additive to improve the absorption of fat in poultry is prepared differently from that in Example 6 in that step S1 does not add the modified lipase prepared in Example 3, but uses unmodified Lipozyme 435.

[0087] Comparative Example 5

[0088] A feed additive that improves the absorption of fat in poultry is prepared differently from that in Example 6 in that tea polyphenols are not added in step S2.

[0089] Experiment Example 1: In Vitro Simulated Digestion Experiment

[0090] (1) The feed additives prepared in Examples 4 to 6 and Comparative Examples 1 to 5 were mixed with the basic feed (the mass ratio of feed to feed additive was 1:0.4). The feed containing the additive was dispersed in deionized water to prepare a 10% suspension. The mixture was stirred for 30 minutes to ensure uniform dispersion. The blank control group consisted of the basic feed without the additive, which was dispersed in deionized water to prepare a 10% suspension. The mixture was stirred for 30 minutes to ensure uniform dispersion.

[0091] (2) Preparation of digestive solution:

[0092] Crop digestion solution: Prepare a dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution with a pH of 6.0–7.5. Add 0.58 g of NaCl, 0.11 g of CaCl2, and 0.2 g of bovine serum albumin to the buffer solution and stir until clear. Weigh out α-amylase, dissolve it in deionized water, and add it to the above solution. Add dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution to a volume of 1 L, so that the final concentration of amylase is 0.5 mg / mL. Filter through a 0.22 μm sterile filter membrane and store at 4°C. Before use, restore the temperature to 37°C.

[0093] Gizzard digestive fluid: Simulates the acidic environment of the gizzard (pH 2.0-3.0). Weigh pepsin, dissolve it in 0.1 mol / L hydrochloric acid solution to make the pepsin concentration 1.0 mg / mL, add 0.5% sodium chloride solution, store at 4°C, and preheat to 37°C before use.

[0094] Small intestinal digestive fluid: Simulates the neutral to slightly alkaline environment of the small intestine (pH 6.5–7.5). Weigh pancreatic lipase and bile salts, dissolve them in 0.1 mol / L phosphate buffer to make the pancreatic lipase concentration 2.0 mg / mL and the bile salt concentration 10 mg / mL. Store at 4°C and preheat to 37°C before use.

[0095] (3) Take 10 mL of the suspension prepared in (1) and add it to a 50 mL centrifuge tube. Add 10 mL of the crop digestion solution prepared in (2). Place the centrifuge tube in a 37°C constant temperature water bath shaker and shake for 2 hours. After the crop digestion is completed, add 15 mL of gizzard digestion solution to the centrifuge tube and continue to shake for 3 hours in a 37°C constant temperature water bath shaker. After the gizzard digestion is completed, adjust the pH of the system in each centrifuge tube to 7.0 with 0.1 mol / L sodium hydroxide solution. Add 20 mL of small intestine digestion solution and continue to shake for 6 hours in a 37°C constant temperature water bath shaker. During this period, take 1 mL of sample every 1 hour for subsequent index detection. After sampling, add an equal amount of the corresponding digestion solution preheated at 37°C to maintain the stability of the system volume.

[0096] (4) Determination of core indicators: In in vitro fat digestion experiments, glycerol content is usually used together with free fatty acid content as core indicators.

[0097] Fat hydrolysis efficiency: Free fatty acid (FFA) content: Samples were taken at 1, 2, 3, 4, 5, and 6 hours of small intestinal digestion. The FFA content in the samples was determined by titration or colorimetric method. The FFA production and hydrolysis rate were calculated (hydrolysis rate = FFA production / initial total fat × 100%). The results are as follows: Figure 2 As shown.

[0098] Glycerol content: The amount of glycerol produced in the sample is determined by enzyme-linked immunosorbent assay (ELISA). The more glycerol produced, the more complete the fat hydrolysis.

[0099] Free fatty acids are one of the products of enzymatic breakdown of fats during digestion. A high content of free fatty acids indicates more complete fat hydrolysis, making the lipid matrix easier to digest and absorb, and thus increasing its nutrient absorption potential. According to... Figure 2 As shown, the free fatty acid content in experimental groups 1 to 3 was significantly higher than that in other experimental groups, indicating that the fat in the experimental groups 1 to 3 was more fully hydrolyzed, and the lipids were more easily absorbed in the body.

