Bioemulsifier compositions, methods of making and use thereof

Abstract

The application provides a biological emulsifier composition, a preparation method and application thereof, wherein the biological emulsifier composition comprises a surface active peptide; the surface active peptide comprises C13-C15 surface active peptide homologues; the total mass content of the C13-C15 surface active peptide homologues is greater than or equal to 80% based on the total mass of the surface active peptide, and the mass content of the C14 surface active peptide homologue is 50%-80%. The biological emulsifier composition, the preparation method and the application thereof can form an emulsified system with smaller particle size, more uniform distribution and better stability in a fat system, increase the dispersion degree and the interface area of fat droplets, and make lipase more fully contact the fat interface, so that the enzymolysis efficiency of lipase on fat is improved, and the release of fatty acids is promoted.

Description

Technical Field

[0001] This application relates to the field of animal feed additives, and in particular to bio-emulsifier compositions, preparation methods and applications. Background Technology

[0002] To improve the digestion and absorption of fats and oils in animals, exogenous emulsifiers are usually added to feed. However, existing emulsifiers have significant limitations. On the one hand, existing emulsifiers lack stability in the complex environment of the digestive tract and are easily affected by factors such as pH value and ionic strength, leading to a decrease in emulsification efficiency. On the other hand, they suffer from limited sources, high production costs, relatively limited functions, and poor adaptability to different animal species, dietary structures, and feed processing conditions. Currently, there is a lack of an emulsifier system that can simultaneously satisfy high emulsification efficiency, good digestive tract stability, and feed processing adaptability. Summary of the Invention

[0003] In view of this, the purpose of this application is to provide a bio-emulsifier composition, preparation method and application thereof, in order to solve the above-mentioned technical problems.

[0004] A first aspect of this application provides a bio-emulsifier composition comprising a surfactant peptide;

[0005] The surfactant peptides include C13-C15 surfactant peptide homologues; based on the total mass of the surfactant peptides, the total mass content of the C13-C15 surfactant peptide homologues is greater than or equal to 80%, and the mass content of the C14 surfactant peptide homologues is 50%-80%; preferably, the mass content of the C13-C15 surfactant peptide homologues is greater than or equal to 90%, and the mass content of the C14 surfactant peptide homologues is 60%-75%.

[0006] Furthermore, it also includes: carriers, stabilizers, and excipients; The total mass of the surfactant peptides accounts for 0.5% to 20% of the total mass of the bio-emulsifier combination; preferably, the total mass of the surfactant peptides accounts for 3% to 10% of the total mass of the bio-emulsifier combination; more preferably, the total mass of the surfactant peptides accounts for 4% to 6% of the total mass of the bio-emulsifier combination.

[0007] Furthermore, the carrier includes one or more of maltodextrin, corn starch, modified starch, soluble starch, wheat bran powder, and rice bran powder; The stabilizer includes one or more of the following: sodium carboxymethyl cellulose, xanthan gum, sodium alginate, chitosan, soybean polysaccharide, agar, guar gum, gum arabic, yeast cell wall, xylooligosaccharide, and sodium polyacrylate. The excipients include one or more of the following: stone powder, sodium sulfate, talc, silica, zeolite powder, bentonite, maifanite, and calcium carbonate.

[0008] Furthermore, the bio-emulsifier composition is in granular form, wherein the particle size is 0.1~1.0 mm; preferably, the particle size is 30~80 mesh.

[0009] A second aspect of this application provides a method for preparing a bio-emulsifier composition, comprising: Bacillus subtilis with accession number CGMCC No.24947 was fermented to obtain Bacillus subtilis fermentation broth; A bio-emulsifier composition was prepared using the Bacillus subtilis fermentation broth.

[0010] Optionally, the preparation of the bio-emulsifier composition using the Bacillus subtilis fermentation broth includes: The inactivated Bacillus subtilis fermentation broth was added to a carrier for mixing and adsorption to obtain an adsorption solution. The adsorbent solution was spray-dried to obtain a powdered product; The powdered product is compounded with a carrier, stabilizer and excipient to obtain a bio-emulsifier composition.

[0011] Furthermore, it also includes: The compounded bio-emulsifier composition was granulated to obtain a granular bio-emulsifier composition.

[0012] A third aspect of this application provides an application of a bio-emulsifier composition, wherein the bio-emulsifier composition described in the first aspect is applied to animal feed.

[0013] Optionally, the animal feed may also include vegetable oils; The vegetable oils include one or more of soybean oil, rapeseed oil, corn oil, and palm oil.

[0014] Optionally, the bio-emulsifier composition is added to the animal feed at an amount of 50–500 g / t.

[0015] As can be seen from the above, this application provides a bio-emulsifier composition, preparation method, and application thereof. It utilizes surfactant peptides with a specific chain length distribution, including C13-C15 surfactant peptide homologues, with the total mass content of the C13-C15 surfactant peptide homologues being greater than or equal to 80%, and the mass content of the C14 surfactant peptide homologues being 50%-80%. The resulting bio-emulsifier composition can form an emulsion system with smaller particle size, more uniform distribution, and better stability in oil systems, increasing the dispersion degree and interfacial area of ​​oil droplets, allowing lipases to more fully contact the oil interface, thereby improving the enzymatic hydrolysis efficiency of lipases and promoting fatty acid release. In an in vitro lipase hydrolysis model, the bio-emulsifier composition of this application exhibits a faster acid value release rate and a higher hydrolysis promotion effect, significantly outperforming existing conventional emulsifiers such as bile acid emulsifiers. Therefore, when the bio-emulsifier composition of this application is applied to animal feed, it can improve the dispersion and enzymatic hydrolysis efficiency of dietary oils in the animal's digestive tract, promote the release and absorption of fatty acids, thereby improving feed energy utilization, improving feed conversion efficiency, and reducing the adverse effects of undigested oils on the intestinal environment. It is especially suitable for feed addition scenarios for young animals, high-oil diets, vegetable oil diets, or animals with insufficient fat digestion capacity, and has application value in improving fat digestibility and improving animal lipid metabolism. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in this application or related technologies, the drawings used in the description of the embodiments or related technologies will be briefly introduced below. Obviously, the drawings described below are only embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a chromatogram of HPLC measurement results of surface-active peptides in the embodiments of this application; Figure 2 This is a chromatogram showing the LC-MS identification results of the surface-active peptides in the embodiments of this application; Figure 3 This is the QE-MS / MS identification spectrum of the C13 surfactant peptide homologue in the embodiments of this application; Figure 4 This is the QE-MS / MS identification spectrum of C14 surfactant peptide homologue I in the surfactant peptides of this application; Figure 5 This is the QE-MS / MS identification spectrum of C14 surfactant peptide homologue II in the surfactant peptides of this application; Figure 6This is the QE-MS / MS identification spectrum of the C15 surfactant peptide homologue in the embodiments of this application; Figure 7 This is a graph showing the detection of liver fat content in grass carp fed with different diets in Experiment 9 of this application; Figure 8 This is a graph showing the detection of intestinal lipase activity in grass carp fed with different diets in Experiment 9 of this application. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with specific embodiments and the accompanying drawings.

[0019] It should be noted that, unless otherwise defined, the technical or scientific terms used in the embodiments of this application should have the ordinary meaning understood by one of ordinary skill in the art to which this application pertains. The terms "first," "second," and similar terms used in the embodiments of this application do not indicate any order, quantity, or importance, but are merely used to distinguish different components. Terms such as "comprising" or "including" mean that the element or object preceding the word encompasses the elements or objects listed after the word and their equivalents, without excluding other elements or objects. Terms such as "connected" or "linked" are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect. Terms such as "upper," "lower," "left," and "right" are only used to indicate relative positional relationships; when the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0020] In modern intensive livestock farming, oils are widely added to animal diets to improve feed energy levels. On the one hand, oils can significantly increase dietary energy concentration, meeting the energy needs of rapid animal growth without significantly increasing feed intake. On the other hand, oils also provide essential fatty acids, promote the absorption of fat-soluble vitamins, and improve feed palatability and pellet processing performance. This is especially important for young animals, which are in a rapid growth phase with high energy requirements per unit body weight but limited feed intake and gastrointestinal capacity; therefore, adding oils to improve dietary energy levels is of great significance.

[0021] However, the digestive system of young animals is not yet fully developed. They have insufficient secretion of endogenous bile acids and low lipase activity, resulting in limited ability to emulsify, digest, and absorb dietary fats, which easily leads to decreased fat utilization. Especially under high-fat diets, fats are difficult to disperse sufficiently in the digestive tract and form a stable emulsion system, easily causing incomplete fat digestion, which in turn leads to problems such as diarrhea, decreased growth performance, and reduced feed conversion ratio.

[0022] To improve the digestibility and absorption of fats and oils, current technologies typically involve adding exogenous emulsifiers to feed, such as bile acids, lecithin, and their derivatives. While these emulsifiers can promote lipid dispersion and emulsification to some extent, they still have certain limitations. For example, some emulsifiers are limited in source and have high production costs; some emulsifiers lack stability in the complex environment of the digestive tract and are easily affected by factors such as pH and ionic strength, leading to a decrease in emulsification efficiency; furthermore, most existing emulsifiers are mainly single-component, with relatively limited functions, resulting in poor adaptability to different animal species, dietary structures, and feed processing conditions, making it difficult to simultaneously achieve emulsification efficiency, digestive tract stability, and processing stability.

[0023] Therefore, current technology lacks an emulsifier system that can simultaneously satisfy high emulsification efficiency, good digestive tract stability, and feed processing compatibility. In particular, how to develop a bio-emulsifier composition that is stable in origin, structurally tunable, and exhibits excellent emulsification performance and application stability both in vivo and in vitro remains a pressing technical problem to be solved in this field.