[0100] In Experiment 5, the additive prepared in Comparative Example 2 was used, and its free fatty acid content was second only to Experiment 2. In Experiment 4, the additive prepared in Comparative Example 1 was used, and its free fatty acid content was significantly lower than that of Experiment 5, but higher than the blank control. In Comparative Example 1, only algal oil was used as the oil, and in Comparative Example 2, only perilla oil was used. Compared with Examples 4-6, which used algal oil and perilla oil or fish oil and flaxseed oil, the MLCT's effect on promoting fat absorption was significantly reduced. However, using perilla oil alone was more effective than using algal oil alone. Perilla oil is rich in α-linolenic acid, and compared with the fatty acids in algal oil, the α-linolenic acid in perilla oil may be more likely to form an MLCT structure with good digestibility and absorption properties with MCT. When the content of long-chain fatty acids at the sn-2 position in triglycerides is high in MLCT, it is beneficial to the digestion and absorption of fat. In MLCT prepared from perilla oil, more than 85% of the long-chain fatty acids are at the sn-2 position in triglycerides. Therefore, the fat hydrolysis effect in experimental group 5 is better than that in experimental group 4.

[0101] In experimental groups 6–8, the content of free fatty acids was significantly lower than that in experimental groups 1–3, but slightly higher than that in experimental group 4. Specifically, the additive prepared in Comparative Example 3 for experimental group 6 used only one type of lipase in its preparation process, without adding lipoxygenase as a coenzyme in the MLCT preparation reaction. The additive prepared in Comparative Example 4 for experimental group 7 used a lipase that was not modified with polysulfonate betaine. The additive prepared in Comparative Example 5 for experimental group 8 did not add tea polyphenols to form a stable complex with MLCT. The experiments demonstrate that the synergistic effect of polysulfonate betaine-modified Lipozyme 435 and lipoxygenase results in higher activity and stability, enabling more efficient catalysis of the binding of long-chain fatty acids in perilla oil and algal oil with MCT to form MLCT with a structure more conducive to digestion and absorption. Adding tea polyphenols can enhance the stability of the complex. The polar groups of tea polyphenols can promote the activity of digestive enzymes and the efficiency of recognizing esters at the sn-1 and sn-3 positions of MLCT, making the long-chain fatty acid at the sn-2 position of MLCT more stably exposed, reducing the masking of enzymatic hydrolysis sites caused by structural disorder, and improving hydrolysis efficiency.

[0102] Table 1. Glycerol content of feed additives in simulated digestive tract

[0103]

[0104]

[0105] The core process of fat digestion is the stepwise hydrolysis of triglycerides. First, pancreatic lipase acts on the sn-1 and sn-3 ester bonds of triglycerides, hydrolyzing them to produce diglycerides and free fatty acids. The second step involves further hydrolysis of diglycerides to produce monoglycerides and free fatty acids. The third step is the final hydrolysis of monoglycerides to produce glycerol and free fatty acids. Throughout this process, glycerol is one of the end products of complete triglyceride hydrolysis; it is only released in the final step of the hydrolysis reaction. Therefore, the production of glycerol is a marker of the depth of fat digestion. Higher glycerol content indicates a higher degree of fat hydrolysis and higher digestive enzyme activity. According to the data in Table 1, the glycerol content in experimental groups 1-3 was above 150 μg / mL, while the glycerol content in experimental groups 4-8 was below 150 μg / mL. The additives prepared in Examples 1-3 were added to experimental groups 1-3, respectively. Experiments show that the additives prepared in Examples 1-3 can effectively promote the hydrolysis, digestion, and absorption of fats.

[0106] Experimental Example 2 Apparent Fat Digestibility

[0107] (1) 180 healthy AA broiler chickens of similar weight and aged 20 days were randomly divided into 9 groups of 20 each and housed in metabolic cages. Experimental groups 1 to 3 were fed a basic diet (5% fat content) with additives prepared in Examples 4 to 6; experimental groups 4 to 8 were fed a basic diet with additives prepared in Comparative Examples 1 to 5; and the control group was fed a basic diet without any additives.