[0024] Based on the above, this application provides a bio-emulsifier composition that can improve the enzymatic hydrolysis efficiency of lipase on oils and fats and promote the release of fatty acids; the bio-emulsifier composition includes surfactant peptides; the surfactant peptides include C13~C15 surfactant peptide homologues; based on the total mass of the surfactant peptides, the total mass content of the C13~C15 surfactant peptide homologues is greater than or equal to 80%, and the mass content of the C14 surfactant peptide homologues is 50%~80%.

[0025] Preferably, the total mass content of the C13-C15 surfactant peptide homologues is greater than or equal to 90%, and the mass content of the C14 surfactant peptide homologues is 60%-75%.

[0026] Surfactin is a cyclic lipopeptide bioactive substance produced by fermentation of Bacillus subtilis and other Bacillus species. Its molecular structure consists of a hydrophilic cyclic peptide structure and a hydrophobic fatty acid chain, possessing both hydrophilic and hydrophobic ends. It can oriented at the oil-water interface, thereby reducing the interfacial tension between oil and water and promoting the dispersion and emulsification of oils in the aqueous phase.

[0027] Compared to traditional chemically synthesized emulsifiers, surfactant peptides are derived from microbial fermentation, offering advantages such as clear biological origin, strong surface activity, and lower dosage. Surfactant peptide molecules contain fatty acid chains of varying lengths; they are not single compounds but rather composed of homologues of various fatty acid chains with different lengths, including C13, C14, and C15 homologues. C13, C14, and C15 represent fatty acid chains containing 13, 14, and 15 carbon atoms, respectively.

[0028] This application reveals that surfactant peptide homologues of different chain lengths exhibit differences in hydrophobicity, aqueous dispersibility, interfacial adsorption capacity, and emulsifying behavior. Therefore, by regulating the proportion of homologues of different chain lengths within surfactant peptides, their hydrophilic-hydrophobic balance and interfacial activity can be modulated, thereby affecting their emulsifying effect on oils and their promoting effect on the enzymatic hydrolysis of oils by lipases.

[0029] When the proportion of C13 to C15 surfactant peptide homologues is high and the C14 surfactant peptide homologues are within the above-mentioned specific content range, the resulting surfactant peptides (i.e., the surfactant peptides of this application) can form a better balance between aqueous phase dispersibility and oil interface adsorption capacity, thereby more effectively reducing oil-water interfacial tension and promoting oil dispersion in the aqueous system.

[0030] The bio-emulsifier composition provided in this application utilizes surfactant peptides with a specific chain length distribution. These surfactant peptides include C13-C15 surfactant peptide homologues, with the total mass content of the C13-C15 surfactant peptide homologues being greater than or equal to 80%, and the mass content of the C14 surfactant peptide homologues being 50%-80%. The resulting bio-emulsifier composition can form an emulsion system with smaller particle size, more uniform distribution, and better stability in oil systems. This increases the dispersion of oil droplets and the interfacial area, allowing lipases to more fully contact the oil interface, thereby improving the enzymatic hydrolysis efficiency of lipases and promoting fatty acid release. In an in vitro lipase hydrolysis model, the bio-emulsifier composition of this application exhibits a faster acid value release rate and a higher hydrolysis promotion effect, significantly outperforming existing conventional emulsifiers such as bile acid emulsifiers. Therefore, when the bio-emulsifier composition of this application is applied to animal feed, it can improve the dispersion and enzymatic hydrolysis efficiency of dietary oils in the animal's digestive tract, promote the release and absorption of fatty acids, thereby improving feed energy utilization, improving feed conversion efficiency, and reducing the adverse effects of undigested oils on the intestinal environment. It is especially suitable for feed addition scenarios for young animals, high-oil diets, vegetable oil diets, or animals with insufficient fat digestion capacity, and has application value in improving fat digestibility and improving animal lipid metabolism.

[0031] Further animal experiments showed that the bio-emulsifier composition provided in this application, when added to low-oil or vegetable oil diets, can improve feed conversion efficiency of grass carp and broilers, reduce serum triglyceride levels, increase lipase activity, and reduce liver fat accumulation, thus having application value in improving the digestibility and utilization of animal fats and lipid metabolism.

[0032] In some embodiments, it further includes: a carrier, a stabilizer, and an excipient; The total mass of the surface-active peptides accounts for 0.5% to 20% of the total mass of the bio-emulsifier combination.

[0033] Preferably, the total mass of the surfactant peptides accounts for 3% to 10% of the total mass of the bio-emulsifier combination; more preferably, the total mass of the surfactant peptides accounts for 4% to 6% of the total mass of the bio-emulsifier combination.

[0034] Specifically, the carrier is used to hold and disperse the surfactant peptides, enabling them to be uniformly distributed in the composition and facilitating subsequent mixing with feed ingredients; the stabilizer is used to improve the stability of the composition during storage and processing, reducing the release, aggregation, or decrease in activity of active ingredients; the excipient is used to improve the shapeability, particle strength, and processing suitability of the composition, enabling it to form granular formulations suitable for feed processing and addition. By controlling the surfactant peptide content within the above-mentioned range and combining it with specific proportions of carrier, stabilizer, and excipient, it is possible to ensure that the composition has sufficient emulsifying active ingredient content while improving the dispersion uniformity, storage stability, and processing performance of surfactant peptides in solid dosage forms.

[0035] Adding the carrier, stabilizer, and excipient according to the proportions in this embodiment can reduce problems such as uneven dispersion, hygroscopic clumping, poor flowability, or insufficient processing adaptability that may occur when surfactant peptides are added directly. This makes the resulting bio-emulsifier composition more suitable for addition to animal feed in the form of premixes, additives, or granular formulations. Therefore, the bio-emulsifier composition maintains the emulsifying activity of surfactant peptides while exhibiting good mixing uniformity, flowability, granulation processing adaptability, and storage stability, making it suitable for industrial application in livestock, poultry, and aquatic compound feeds.

[0036] In some embodiments, the carrier includes one or more of maltodextrin, corn starch, modified starch, soluble starch, wheat bran powder, and rice bran powder; the stabilizer includes one or more of sodium carboxymethyl cellulose, xanthan gum, sodium alginate, chitosan, soybean polysaccharide, agar, guar gum, gum arabic, yeast cell wall, xylooligosaccharide, and sodium polyacrylate. The excipients include one or more of the following: stone powder, sodium sulfate, talc, silica, zeolite powder, bentonite, maifanite, and calcium carbonate.

[0037] The carrier, stabilizer, and excipients are all selected from feedable adjuvants suitable for animal feed or feed additive products. Since the bio-emulsifier composition of this application is used in feed for livestock, poultry, aquatic animals, etc., the selection of the above-mentioned adjuvants must take into account feed safety, processing suitability, and storage stability, and comply with the relevant national requirements for feed ingredients, feed additives, and their use.

[0038] In some embodiments, the bio-emulsifier composition is granular, wherein the particle size is 0.1-1.0 mm; preferably, the particle size is 30-80 mesh. When the particle size is controlled within 0.1-1.0 mm, preferably 30-80 mesh, the resulting particles have a certain specific surface area, facilitating the release and emulsification of surfactant peptides in the feed system, while maintaining good particle strength and processing stability. This avoids pulverization, dust generation, or mixing losses due to excessively fine particles, and also avoids decreased feed mixing uniformity due to excessively coarse particles. Therefore, the granular bio-emulsifier composition exhibits good mixing uniformity, flowability, granulation processing adaptability, and storage stability, making it suitable for industrial addition and application in livestock, poultry, and aquatic compound feeds.

[0039] In some embodiments, the surfactant peptide is prepared using Bacillus subtilis with accession number CGMCC No. 24947.

[0040] Surfactant peptides are typically mixtures of homologues of various fatty acids with different chain lengths. Their specific composition is easily affected by factors such as strain type, fermentation conditions, culture medium composition, and post-treatment methods. Therefore, the content distribution of C13, C14, and C15 homologues may vary among surfactant peptides from different sources. This application utilizes *Bacillus subtilis* (accession number CGMCC No. 24947) for fermentation, which can reliably obtain surfactant peptides with the specific chain length distribution described in this application. Specifically, the total mass content of C13-C15 surfactant peptide homologues in the obtained surfactant peptides is greater than or equal to 80%, and the mass content of C14 surfactant peptide homologues is 50%-80%. This improves the stability and batch consistency of the surfactant peptide raw material composition, resulting in a more stable emulsifying performance and lipolysis-promoting effect in the subsequently prepared bio-emulsifier composition.

[0041] Based on the same inventive concept, this application also provides a method for preparing a bio-emulsifier composition for preparing the bio-emulsifier composition described in any of the above embodiments.

[0042] The method includes: Bacillus subtilis with accession number CGMCC No.24947 was fermented to obtain Bacillus subtilis fermentation broth; A bio-emulsifier composition was prepared using the Bacillus subtilis fermentation broth.

[0043] Specifically, Bacillus subtilis with the preservation number CGMCC No. 24947, stored at -80℃, was streaked on LB solid plates to form single colonies. These single colonies were then picked and inoculated into LB liquid medium for expansion to OD4-6. The resulting Bacillus subtilis seed culture was then inoculated into a fermenter and cultured using an optimized industrial fermentation medium to obtain Bacillus subtilis fermentation broth. This Bacillus subtilis fermentation broth can then be used to prepare a bio-emulsifier composition.

[0044] The resulting bio-emulsifier composition can effectively reduce the interfacial tension between oil and water in an oil-water system, promote oil dispersion and form an emulsion system with smaller particle size and uniform distribution, thereby increasing the contact area between oil and lipase, promoting oil enzymatic hydrolysis and fatty acid release, and thus improving the digestibility and utilization efficiency of dietary oil in animals.

[0045] In some embodiments, the preparation of the bioemulsifier composition using the Bacillus subtilis fermentation broth includes: The inactivated Bacillus subtilis fermentation broth was added to a carrier for mixing and adsorption to obtain an adsorption solution. The adsorbent solution was spray-dried to obtain a powdered product; The powdered product is compounded with a carrier, stabilizer and excipient to obtain a bio-emulsifier composition.