[0108] (2) After feeding for 7 days to allow the poultry to adapt to the feed and rearing environment, the formal experiment began. After the formal experiment began, 120g of feed was accurately fed daily. If there was any leftover feed, it was weighed and recorded before the next feeding, and the actual intake was calculated. For 7 days of feeding, starting from the morning of the first day, all feces excreted by each poultry were collected using the feces collection device of the metabolic cage. The feces were placed in an oven and dried to constant weight at 65-70℃. The total dried weight of the feces was recorded. The feces were then pulverized into powder using a grinder, passed through a 40-mesh sieve, and the fecal fat content was determined by near-infrared spectroscopy. The following calculations were made: Fat intake = (Feed intake × Feed fat content %); Total fecal fat excretion = (Total dried fecal weight × Fecal fat content); Apparent fat digestibility (%) = (Total fat intake - Fecal fat excretion) / Total fat intake × 100%. Apparent fat digestibility data are as follows: Figure 3 As shown.

[0109] A high apparent fat digestibility generally means that the body's digestion, absorption, and utilization of ingested fat are more efficient. A higher apparent fat digestibility indicates more complete fat hydrolysis and absorption, and a higher utilization rate of fat by the body. According to... Figure 3 As shown, after 7 days, the apparent fat digestibility of chicks fed in experimental groups 1 to 3 was all above 85%. The apparent fat digestibility of chicks fed in experimental groups 4 to 8 was lower than that of experimental groups 1 to 3 after 7 days. Specifically, the apparent fat digestibility of chicks fed in experimental groups 4 and 8 was below 75% after 7 days, significantly lower than the other groups. This experiment demonstrates that the chicks fed in experimental groups 1 to 3 have a higher utilization rate of fat, thus indicating that the additives prepared in Examples 4 to 6 can effectively promote the digestion and absorption of fat in poultry.

[0110] Experimental Example 3: Serum Lipid Index Measurement

[0111] Seven days after feeding, blood was collected from the hindwing veins of experimental groups 1 to 8 after fasting for 12 hours to measure serum triglyceride (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) levels.

[0112] Table 2 Serum biochemical indicators of chicken fat absorption

[0113] Sample number TG (mmol / L) TC (mmol / L) HDL-C (mmol / L) Experimental group 1 0.6513±0.08 2.76±0.23 0.98±0.12 Experimental group 2 0.6675±0.07 2.58±0.18 1.02±0.11 Experimental group 3 0.7125±0.11 2.97±0.21 1.08±0.18 Experimental group 4 0.4216±0.09 1.35±0.20 0.43±0.16 Experimental group 5 0.6078±0.14 2.35±0.11 0.78±0.15 Experimental group 6 0.5548±0.10 2.06±0.16 0.64±0.20 Experimental group 7 0.4156±0.08 1.85±0.21 0.55±0.16 Experimental group 8 0.4006±0.06 1.64±0.18 0.51±0.15 Blank control 0.3328±0.11 1.30±0.22 0.40±0.11

[0114] Lipid levels can indirectly reflect fat absorption efficiency and assess the overall impact of additives on poultry lipid metabolism. Fatty acids and glycerol produced after fat digestion are absorbed into the bloodstream through the intestines. By measuring serum triglycerides (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C), it was found that if the additive promoted fat absorption, serum TG and other indicators would show a corresponding increase (within the normal physiological range). According to the data in Table 2, the TG, TC, and HDL-C levels of chicks in experimental groups 1-3 were significantly higher than those in experimental groups 4-8. The experiment demonstrates that the additives prepared in Examples 4-6 can effectively improve fat absorption in chicks.

[0115] Experiment Example 4: Determination of Intestinal Fat Absorption-Related Enzyme Activity

[0116] After 7 days of feeding in experimental groups 1–8, and following serum collection, the experimental chicks were euthanized, and the duodenum and jejunum were separated. The intestines were cut open, and intestinal mucosal tissue was scraped and placed in pre-cooled culture dishes. PBS (at a weight-to-volume ratio of 1:9, i.e., 1g tissue to 9mL PBS) was added, and homogenization was performed under ice bath conditions to prepare a 10% tissue homogenate. The homogenate was centrifuged at 4000r / min for 15min at 4℃, and the supernatant was collected to prepare the intestinal mucosal enzyme extract. The activities of lipase and cholesterol esterase were measured according to the instructions of the lipase and cholesterol esterase assay kits.