[0046] The carrier includes one or more of maltodextrin, corn starch, modified starch, soluble starch, wheat bran powder, and rice bran powder; the stabilizer includes one or more of sodium carboxymethyl cellulose, xanthan gum, sodium alginate, chitosan, soybean polysaccharide, agar, guar gum, gum arabic, yeast cell wall, xylooligosaccharide, and sodium polyacrylate; and the excipients include limestone powder, sodium sulfate, talc, silica, zeolite powder, bentonite, maifanite, and calcium carbonate.

[0047] Specifically, the obtained Bacillus subtilis fermentation broth is heated to 90-100℃ and kept at that temperature for 15-30 minutes to inactivate the Bacillus subtilis fermentation broth. The inactivated Bacillus subtilis fermentation broth is then added to a carrier for mixing and adsorption to obtain an adsorbent solution. In this step, the weight ratio of the carrier to the fermentation broth is 1:4-6. The thoroughly mixed adsorbent solution is then subjected to centrifugal spray drying, with the controlled parameters being an inlet air temperature of 150-170℃ and an outlet air temperature of 60-90℃, to obtain a powdered product with a moisture content of less than 5%. Finally, the powdered product is compounded with the carrier, stabilizer, and excipient in the following proportions: 200-400 parts of powdered product, 200-400 parts of carrier, 30-100 parts of stabilizer, and 100-400 parts of excipient. Thus, the bio-emulsifier composition described in any of the aforementioned embodiments is prepared.

[0048] In some embodiments, it also includes: The compounded bio-emulsifier composition was granulated to obtain a granular bio-emulsifier composition.

[0049] Specifically, granulation can be performed using either wet or dry granulation, with dry granulation being preferred. Wet granulation involves adding an appropriate amount of water or other feedable binder to the compounded powder material, wetting the material and forming a wet material with a certain degree of binding properties. This wet material is then stirred, extruded, sieved, dried, and granulated to obtain granular products. Wet granulation improves granule formability and uniformity, but requires drying, which may increase energy consumption and potentially affect heat-sensitive or water-sensitive active ingredients. Dry granulation involves directly compacting the compounded powder material into blocks, flakes, or strips through methods such as tableting, rolling, or extrusion, without or with minimal addition of liquid binders. This is followed by crushing, sieving, and granulation to obtain granular products within the target particle size range. The dry granulation process is relatively simplified, avoiding wetting and drying processes, which helps reduce the impact of moisture and high temperatures on the activity of surface-active peptides and improves granulation efficiency and product storage stability.

[0050] Finally, granulation yields a bio-emulsifier composition with a particle size of 0.1–1.0 mm or 30–80 mesh.

[0051] Based on the same inventive concept, this application also provides an application of a bio-emulsifier composition, wherein the bio-emulsifier composition described in any of the foregoing embodiments is applied to animal feed.

[0052] Animal feed containing the bio-emulsifier composition described in this application can promote the dispersion and emulsification of oils in the diet, improve the contact efficiency between lipase and the oil interface, thereby improving the enzymatic hydrolysis and absorption of oils, and improving animal growth performance and lipid metabolism. It is especially suitable for young animals, diets with high vegetable oil content, or feeding scenarios with low oil utilization efficiency.

[0053] Animal experiments have shown that animal feeds obtained by adding the bio-emulsifier composition described in this application to low-oil diets or vegetable oil diets can improve the feed conversion efficiency of grass carp and broilers, reduce serum triglyceride levels, increase lipase activity, and reduce liver fat accumulation, effectively promote the digestion and absorption of dietary oils by animals, improve oil utilization, and have application value in improving lipid metabolism.

[0054] In some embodiments, the animal feed also includes vegetable oils; The vegetable oils include one or more of soybean oil, rapeseed oil, corn oil, and palm oil.

[0055] Vegetable oils, as an energy source in animal feed, can increase dietary energy levels and provide animals with nutrients such as fatty acids. By adding the bio-emulsifier composition described in this application to animal feed containing vegetable oils, the dispersion and emulsification effect of vegetable oils in the digestive tract can be improved, increasing the specific surface area of ​​oil droplets and allowing lipases to act more fully on the oil interface, thereby improving the digestibility and utilization rate of vegetable oils. This is especially suitable for feeding young animals, diets high in vegetable oil, or feeding scenarios where the utilization efficiency of vegetable oils is low.

[0056] In some embodiments, the amount of the bio-emulsifier composition added to the animal feed is 50-500 g / t, preferably 100-300 g / t, and more preferably 200 g / t.

[0057] By controlling the amount of the bio-emulsifier composition added within the above-mentioned range, it is possible to ensure its emulsification promoting effect while avoiding insufficient oil emulsification and enzymatic hydrolysis promotion due to excessively low addition, and also to avoid excessive addition leading to increased formulation costs or affecting the stability of the feed system.

[0058] The following experiments further verify the technical effects of this application.

[0059] 1. Preparation of bio-emulsifier composition.

[0060] Before conducting specific experiments, the bio-emulsifier composition was prepared according to the following method: (1) Strains and culture: Bacillus subtilis CGMCC No.24947, which was frozen at -80℃, was streaked on LB solid plates. Single colonies were picked and inoculated into LB liquid medium to expand the culture to OD4-6. The obtained Bacillus subtilis seed culture was then inoculated into a fermenter to obtain Bacillus subtilis fermentation broth. The fermentation liquid culture medium consisted of the following components: 70 g / L carbon source, 1.25 g / L yeast extract, 5 g / L urea, 0.333 g / L KH2PO4, 1 g / L Na2HPO4•12H2O, 0.15 g / L MgSO4•7H2O, 7 mg / L CaCl2 (added to 100 μL of mother liquor), 6 mg / L MnSO4•H2O (added to 100 μL of mother liquor), 6 mg / L FeSO4•7H2O (added to 100 μL of mother liquor), and 50 g / L soybean oil, dissolved in water and adjusted to pH 7.0.

[0061] (2) Inactivation of fermentation broth: The Bacillus subtilis fermentation broth obtained in the previous step is heated to 90~100℃ and kept warm for 15~30min to obtain inactivated Bacillus subtilis fermentation broth.

[0062] (3) Mixed adsorption: The inactivated Bacillus subtilis fermentation broth was added to the carrier for mixed adsorption to obtain an adsorbent solution. The weight ratio of the carrier to the Bacillus subtilis fermentation broth was 1:4~6.

[0063] (4) Spray drying: The well-mixed adsorbent liquid is centrifugally spray dried, with the control parameters being an inlet air temperature of 150~170℃ and an outlet air temperature of 60~90℃, to obtain a powdered product with a moisture content of less than 5%.

[0064] (5) Compounding: Compounding is carried out according to the following weight parts: 200-400 parts of powdered product, 200-400 parts of carrier, 30-100 parts of stabilizer, and 100-400 parts of excipient, thereby obtaining a bio-emulsifier composition.

[0065] (6) Granulation: The compounded bio-emulsifier composition is granulated to form uniform granules / granulated products with a particle size of 30~80 mesh, thus obtaining a granular bio-emulsifier composition.

[0066] The bio-emulsifier composition was sieved and packaged, and then packaged in aluminum foil bags to prevent moisture absorption and deterioration, in preparation for subsequent experiments.

[0067] The granular bio-emulsifier composition thus prepared is referred to as the product of Example 1 and was used in subsequent experiments.

[0068] 2. Experiment 1: Characterization of surface-active peptide chain length distribution in bio-emulsifier compositions.

[0069] (1) Purpose of the experiment The chain length distribution of the surfactant peptides used in the bioemulsifier composition of this application was characterized by liquid chromatography-mass spectrometry (LC-MS) or high performance liquid chromatography (HPLC).

[0070] (2) Test materials Sample preparation: The surfactant peptide was prepared using Bacillus subtilis strain CGMCC No. 24947. The preparation process followed the steps in "1. Preparation of Bioemulsifier Composition" ("(1) Strains and Culture") to obtain the Bacillus subtilis fermentation broth. The resulting Bacillus subtilis fermentation broth is the surfactant peptide of this application. The surfactant peptide solution of this application was diluted and pretreated to make it suitable for LC-MS or HPLC testing. Equipment: LC-MS system, used for qualitative and quantitative analysis of surfactant peptide homologues with different chain lengths, model Ultimate 3000-Q / Exactive-MS; HPLC system, used to confirm the chain length distribution of surfactant peptides, model Shimadzu LC2050.

[0071] (3) Experimental analysis conditions 1) LC-MS analysis conditions Chromatographic column: C18 reversed-phase column (250mm x 4.6mm, 5μm); Mobile phase: A mixture of aqueous phase and organic solvent (acetonitrile or methanol); Flow rate: 0.8 mL / min; Detection wavelength: The characteristic wavelength applicable to surface-active peptides.

[0072] 2) HPLC analysis conditions Analysis was performed using a reversed-phase column (C18).

[0073] By monitoring the retention time of surfactant peptide homologues, homologues of different chain lengths are separated and identified, and their contents are quantitatively analyzed based on the corresponding chromatographic peak areas, thereby determining the distribution of homologues of different chain lengths in surfactant peptides.

[0074] (4) Experimental results and data analysis The HPLC measurement results of the surface-active peptides in this application are as follows: Figure 1 As shown, the LC-MS identification results are as follows: Figure 2 As shown. Figure 2 In the graph, the horizontal axis represents the mass-to-charge ratio (m / z), and the vertical axis represents the percentage (%).