[0117] Table 3. Intestinal lipase activity test data

[0118]

[0119] The digestion and absorption of fat depends on the action of digestive enzymes. Lipases can break down triglycerides into fatty acids and monoglycerides, while cholesterol esterases can promote the hydrolysis of cholesterol esters. Additives that can increase the activity of these enzymes can enhance the efficiency of fat digestion, thereby promoting absorption. According to the data in Table 3, the levels of duodenal lipase, duodenal cholesterol esterase, jejunal lipase, and jejunal cholesterol esterase in chickens in experimental groups 1-3 were all higher than those in experimental groups 4-8. The experiments demonstrate that the additives prepared in Examples 4-6 can effectively promote the absorption of fat in chickens.

[0120] Experimental Example 5: Determination of Tissue Fat Deposition

[0121] After feeding experimental groups 1 to 8 for 7 days, the experimental chicks were euthanized after serum collection. Liver, pectoral muscle and leg muscle tissue samples were collected. The surface bloodstains were washed off with physiological saline, and the tissue samples were homogenized into a fine paste using a tissue homogenizer. 3g of the paste was placed in a dry Soxhlet extraction flask.

[0122] Add 80 mL of diethyl ether to the flask, connect the flask to a Soxhlet extractor, and heat in a water bath to reflux the ether for 8–12 hours until the ether in the extraction tube is colorless, indicating complete fat extraction. Remove the flask, recover the ether using a rotary evaporator, and then dry the flask in a 105°C oven for 2 hours. After drying, place it in a desiccator to cool for 30 minutes and weigh the flask. The formula for calculating fat content is: Tissue fat content (%) = (Weight of flask after extraction - Weight of flask before extraction) / Weight of tissue sample × 100%. Experimental results are as follows: Figure 4 As shown.

[0123] like Figure 4 As shown, the fat content in the leg muscles, pectoral muscles, and livers of chickens in experimental groups 1-3 was significantly higher than that in other experimental groups and the control group. The leg muscles had the highest fat content, followed by the liver, while the pectoral muscles had the lowest. Fat was mainly deposited in the leg muscles, which is related to the metabolic needs of the leg muscles. The liver had the second highest fat deposition; the liver is the core organ for fat metabolism, responsible for fat synthesis, decomposition, and transport. The additives promoted fat absorption, and some fat temporarily accumulated in the liver. The pectoral muscles had the lowest fat deposition; as the main moving muscles of chickens, the pectoral muscles had lower fat accumulation than the leg muscles and liver. The differences in fat content in different tissues indicate that the additives increase fat deposition in the body by improving fat absorption rate, and their effect is tissue-targeted, rather than simply promoting fat synthesis. Therefore, the experiment proves that the additives prepared in Examples 4-6 can effectively promote the rational absorption and utilization of fat in chickens.

[0124] Experiment Example 6: Determination of Key Growth Indicators

[0125] The experimental groups 1 to 8 were fed for 7 days. The feed intake and daily weight gain of each group were recorded and the average value was calculated.

[0126] Table 4. Measurement of growth indicators

[0127]

[0128]

[0129] As shown in Table 4, the average daily feed intake of chicks in experimental groups 1-8 and the blank control group was around 100g within 7 days, with no significant difference. However, there were significant differences in daily weight gain. The daily weight gain of chicks in experimental groups 1-3 was over 40g, and the daily weight gain of chicks in experimental group 5 was also over 40g, although the daily weight gain of chicks in experimental group 5 was slightly lower than that of chicks in experimental groups 1-3. The daily weight gain of chicks in experimental groups 6-8 was between 35-40g, while the daily weight gain of chicks in experimental group 4 was less than 35g. Under the same growth environment and with similar feed intake, the daily weight gain of the chicks in the experimental groups differed significantly. This experiment demonstrates that chicks in experimental groups 1-3 had better fat absorption capacity, followed by chicks in experimental group 5, while chicks in experimental groups 6-8 had poor fat absorption capacity. Based on the experimental data of apparent fat digestibility, it can be seen that when the fat intake of chickens in experimental groups 1 to 8 and the blank control group is similar, the higher the apparent fat digestibility, the better the body's absorption of fat and the higher the daily weight gain. Therefore, the additives prepared in Examples 4 to 6 have a better effect on promoting fat absorption.