[0075] Figure 1 This is an HPLC chromatogram of a surfactant peptide. Figure 2 yes Figure 1 The corresponding LC-MS spectra were analyzed by mass spectrometry, with peaks at retention times of 11.15, 13.57, 15.13, 16.10, 17.29, and 20.68 min. Figures 1-2 It can be seen that the surfactant peptides prepared in this application include homologues of various fatty acid chain lengths. The QE-MS / MS spectra of the C13 surfactant peptide homologues are shown below. Figure 3 As shown, the QE-MS / MS spectra of the C14 surfactant peptide homologues are as follows: Figure 4 , 5 As shown, the QE-MS / MS spectra of the C15 surfactant peptide homologues are as follows: Figure 6 As shown. Among them, Figure 4 and Figure 5 These correspond to C14 surfactant peptide homologues I and II, respectively. It should be noted that C14 surfactant peptide homologues are not necessarily a single structural component; they can include multiple structural variants where the fatty acid chains all have 14 carbon atoms, but the amino acid composition or arrangement in the peptide ring differs. In this experiment, the surfactant peptides detected included two structural variants of the C14 surfactant peptide homologue, denoted as C14 surfactant peptide homologue I and C14 surfactant peptide homologue II, respectively.

[0076] exist Figures 3-6 In the diagram, b1–b6 and y1–y5 represent fragment ions generated after peptide chain breakage. In secondary mass spectrometry, peptide bond breakage produces fragment ions in different orientations. b ions represent fragments retained from the N-terminus, and y ions represent fragments retained from the C-terminus.

[0077] exist Figures 3-6 In the text, b5-H2O represents the dehydrated ion after the b5 fragment ion further loses a water molecule; H2O represents a water molecule, indicating that the b5 fragment underwent neutral loss during the mass spectrometry collision process, losing one H2O molecule and reducing its mass by 18 Da.

[0078] exist Figures 3-6 In the middle, [M+Na] + Indicates: The measured molecule M reacts with a sodium ion Na + The sodium ion peak formed by the combination.

[0079] Figure 3 The image shows the QE-MS / MS spectrum of C13 surfactant peptide homologues. Therefore, the measured molecule M is the C13 surfactant peptide homologue. Figure 3 In this context, M represents a C13 surfactant peptide homologue. The mass-to-charge ratio (m / z) of the C13 surfactant peptide homologue is 1030, and it contains a peptide of glutamic acid-leucine-leucine-valine-aspartic acid-leucine-leucine (Glu-Leu-Leu-Val-Asp-Leu-Leu) and a C13 β-hydroxy fatty acid chain.

[0080] Figure 4 The image shows the QE-MS / MS spectrum of C14 surfactant peptide homologue I. Therefore, the measured molecule M is C14 surfactant peptide homologue I. Figure 4 In this context, M represents C14 surfactant peptide homologue I. C14 surfactant peptide homologue I has a mass-to-charge ratio (m / z) of 1044 and contains a C13 β-hydroxy fatty acid chain.

[0081] Figure 5 The image shows the QE-MS / MS spectrum of C14 surfactant peptide homologue II. Therefore, the measured molecule M is C14 surfactant peptide homologue II. Figure 4 In this context, M represents C14 surfactant peptide homologue II. C14 surfactant peptide homologue I has a mass-to-charge ratio (m / z) of 1030 and contains a peptide of glutamic acid-valine-leucine-leucine-aspartic acid-leucine-valine (Glu-Val-Leu-Leu-Asp-Leu-Val) and a C14 β-hydroxy fatty acid chain.

[0082] Figure 6The image shows the QE-MS / MS spectrum of C15 surfactant peptide homologues. Therefore, the measured molecule M is the C15 surfactant peptide homologue. Figure 6 In this context, M represents a C15 surfactant peptide homologue. The mass-to-charge ratio (m / z) of the C15 surfactant peptide homologue is 1058, and it contains a C15 β-hydroxy fatty acid chain.

[0083] The parent ion [M+Na] + After collision-induced cleavage, the peptide chain breaks, forming a series of b and y ions; the amino acid sequence and fatty acid chain length of the surface-active peptide can be determined by the mass difference of these fragment ions.

[0084] The mass percentage of each homologue was calculated based on the total mass of the surfactant peptides using LC-MS and HPLC. This yielded the total mass content of C13-C15 surfactant peptide homologues and the mass content of C14 surfactant peptide homologues. The chain length distribution of the surfactant peptides in this application is shown in the table below. Table 1. Chain length distribution of surface-active peptides

[0085] (5) Results Analysis and Conclusions According to the LC-MS and HPLC test results, the fatty acid chain lengths of the surfactant peptides are mainly concentrated in C13, C14, and C15, that is, the surfactant peptides are mainly C13~C15 surfactant peptide homologues. Based on the total mass of the surfactant peptides, the total mass content of C13~C15 surfactant peptide homologues reaches 92.5%, and the mass content of C14 surfactant peptide homologues is 70.6%.

[0086] The above results indicate that the surfactant peptides of this application have a relatively concentrated chain length distribution, with C14 homologues as the main components. The surfactant peptides of the above components have a relatively ideal chain length distribution, which is conducive to forming a suitable hydrophilic-hydrophobic balance and interfacial adsorption capacity. They can be used stably and efficiently for oil emulsification, have good emulsification stability, and can stably play an emulsifying role in feed, thereby improving the digestibility and utilization efficiency of oils.

[0087] 3. Experiment 2: Effect of different fatty acid chain length distributions of surfactant peptides on their emulsifying properties and fat digestibility.

[0088] (1) Experimental materials Three surfactant peptides with different fatty acid chain length distributions were selected for comparative experiments. They are: 1) Experimental group The surfactant peptides obtained by fermentation culture of Bacillus subtilis with accession number CGMCC No.24947 had a total mass ratio of C13~C15 surfactant peptide homologues (based on the total mass of surfactant peptides) of 93.2% and a mass ratio of C14 surfactant peptide homologues (based on the total mass of surfactant peptides) of 74.6%.

[0089] 2) Comparative Example A Comparative Example A is the prepared surfactant peptide product A, in which the total mass percentage of C13~C15 surfactant peptide homologues (based on the total mass of surfactant peptides) is 66.4%, and the mass percentage of C14 surfactant peptide homologues (based on the total mass of surfactant peptides) is 30.3%.

[0090] 3) Comparative Example B Comparative Example B is the prepared surfactant peptide product B, in which the total mass percentage of C13~C15 surfactant peptide homologues (based on the total mass of surfactant peptides) is 87.5%, and the mass percentage of C14 surfactant peptide homologues (based on the total mass of surfactant peptides) is 26.7%.

[0091] The content of surfactant peptides in each group was determined by HPLC to ensure that the effective content remained consistent, thus eliminating the influence of differences in the amount added on the experimental results.

[0092] (2) Experimental content and methods 1) Emulsification performance test Oil-water emulsions were prepared using a high-speed homogenization method, and the droplet size (D50) and emulsion stability were determined.

[0093] 2) In vitro digestion experiment: A simulated digestion system was used to determine the change in acid value during the hydrolysis of fats and oils, in order to characterize the efficiency of lipolysis. Acid value was calculated based on the amount of KOH consumed to neutralize free fatty acids, expressed in mg KOH / g.

[0094] In this experiment, the acid value enhancement factor was used to evaluate the promoting effect of emulsifiers on the enzymatic hydrolysis of fats and oils. The calculation method is as follows: Acid value increase factor = Acid value increase of added surfactant peptides / Acid value increase of blank control group; The increase in acid value = the final acid value of the reaction - the initial acid value of the reaction.

[0095] (3) Experimental results The performance test results of the experimental group, comparative example A, and comparative example B are shown in the table below: Table 2 Performance test results of surfactant peptides with different chain length distributions

[0096] Note: Different lowercase letters in the subscript of data in the same column indicate significant differences (P<0.05); the same subscript indicates no significant difference. Data are expressed as mean ± standard deviation.

[0097] In the table above, the total mass percentage of C13~C15 and the mass percentage of C14 are both calculated based on the total mass of surfactant peptides.

[0098] (4) Results Analysis As shown in Table 2, under the condition that the effective content of surfactant peptides in each group is kept consistent, there are significant differences in emulsification performance and lipid enzymatic hydrolysis promotion effect among the experimental group, comparative example A and comparative example B.

[0099] Specifically, in the experimental group, the total mass proportion of C13-C15 surfactant peptide homologues was 93.2%, and the proportion of C14 surfactant peptide homologues was 74.6%. Their droplet diameter (D50) was 1.81±0.13 μm, significantly lower than that of Comparative Example A (3.52±0.19 μm) and Comparative Example B (3.02±0.22 μm). Simultaneously, the acid value increase factor in this application group was 2.23±0.18, significantly higher than that of Comparative Example A (1.26±0.12) and Comparative Example B (1.32±0.14). All of the above differences reached a statistically significant level (P<0.05).

[0100] In Comparative Example A, the total mass percentage of C13-C15 surfactant peptide homologues was 66.4%, and the mass percentage of C14 surfactant peptide homologues was 30.3%, which was significantly lower than the range defined in this application. The droplet size was significantly increased, and the acid value increase factor was significantly decreased. This indicates that when the proportions of C13-C15 surfactant peptide homologues and 145 surfactant peptide homologues both deviate from the chain length range of the surfactant peptides selected in this application (i.e., the total mass content of C13-C15 surfactant peptide homologues is greater than or equal to 80%, and the mass content of C14 surfactant peptide homologues is 50%-80%), the emulsifying performance and the ability to promote lipolysis are significantly reduced.

[0101] In Comparative Example B, although the total mass percentage of C13-C15 surfactant peptide homologues reached 87.5%, close to the chain length range of the surfactant peptides selected in this application, its C14 surfactant peptide homologues accounted for only 26.7%, and its droplet size and acid value enhancement factor were still significantly inferior to those of the present invention. This result indicates that simply increasing the proportion of C13-C15 surfactant peptide homologues is not sufficient to achieve the best emulsification effect; when the proportion of C14 surfactant peptide homologues is insufficient, the emulsification performance and lipolysis promotion ability still cannot reach the level of this application.