[0130] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a feed additive that improves fat absorption in poultry, characterized in that, include: Step S1: Mix 1 / 2 of MCT with oil, add modified lipase and coenzyme, and after the first stage reaction, add the remaining 1 / 2 of MCT, and after the second stage reaction, react at room temperature to obtain crude MLCT product, which is then purified to obtain MLCT. Step S2: Mix MLCT with antioxidants, add lecithin and deionized water, and homogenize to obtain MLCT emulsion; Step S3: Disperse water-soluble dietary fiber in deionized water, add MLCT emulsion, adjust pH to 2.5-5, add cross-linking agent, stir to gel the emulsion, and freeze-dry to obtain modified MLCT; Step S4: Mix the modified MLCT, compound vitamins, compound amino acids, antifungal agent and binder to obtain the feed additive.

2. The method for preparing a feed additive to improve poultry fat absorption according to claim 1, characterized in that, In step S1, the oil is any one or more of a mixture of algal oil and perilla oil or a mixture of fish oil and flaxseed oil, with the mass ratio of algal oil to perilla oil being (4-7):1 and the mass ratio of fish oil to flaxseed oil being (3-5):1; the coenzyme is any one or more of lipoxygenase or phospholipase A; and the mass ratio of MCT, oil, modified lipase and coenzyme is (1-3):1:(0.05-0.1):(0.01-0.03).

3. The method for preparing a feed additive to improve poultry fat absorption according to claim 1, characterized in that, In step S1, the temperature of the first stage reaction is 30-40°C and the reaction time is 0.5-1h; the temperature of the second stage reaction is 25-35°C and the reaction time is 1-3h; the reaction time at room temperature is 3-5h.

4. The method for preparing a feed additive to improve poultry fat absorption according to claim 1, characterized in that, In step S2, the antioxidant is any one or more of tea polyphenols or curcumin; the mass ratio of MLCT, antioxidant, lecithin and deionized water is 1:(0.055-0.15):(0.12-0.25):(2-3).

5. The method for preparing a feed additive to improve poultry fat absorption according to claim 1, characterized in that, In step S3, the water-soluble dietary fiber is any one or more of pectin, sodium alginate, and guar gum; the crosslinking agent is calcium chloride solution, and the concentration of the calcium chloride solution is 0.5% to 1%; the stirring temperature is 30 to 45°C, and the stirring time is 1 to 3 hours; the freeze-drying operation is as follows: -40°C to -20°C, holding for 2 to 4 hours; -30°C to -10°C, holding for 2 to 4 hours; 20°C to 30°C, holding for 6 to 8 hours; the mass ratio of the water-soluble dietary fiber, deionized water, MLCT emulsion, and crosslinking agent is 1:(5 to 10):(2 to 4):(0.12 to 0.2).

6. A method for preparing a feed additive to improve poultry fat absorption according to claim 1, characterized in that, In step S4, the complex vitamins include vitamin A, vitamin D, vitamin E, and vitamin K; the mass ratio of vitamin A, vitamin D, vitamin E, and vitamin K in the complex vitamins is 1:(1-1.5):(0.75-1.2):(0.5-0.75).

7. The method for preparing a feed additive to improve poultry fat absorption according to claim 1, characterized in that, In step S4, the composite amino acid includes cysteine, arginine, and leucine; the mass ratio of cysteine, arginine, and leucine in the composite amino acid is 1:(1-1.2):(1-1.1); the antifungal agent is any one or more of potassium sorbate, sodium sorbate, and sodium benzoate; the binder is sodium tripolyphosphate; the mass ratio of the modified MLCT, composite vitamin, composite amino acid, antifungal agent, and binder is 1:(0.1-0.3):(0.05-0.12):(0.05-0.075):(0.005-0.02).

8. A method for preparing a feed additive to improve poultry fat absorption according to claim 1, characterized in that, In step S1, the method for preparing the modified lipase includes: Step A1: Disperse polysulfonate betaine in MES buffer solution, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and stir to obtain activated polysulfonate betaine; Step A2: Mix activated polysulfonate betaine and lipase, disperse in phosphate buffer at pH 7.0, and stir to obtain modified lipase.

9. A method for preparing a feed additive to improve poultry fat absorption according to claim 8, characterized in that, In step A1, the mass ratio of polysulfonate betaine, MES buffer solution, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:(5-10):(1-1.2):(1-1.2); the stirring time is 1-2 hours. In step A2, the lipase is Lipozyme 435; the mass ratio of activated polysulfonate betaine, phosphate buffer, and lipase is 1:(5-10):(0.05-0.2); the stirring temperature is 25-30°C, and the stirring time is 1-2 hours.

10. A feed additive for improving the absorption of poultry fat, prepared by the preparation method according to any one of claims 1-9.

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