[0102] Therefore, it can be seen that the fatty acid chain length distribution of surfactant peptides has a significant impact on emulsifying performance and oil hydrolysis ability. Among them, the C13-C15 surfactant peptide homologues and the C14 surfactant peptide homologues have a synergistic effect, which is a key technical feature determining emulsifying performance and oil hydrolysis ability. Only when the mass proportions of C13-C15 surfactant peptide homologues and C14 surfactant peptide homologues are both within the range defined in this application, the resulting emulsion system has a smaller droplet size and a higher acid value enhancement factor, thereby obtaining a chyle system with smaller particle size and better dispersibility, and significantly improving the hydrolysis efficiency of lipase on oils.

[0103] 4. Experiment 3: Comparative test on the promoting effect of bio-emulsifier composition and bile acid on lipase hydrolysis of fats.

[0104] (1) Experimental objective This study investigated the promoting effect of the bio-emulsifier composition of this application on the enzymatic hydrolysis of oils by lipase. Using the product of Example 1 prepared in "1. Preparation of Bio-emulsifier Composition" as the experimental sample, and bile acids as a control, the effects of both on promoting the enzymatic hydrolysis of soybean oil by lipase under in vitro conditions were compared.

[0105] (2) Experimental materials Example 1 product, bile acids, soybean oil, lipase, petroleum ether-ethanol solution (2:1), KOH ethanol solution, phenolphthalein, PBS buffer, 250mL Erlenmeyer flask, etc.

[0106] (3) Experimental methods Fifteen 250mL Erlenmeyer flasks were divided into three groups: a control group, an experimental group, and a bile acid group, with five flasks in each group. 50mL of PBS buffer was added to each flask; the experimental group received 0.2g of the product from Example 1, and the bile acid group received 0.2g of bile acid. The Erlenmeyer flasks were sealed and placed in a shaker at 37°C for enzymatic hydrolysis. Samples were taken periodically to determine the acid value. A total of five experiments were conducted, numbered 3-1, 3-2, 3-3, 3-4, and 3-5, using different lipase and oil addition conditions (see Table 3 for details).

[0107] Acid value determination: Add 50 mL of petroleum ether-ethanol solution (2:1) to the enzymatic hydrolysis flask, add 2-3 drops of phenolphthalein, and titrate with KOH ethanol solution until the solution turns slightly pink. Calculate the acid value based on the volume of KOH consumed to neutralize free fatty acids. The higher the acid value, the greater the degree to which the lipase hydrolyzes the oil into fatty acids, and the better the enzymatic hydrolysis effect.

[0108] (4) Enzyme and oil addition conditions for different experiments Table 3. Conditions for lipase and oil addition in different experiments

[0109] (5) Experimental results To systematically evaluate the effect of the bio-emulsifier composition of this application on promoting the enzymatic hydrolysis of oils by lipase, the changes in the acid value of oils during enzymatic hydrolysis were measured under different lipase addition amounts and oil load conditions. The results are shown in Tables 4 to 8.

[0110] Table 4. Acid value of oil after enzymatic hydrolysis in Experiment 3-1 (enzyme addition 0.01g, oil addition 3g).

[0111] Table 5. Acid value of oil after enzymatic hydrolysis in Experiment 3-2 (enzyme addition 0.02g, oil addition 3g).

[0112] Table 6. Acid value of oil after enzymatic hydrolysis in Experiment 3-3 (enzyme addition 0.01g, oil addition 5g).

[0113] Table 7. Acid value of oils after enzymatic hydrolysis in Experiments 3-4 (enzyme addition 0.02g, oil addition 5g).

[0114] Table 8. Acid value of oils after enzymatic hydrolysis in Experiments 3-5 (enzyme addition 0.04g, oil addition 5g).

[0115] (6) Results Analysis and Conclusions The experimental results show that, in all experiments, the acid value of the experimental group's bio-emulsifier composition was significantly higher than that of the control group and the bile acid group at all time points (especially 1h and 2h), indicating that the bio-emulsifier composition of this application has a more significant promoting effect on lipase hydrolysis of oils. The promoting effect was most pronounced under the conditions of Experiment 3-3 (0.01g enzyme, 5g oil), effectively promoting the contact between lipase and oil at the interface and accelerating oil hydrolysis.

[0116] 5. Experiment 4: Laser particle size analyzer was used to determine the particle size of chylomicrons to evaluate emulsifying ability.

[0117] (1) Purpose of the experiment The particle size distribution characteristics of oil droplets (chylomicrons) in the chyle system were determined by laser particle size analyzer. Parameters such as D10, D50, D90, volume average diameter D[4,3], and span were used as evaluation indicators to compare the particle size refinement and distribution concentration of chyle systems formed by different emulsifiers, thereby evaluating their emulsifying ability. Generally speaking, the smaller the particle size and the more concentrated the distribution of chylomicrons, the better the emulsification effect, which is beneficial to the stable existence and further digestion and absorption of oils in the digestive system.

[0118] (2) Test materials Soybean oil; deionized water or PBS buffer; The product from Example 1 was used in the experiment.

[0119] (3) Method for preparing chyme Take 3-5 g of soybean oil and add it to 50-100 mL of aqueous phase. Then add the product of Example 1 to make its concentration in the system 0.2 g / 100 mL. After shaking to form a pre-emulsified system, prepare a uniform chyme system by ultrasonic and circulating dispersion.

[0120] During the particle size test, the chyme sample remained uniform under the conditions of cyclic dispersion and ultrasound in a laser particle size analyzer, with a cyclic speed of 1200 rpm, an ultrasonic power of 35W, and a dispersion time of 180s.

[0121] (4) Particle size determination method Wet particle size distribution analysis was performed using a Bettersize 2600 laser particle size analyzer, with a test range of 0.02–2600 μm. The particle size distribution type was volumetric distribution, and the Mie model was used for analysis. The medium was water with a refractive index of 1.333, a refractive index of 1.52, and an absorption rate of 0.1. The D10, D50, D90, volume average diameter D[4,3], area average diameter D[3,2], span, and peak particle size of the chylomicrons were recorded.

[0122] (5) Test results The chyle samples prepared in this experiment were tested, and the results showed that: D10 was 0.749 μm, D50 was 1.934 μm, D90 was 9.700 μm, the volume average diameter D[4,3] was 4.183 μm, the area average diameter D[3,2] was 0.761 μm, the peak particle size was 1.896 μm, and the span was 4.627.

[0123] Particle size distribution analysis showed that the chylomicrons were mainly concentrated in the range of 0.2 to 5.0 μm, with a volume fraction of approximately 84.19%. This indicates that under the emulsification effect of the bio-emulsifier components in this application, soybean oil can form a chylomicron system with small particle size and relatively concentrated distribution.

[0124] 6. Experiment 5: Performance comparison experiment of the bio-emulsifier composition of this application and common feed emulsifiers.

[0125] To further verify the technical effectiveness of the bio-emulsifier composition of this application, a comparative experiment was conducted with common feed emulsifiers. Without changing the ratio of the oil phase and water phase, or the preparation and testing conditions, bile acids, glyceryl monostearate, and lysophosphatidylcholine were used to replace the bio-emulsifier composition of this application to prepare chyme, and the same laser particle size analyzer parameters were used for particle size testing and emulsion stability; the change in acid value during oil hydrolysis was also measured.

[0126] (1) Experimental materials and grouping The groups are divided as follows: Experimental group: Product of Example 1; Control group 1: Added with conventional feed bile acid preparations; Control group 2: Added glyceryl monostearate emulsifier; Control group 3: Added lysophosphatidylcholine emulsifier; Control group 4: As a blank group, no emulsifier was added.

[0127] The amount of emulsifier added in each of the above groups was kept consistent based on the effective ingredient, and the oil source was the same to eliminate interference from non-target factors.

[0128] (2) Experiment content 1) Emulsification performance test: Determine the droplet size (D50) and emulsion stability; 2) In vitro digestion experiment: Determine the change in acid value during the hydrolysis of fats and oils.

[0129] (3) Results Analysis Table 9 Comparison of emulsifying properties and enzymatic hydrolysis promoting effects of the bio-emulsifier composition of this application and common feed emulsifiers.

[0130] Note: Different lowercase letters in the subheadings of data in the same column indicate significant differences (P<0.05); no subheadings or identical subheadings indicate no significant differences. Data are expressed as mean ± standard deviation, n=3.

[0131] From the table above, we can see that: 1) Compared with the emulsifiers in all control groups, the experimental group formed the smallest droplet size, indicating that the bio-emulsifier combination provided in this application has better oil-water dispersion ability; 2) In the in vitro digestion experiment, the experimental group showed the highest increase in acid value, indicating that the combination of bio-emulsifiers provided in this application has a significant promoting effect on lipase hydrolysis; 3) Although traditional emulsifiers (such as bile acids and lysophosphatidylcholine) have certain emulsifying abilities, they are inferior to the experimental group in terms of emulsion particle size control and enzymatic hydrolysis promotion. 4) The control group 4 (i.e. blank group) had the worst performance in all indicators, which further illustrates the necessity of emulsifiers in the utilization of oils.

[0132] (4) Conclusion This experiment demonstrates that the bio-emulsifier composition containing a specific chain length distribution provided in this application is significantly superior to existing common feed emulsifiers in terms of emulsion particle size control, emulsion stability, and fat digestion promotion.

[0133] To further clarify, the bio-emulsifier composition containing a specific chain length distribution provided in this application is not a simple replacement for existing emulsifiers, but rather achieves a synergistic improvement in emulsification performance and bioavailability efficiency based on molecular structure regulation.

[0134] Existing feed emulsifiers typically include bile acids, lecithin, and their derivatives. They primarily function by promoting oil dispersion or improving interfacial conditions, but their mechanisms are singular and lack precise structural control. None of them regulate emulsifying performance from the perspective of fatty acid chain length distribution of surfactant molecules, nor do they achieve simultaneous optimization of emulsifying performance and oil digestibility. This application, by limiting the content distribution of specific fatty acid chain length homologues in surfactant peptides, especially controlling the total content of C13-C15 homologues and the proportion of C14 homologues, results in a bio-emulsifier composition that combines better interfacial activity, emulsion stability, and oil enzymatic hydrolysis promotion, thus achieving superior overall performance compared to common feed emulsifiers.

[0135] To further verify the feasibility of the bio-emulsifier composition provided in this application in industrial production and actual feed application, the retention rate of effective ingredients before and after granulation and storage stability were evaluated, as detailed in Experiments 6-8.

[0136] 7. Experiment 6: Verification experiment on the retention rate of effective ingredients before and after granulation.

[0137] (1) Experimental objective The content of active surface-active peptides and the retention of emulsifying function of the bio-emulsifier composition provided in this application before and after granulation are evaluated to verify the product's suitability for the granulation process.

[0138] (2) Test materials The reagents used in the test include chromatographically pure reagents such as methanol, acetonitrile, formic acid, and water.

[0139] The testing equipment includes HPLC or LC-MS analysis systems, laser particle size analyzers, and conventional moisture determination devices.

[0140] (3) Experimental grouping Set up the following groups: 1) Control group: The ungranulated bio-emulsifier composition was used as a sample control before granulation. The control group sample was prepared according to the steps of “(1) strain and culture”, “(2) fermentation broth inactivation”, “(3) mixed adsorption”, “(4) spray drying” and “(5) compounding” in “1. Preparation of bio-emulsifier composition”, but without subsequent granulation treatment.

[0141] 2) Experimental Group 1: The bio-emulsifier composition of the control group was dry granulated to obtain the dry granulated sample used in this group.

[0142] 3) Experimental Group 2: The bio-emulsifier composition of the control group was wet-granulated to obtain the wet-granulated sample used in this group.

[0143] Among them, the dry granulation process of experimental group 1 and the wet granulation process of experimental group 2 were prepared using conventional feed additive granulation processes.

[0144] (4) Test methods The content of surfactant peptides in samples from experimental groups 1 and 2 and the control group was determined by HPLC, and the retention rate of surfactant peptides after granulation was calculated. The retention rate was calculated using the following formula: Effective ingredient retention rate (%) = surfactant peptide content after granulation / surfactant peptide content before granulation × 100%.

[0145] Simultaneously, the enzymatic hydrolysis-promoting function of the samples before and after granulation was evaluated. Specifically, the change in acid value was determined by in vitro lipase hydrolysis, and the retention rate of the enzymatic hydrolysis-promoting function was calculated according to the following formula: Enzymatic hydrolysis-promoted function retention rate (%) = acid value increase of sample after granulation / acid value increase of sample before granulation × 100%; The increase in acid value = the final acid value of the reaction - the initial acid value of the reaction.

[0146] In addition, the moisture content, particle size distribution, flowability, and particle integrity of the samples were tested.

[0147] (5) Results Table 10. Content of surface active peptides, retention rate of effective ingredients, and retention rate of enzymatic hydrolysis-promoted function before and after granulation.

[0148] In the table above, the retention rate of enzymatic hydrolysis promotion function is calculated based on the in vitro enzymatic hydrolysis promotion capacity.

[0149] The results showed that the surface active peptide retention rate was about 97.6% after dry granulation and about 91.0% after wet granulation, both maintaining a high level. At the same time, the enzymatic hydrolysis promoting function retention rate of the granulated sample was over 90%, indicating that the bio-emulsifier composition of this application can still maintain good functional activity under granulation processing conditions.

[0150] (6) Conclusion Experiments have shown that the bio-emulsifier composition provided in this application exhibits minimal changes in surface-active peptide content before and after granulation, and maintains stable emulsification function, indicating good compatibility with granulation processes and suitability for industrial production and practical feed applications.

[0151] 8. Experiment 7: Feed pelleting temperature tolerance test.

[0152] (1) Purpose of the experiment To further evaluate the stability of the bio-emulsifier composition of this application under actual pelleting processing conditions in a feed mill, this embodiment simulates short-term high-temperature treatment conditions during feed pelleting and examines the effects of different temperatures and treatment times on the retention rate of effective surfactant peptide components and the retention rate of enzymatic hydrolysis-promoting function.

[0153] (2) Test materials Using the product from Example 1 as the test sample, it was determined that the total mass of its surface-active peptides accounted for approximately 5% of the total mass of the bio-emulsifier combination.

[0154] (3) Test methods Ten groups were set up. One group served as a control group, receiving no heat treatment. The remaining nine groups underwent short-time heat treatment under simulated granulation heat treatment conditions. Experimental groups 1-3 were treated at 75℃ for 30, 60, and 90 seconds, respectively; experimental groups 4-6 were treated at 85℃ for 30, 60, and 90 seconds, respectively; and experimental groups 7-9 were treated at 90℃ for 30, 60, and 90 seconds, respectively. After all experimental groups have been processed, the samples were immediately cooled to room temperature and sealed for later testing.

[0155] Test items: retention rate of active ingredients, retention rate of enzymatic hydrolysis function, and detection of appearance and physical properties.

[0156] Effective ingredient retention rate (%) = surface active peptide content after granulation heat treatment / surface active peptide content before granulation heat treatment × 100%.

[0157] Enzymatic hydrolysis-promoted function retention rate (%) = acid value increase of sample after granulation heat treatment / acid value increase of sample before granulation heat treatment × 100%, acid value increase = reaction endpoint acid value - initial reaction acid value, and the sample before granulation heat treatment is the control group sample.

[0158] Appearance and physical properties: Observe the color, odor, clumping and flowability of the sample after heat treatment, and measure the change in moisture content of the sample.

[0159] (4) Results Analysis Table 11: Retention rate of effective ingredients, retention rate of enzymatic hydrolysis-promoting function, and appearance changes after simulated feed pelleting temperature

[0160] As can be seen from the table, under the conditions of treatment at 75℃ for 30-90 seconds and treatment at 85℃ for 30-90 seconds, the effective component retention rate of surface active peptides in the bio-emulsifier composition of this application can still reach more than 98%, and the functional activity retention rate can still reach more than 97%. This indicates that the product of this application has good thermal stability under conventional pelleting temperature conditions and can adapt to the common pelleting processing conditions of most feed mills.

[0161] Under the condition of treatment at 90℃ for 30 to 90 seconds, the content of surface active peptides and emulsifying activity decreased slightly with the extension of treatment time. However, after treatment at 90℃ for 90 seconds, the retention rate of effective ingredients can still reach more than 95%, and the retention rate of functional activity can still reach more than 94%, indicating that the product of this application still has good tolerance under high temperature and short time granulation conditions.

[0162] (5) Conclusion The above results indicate that the bio-emulsifier composition of this application exhibits high retention rates of effective components and emulsifying activity under simulated feed pelleting temperature conditions. This product can withstand short-term heat treatment processes of 75℃~90℃ for 30~90 seconds, making it suitable for industrial feed pelleting processes.

[0163] This demonstrates that the bio-emulsifier composition of this application not only has good emulsifying properties and the ability to promote the digestion and utilization of oils, but also has good adaptability to feed processing and stability in industrial applications.

[0164] 9. Experiment 8: Storage stability test.

[0165] (1) Purpose of the experiment The changes in surface-active peptide content, physical properties, and emulsifying function of the bio-emulsifier composition of this application under different storage conditions were evaluated to verify its storage stability.

[0166] (2) Test materials The product from Example 1 was sealed in an aluminum foil bag.

[0167] (3) Test conditions Set the following storage conditions: Long-term storage conditions: 25℃±2℃, relative humidity 60%±5%; Accelerated storage conditions: 40℃±2℃, relative humidity 75%±5%; Kaifeng exposure conditions: 25℃, relative humidity 75%; The long-term storage testing time points were selected as 0, 1, 3, 6, 9, and 12 months; Accelerated storage testing time points were selected as 0, 7, 14, 30, 60, and 90 days; The exposure detection time points in Kaifeng were selected as 0, 3, 7, 14, and 30 days.

[0168] (4) Detection indicators At each storage time point, the samples were tested as follows: Active ingredient detection: The content of surface-active peptides was determined by HPLC; Physical property testing: including appearance, color, odor, moisture, agglomeration rate, flowability, and particle size change; Functional testing: Its ability to promote the enzymatic hydrolysis of fats and oils was evaluated using an in vitro enzymatic hydrolysis method.

[0169] Storage retention rate is calculated using the following formula: Storage retention rate (%) = content of surfactant peptides at a certain time point / initial content of surfactant peptides × 100%.

[0170] (5) Results Under long-term storage, accelerated storage, and open-pack exposure conditions, the content of surfactant peptides, moisture, clumping, and emulsifying function were tested according to the detection time under each storage condition. The results are summarized in the table below: Table 12 Product stability test results under long-term storage conditions at room temperature

[0171] Table 13 Product stability test results under accelerated storage conditions

[0172] The results showed that after 12 months of sealed storage at room temperature, the surface-active peptide content in the bio-emulsifier composition of this application could still reach about 98.4%, and the function retention rate was about 98.1%; after 90 days of accelerated storage, the content retention rate could still reach about 96.7%, showing good storage stability.

[0173] (6) Conclusion The above results indicate that the bio-emulsifier composition of this application has good component stability and functional stability under long-term storage at room temperature, accelerated storage, and exposure after opening. Moreover, the particles are not prone to moisture absorption and clumping, indicating that the composition has good shelf life suitability and end-use stability, as well as good storage stability and commercial application prospects.

[0174] Experiments 6-8 demonstrate that the bio-emulsifier composition provided in this application not only improves oil emulsification and promotes oil enzymatic hydrolysis, but also exhibits high retention rates of effective components and functions during pelleting, simulated high-temperature short-time pelleting treatment, and storage. Therefore, the product of this application combines emulsification advantages, processing adaptability, and storage stability, meeting the actual production, storage, transportation, and end-use requirements of the feed industry.

[0175] 10. Experiment 9: Effects of adding the bio-emulsifier composition of this application to feed on grass carp growth, feed utilization and lipid metabolism.

[0176] (1) Experimental methods This experiment designed five groups of feeds: a high-grade control (HC), a low-grade high-oil control (LHC), a low-grade low-oil control (LLC), a low-grade low-oil bio-emulsifier 1 (LLH5), and a low-grade low-oil bio-emulsifier 2 (LLH1). Five isonitrogenous and isoenergetic experimental feeds were prepared and a 9-week breeding trial was conducted.

[0177] (2) Experimental feed Five feed groups were designed: high-grade control (HC), low-grade high-oil control (LHC), low-grade low-oil control (LLC), low-grade low-oil emulsifier 1 (LLH5), and low-grade low-oil emulsifier 2 (LLH1). The feed formulations are shown in Table 14.

[0178] Table 14 Feed Formulation Table

[0179] (3) Experimental grouping and aquaculture management The experiment was conducted in an indoor recirculating aquaculture system at the Biotechnology Research Institute of Beijing Yinghuier Biotechnology Co., Ltd. Before the experiment, the fish were temporarily held in the aquaculture system for two weeks, during which time they were fed the control group feed. Healthy, uniformly sized grass carp (initial weight 46±0.00g) were randomly selected. Each treatment had four replicates, with 30 fish per replicate. Each tank was fed four times daily to full capacity for a nine-week period. The daily feed intake was recorded.

[0180] (4) Detection indicators Test the following indicators: Growth indicators: final average weight, weight gain rate, specific growth rate, feed intake rate, feed conversion ratio, survival rate; Body shape indicators: fatness, visceral-to-body ratio, liver-to-body ratio; Serum biochemical indicators: total protein (TP), albumin (ALB), triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). Liver fat content; Intestinal lipase (LPS) activity.

[0181] (5) Data statistics Experimental data are expressed as mean ± standard error and analyzed using one-way ANOVA (SPSS 17.0). When differences were significant, Duncan's method was used for multiple comparisons, with P < 0.05 as the criterion for significance.

[0182] (6) Test results 1) The effect of the bio-emulsifier composition of this application on the growth performance of grass carp The effects of adding the bio-emulsifier composition of this application to the feed on the body morphology indicators of grass carp are shown in Table 15. As can be seen from Table 15, compared with the high-grade feed and low-grade high-oil feed groups, the low-grade low-oil feed group showed significantly lower final average weight, weight gain rate, and specific growth rate (P<0.05). Compared with the high-grade feed group, the low-grade low-oil feed group showed significantly higher feed intake and feed conversion ratio (P<0.05). Adding the bio-emulsifier composition of this application significantly promoted growth and reduced the feed conversion ratio (P<0.05), with no significant difference compared to the high-grade feed group (P>0.05). There was no significant difference in survival rate among the groups (P>0.05).

[0183] Table 15 Summary of the effects of adding the bio-emulsifier composition of this application to the feed on the body shape indicators of grass carp

[0184] Note: Different lowercase letters in the same row of data indicate significant differences (P<0.05); no subscript or the same subscript indicates no significant differences (P>0.05).

[0185] The calculation formulas for each indicator in Table 15 are as follows: Weight gain rate = 100 × (Fish final weight - Fish initial weight) / Fish initial weight; Specific growth rate = 100 × (LN(final average weight) - LN(initial average weight)) / number of days; Feeding rate = 100 × feed intake / [(initial weight of fish + final weight of fish) / 2] / number of days; Feed conversion ratio = feed intake / (final body weight of fish - initial body weight of fish); Survival rate = 100 × final number of fish / initial number of fish.

[0186] 2) The effect of the bio-emulsifier composition of this application on the body shape index of grass carp Experiments showed that adding the bio-emulsifier composition of this application to the feed had no significant effect on the body shape indicators of grass carp (P>0.05), and the specific results are shown in the table below.

[0187] Table 16 Summary of the effects of adding the bio-emulsifier composition of this application to the feed on the body shape indicators of grass carp

[0188] The calculation formulas for each indicator in the table above are as follows: Fatness = 100 × body weight / body length 3; Visceral weight to body weight ratio = 100 × visceral weight / body weight; Liver-to-body weight ratio = 100 × liver weight / body weight.

[0189] 3) The effect of the bio-emulsifier composition of this application on the biochemical indicators of grass carp serum. The effects of adding the bio-emulsifier composition of this application to the feed on the serum biochemical indicators of grass carp are shown in Table 17.

[0190] Table 17 Summary of the effects of adding the bio-emulsifier composition of this application to the feed on the serum biochemical indicators of grass carp.

[0191] Note: Different lowercase letters in the same row of data indicate significant differences (P<0.05); no subscript or the same subscript indicates no significant differences (P>0.05).

[0192] As shown in the table above, different feed intakes had no significant effect on serum TP, ALB, TC, and LDL-C levels in grass carp (P>0.05). Compared with the high-grade feed group, the serum TG and HDL-C levels in the low-grade, low-oil feed group were significantly lower (P<0.05). After adding the bio-emulsifier composition of this application, the TG level further decreased, and the HDL-C level increased, showing no significant difference compared with the high-grade feed group (P>0.05).

[0193] 4) The effect of the bio-emulsifier composition of this application on the fat content of grass carp liver. The fat content of grass carp liver was determined, and the test results are as follows: Figure 7 As shown.

[0194] from Figure 7As can be seen, the addition of the bio-emulsifier composition of this application to the feed has a significant effect on the liver fat content of grass carp (P<0.05). Grass carp fed with the bio-emulsifier composition of this application (LLH5 and LLH1 groups) had significantly lower liver fat content than the high-grade feed group (HC) and the low-grade high-oil feed group (LHC) (P<0.05).

[0195] 5) The effect of the bio-emulsifier composition of this application on the intestinal lipase activity of grass carp. The intestinal lipase activity of grass carp was measured, and the test results are as follows: Figure 8 As shown. Figure 8 In the graph, the vertical axis represents the enzyme activity level per unit amount of protein. From... Figure 8 As can be seen, the lipase activity in LLH1 was significantly higher than that in other groups (P<0.05), indicating that adding 0.1% of the bio-emulsifier composition of this application to the feed can significantly improve the intestinal lipase activity of grass carp.

[0196] Therefore, it can be seen that adding the bio-emulsifier composition described in this application to low-grade, low-oil feed can significantly promote the growth of grass carp, reduce the feed conversion ratio, and improve blood lipid-related indicators, manifested as a decrease in serum TG content and an increase in HDL-C content; at the same time, the composition can also reduce fat accumulation in the liver of grass carp and increase intestinal lipase activity, thus having multiple combined effects.

[0197] 11. Experiment 10: Effect of adding the bio-emulsifying composition of this application to feed on the growth performance and lipid metabolism of broilers.

[0198] (1) Experimental objective This study investigates the effects of adding different levels of the bio-emulsifier composition of this application to broiler diets on broiler growth performance and lipid metabolism indicators, thereby evaluating the effect of adding the product of this application to broiler diets.

[0199] (2) Experimental methods The experiment used 648 one-day-old male AA broiler chicks with an initial weight of 42.43 ± 0.25 g, which were randomly divided into 6 treatments, with 6 replicates per treatment and 18 chicks per replicate. The experiment lasted for 42 days, divided into two phases: an early phase (0–21 days) and a late phase (22–42 days). The experiment used a corn-soybean meal basal diet, and each treatment group was supplemented with 0, 50, 100, 150, 200, and 250 mg / kg of the bio-emulsifier composition provided in this application, respectively.

[0200] (3) Basic diet and feeding management The basal diet was formulated according to the nutritional requirements of broilers. The experiment used a corn-soybean meal basal diet, with oil sources being soybean oil / vegetable oil. Feeding conditions and immunization programs were carried out as usual, and feed and water were available freely. The composition of the diet is shown in Table 18.

[0201] Table 18. Composition of Basal Diet (Basal Feeding, %)

[0202] Note:¹ Each kilogram of premixed feed provides: Vitamin A 9000 IU; Vitamin D3 3000 IU; Vitamin E 24 mg; Vitamin K3 1.8 mg; Vitamin B1 2.0 mg; Riboflavin 5.0 mg; Vitamin B6 3.0 mg; Vitamin B... 12 0.1mg; Niacin 40mg; Pantothenic acid 15mg; Folic acid 1.0mg; Biotin 0.05mg; Choline chloride 500mg; Iron 80mg; Copper 20mg; Manganese 90mg; Zinc 90mg; Iodine 0.35mg; Selenium 0.30mg.

[0203] (3) Measurement indicators and methods 1) Growth performance and mortality rate At the start of the experiment, the initial body weight of the broilers was measured. At the end of each feeding stage at 21 days and 42 days, they were weighed on an empty stomach in the morning, and their feed consumption was recorded. The average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (F:G) were calculated for 0-21 days, 22-42 days, and 0-42 days. The growth and mortality of the chickens were observed and recorded daily, and the mortality rate was calculated.

[0204] 2) Lipid metabolism indicators On days 21 and 42 of the experiment, six chickens were randomly selected from each treatment (one chicken per replicate). 10 mL of non-anticoagulated blood was collected from the wing vein using a vacuum blood collection tube. The blood was incubated at room temperature at an angle for 30 min, then centrifuged at 3000 rpm for 10 min to prepare serum. The serum was then stored at -20°C for later use. Lipid metabolism parameters, including total cholesterol (TC), total triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and very low-density lipoprotein cholesterol (vLDL-C), were measured using a fully automated biochemical analyzer (Mindray BS-420, Shenzhen Mindray Bio-Medical Electronics Co., Ltd.). The kits were manufactured by Sinopharm Biotech Co., Ltd.

[0205] (4) Results and Analysis 1) Growth performance and mortality rate The effects of adding different levels of bio-emulsifier compositions to the diet on the growth performance of broilers are shown in Table 19.

[0206] Compared with the control group, the addition of 100-250 mg / kg of the bio-emulsifier composition to the diet significantly reduced the fat-to-growth ratio (F:G) of broilers on day 21 (P<0.01), and the addition of 100-200 mg / kg of the bio-emulsifier composition significantly reduced the F:G of broilers from day 0-42 (P<0.05). With increasing levels of the bio-emulsifier composition in the diet, the F:G of broilers from day 0-21 showed a significant linear and quadratic decrease (P<0.01), and the F:G of broilers from day 0-42 showed a significant quadratic decrease (P<0.01). Throughout the experiment, the mortality rate of broilers in all treatment groups was less than 2%. These results indicate that the addition of 100-200 mg / kg of the bio-emulsifier composition to the diet can significantly improve the growth performance of broilers.

[0207] Table 19. Summary of Results: Effects of Different Levels of Bioemulsifier Compositions Added to Diets on Growth Performance of Broilers¹

[0208] Note: 1 Different lowercase letters in the shoulder labels indicate significant differences (P<0.05). ADG, average daily weight gain; ADFI, average daily feed intake; F:G, feed consumption to weight gain ratio.

[0209] 2) Fat metabolism The effects of adding different levels of bio-emulsifier compositions to the diet on fat metabolism in broilers are shown in Table 20.

[0210] With the increase in the level of biological emulsifier added to the diet, the serum TG content of broilers showed a significant linear decrease on day 21 of the experiment (P<0.05). This indicates that the product of this application can improve the lipid metabolism status of broilers.

[0211] Table 20 Summary of the effects of different levels of bioemulsifier compositions added to diets on fat metabolism in broilers

[0212] (5) Conclusion This experiment shows that the bio-emulsifier composition of this application has the effect of improving growth performance and regulating lipid metabolism (reducing serum TG) in broiler diets, and can be used to improve the utilization efficiency of animal fats and improve production performance.

[0213] Unless otherwise stated, all percentages mentioned in the embodiments of this application are mass percentages; the unit of acid value is mg KOH / g; D10, D50, and D90 are volume distribution particle sizes in μm; the effective component retention rate is the ratio of the surface-active peptide content of the treated sample to the surface-active peptide content of the untreated sample; the enzymatic hydrolysis promoting function retention rate is the ratio of the acid value increase of the treated sample to the acid value increase of the untreated sample; the acid value increase is the difference between the final acid value and the initial acid value. In animal experiments, different lowercase letters in the superscript of the same row of data indicate significant differences (P<0.05).

[0214] Therefore, as demonstrated by the above experiments, the bio-emulsifier composition provided in this application has at least one of the following beneficial effects: (1) By controlling the fatty acid chain length distribution of homologues in surfactant peptides, C13-C15 surfactant peptide homologues become the main components, and C14 surfactant peptide homologues are further made to be in the dominant proportion range. Thus, the hydrophilic-hydrophobic balance and oil-water interface adsorption capacity of surfactant peptides can be optimized at the molecular composition level, so that they form a chyme system with smaller particle size and more uniform distribution in the oil-water phase system, with D50 not higher than 3.0 μm, about 1.5~2.5 μm, for example, it can reach about 1.9 μm, thereby improving the emulsification effect and emulsion stability.

[0215] (2) It can promote the emulsification of oils and the enzymatic hydrolysis process of lipase. In vitro lipase hydrolysis experiments show that after adding the bio-emulsifier composition of this application, the release rate of acid value in the system is accelerated, and the acid value increases by no less than 1.5 times, and even no less than 1.8 times, and up to about 2 times. This indicates that it can increase the contact interface between oils and lipase, improve the efficiency of oil hydrolysis, and thus help improve the digestibility and utilization rate of dietary oils by animals. This effect is particularly suitable for feed systems containing vegetable oils, and preferably suitable for feed systems containing soybean oil.

[0216] (3) Compared with existing common feed emulsifiers such as bile acids, the bio-emulsifier composition of this application exhibits faster enzymatic hydrolysis promotion and better emulsification stability in in vitro models. Its technological contribution is not simply to replace existing emulsifiers, but to regulate interfacial activity at the molecular structure level by limiting the fatty acid chain length distribution of homologues in surfactant peptides, thereby achieving a synergistic improvement in emulsification performance and lipid enzymatic hydrolysis promotion effect.

[0217] (4) The bio-emulsifier composition of this application has shown good feeding effects in animal applications. Animal test results show that adding the bio-emulsifier composition of this invention to grass carp and broiler feed can reduce the feed conversion ratio and improve feed conversion efficiency, for example, the FCR can be reduced by about 3% to 5%; at the same time, it can reduce serum TG levels, increase HDL-C levels, increase lipase activity, and reduce liver fat accumulation. The above results indicate that the bio-emulsifier composition of this application can promote the digestion and absorption of dietary oils in animals, improve lipid metabolism, and support its role in improving oil utilization from a mechanistic perspective.

[0218] (5) This application prepares surfactant peptides with specific chain length distributions into granular feed formulations, giving the product both emulsifying activity and feed processing suitability. Granular products have good flowability, uniform mixing and convenient metering, which can reduce problems such as dust, clumping and uneven local addition, making them suitable for industrial addition in livestock, poultry and aquatic compound feeds by feed mills.

[0219] (6) It has good compatibility with the pelleting process. After pelleting, the effective components of surface-active peptides in the product are retained at a high rate and the loss of emulsifying activity is small, indicating that the bio-emulsifier composition can adapt to the feed pelleting process and maintain good functional stability after processing.

[0220] (7) It has good storage stability. Under normal temperature and accelerated storage conditions, the bio-emulsifier composition can maintain a high retention rate of effective ingredients and functional stability. It is not easy to have obvious moisture absorption and clumping or a decrease in emulsification performance, which is conducive to the stable application of the product in the process of feed production, transportation and storage.

[0221] In summary, this application achieves a superior bio-emulsifier composition with excellent oil-water interfacial activity by selecting surfactant peptides of specific chain lengths, with C13-C15 homologues as the main component and C14 homologues in a dominant proportion. This composition can form smaller, more uniform chylomicrons, improving the enzymatic hydrolysis efficiency of lipases on oily substrates. In animal feed applications, it exhibits effects such as promoting growth, reducing feed conversion ratio, improving lipid metabolism, and reducing hepatic fat accumulation. Furthermore, by preparing this bio-emulsifier composition into a granular feed formulation, it simultaneously possesses emulsifying properties, granulation processing stability, storage stability, and animal application effects, making it suitable for industrial application in livestock, poultry, and aquatic compound feeds.

[0222] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of this application (including the claims) is limited to these examples; within the framework of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of the embodiments of this application as described above, which are not provided in the details for the sake of brevity.

[0223] Furthermore, given that details have been set forth to describe exemplary embodiments of this application, it will be apparent to those skilled in the art that embodiments of this application may be practiced without these details or with variations thereof. Therefore, these descriptions should be considered illustrative rather than restrictive.

[0224] To simplify the description and discussion, and to avoid obscuring the embodiments of this application, well-known power / ground connections to other components may or may not be shown in the provided drawings. Furthermore, the apparatus may be shown in block diagram form to avoid obscuring the embodiments of this application, and this also takes into account the fact that the details of implementation of these block diagram apparatuses are highly dependent on the platform on which the embodiments of this application will be implemented (i.e., these details should be fully understood by those skilled in the art). While specific details have been set forth to describe exemplary embodiments of this application, it will be apparent to those skilled in the art that the embodiments of this application may be implemented without these specific details or with variations thereof. Therefore, these descriptions should be considered illustrative rather than restrictive.

[0225] Although this application has been described in conjunction with specific embodiments thereof, many substitutions, modifications, and variations of these embodiments will be apparent to those skilled in the art from the foregoing description. The embodiments of this application are intended to cover all such substitutions, modifications, and variations falling within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the embodiments of this application should be included within the protection scope of this application.

Claims

1. A bio-emulsifier composition, characterized in that, Including surfactant peptides; The surfactant peptides include C13-C15 surfactant peptide homologues; based on the total mass of the surfactant peptides, the total mass content of the C13-C15 surfactant peptide homologues is greater than or equal to 80%, and the mass content of the C14 surfactant peptide homologues is 50%-80%.

2. The bio-emulsifier composition according to claim 1, characterized in that, Also includes: Carriers, stabilizers, and excipients; The total mass of the surface-active peptides accounts for 0.5% to 20% of the total mass of the bio-emulsifier combination.

3. The bio-emulsifier composition according to claim 2, characterized in that, The carrier includes one or more of maltodextrin, corn starch, modified starch, soluble starch, wheat bran powder, and rice bran powder; The stabilizer includes one or more of the following: sodium carboxymethyl cellulose, xanthan gum, sodium alginate, chitosan, soybean polysaccharide, agar, guar gum, gum arabic, yeast cell wall, xylooligosaccharide, and sodium polyacrylate. The excipients include one or more of the following: stone powder, sodium sulfate, talc, silica, zeolite powder, bentonite, maifanite, and calcium carbonate.

4. The bio-emulsifier composition according to claim 1, characterized in that, The bio-emulsifier composition is in granular form, wherein the particle size is 0.1~1.0 mm.

5. A method for preparing a bio-emulsifier composition, characterized in that, include: Bacillus subtilis with accession number CGMCC No.24947 was fermented to obtain Bacillus subtilis fermentation broth; A bio-emulsifier composition was prepared using the Bacillus subtilis fermentation broth.

6. The preparation method according to claim 5, characterized in that, The preparation of the bio-emulsifier composition using the Bacillus subtilis fermentation broth includes: The inactivated Bacillus subtilis fermentation broth was added to a carrier for mixing and adsorption to obtain an adsorption solution. The adsorbent solution was spray-dried to obtain a powdered product; The powdered product is compounded with a carrier, stabilizer and excipient to obtain a bio-emulsifier composition.

7. The preparation method according to claim 6, characterized in that, Also includes: The compounded bio-emulsifier composition was granulated to obtain a granular bio-emulsifier composition.

8. The application of a bio-emulsifier composition, characterized in that, The bio-emulsifier composition according to any one of claims 1 to 4 is applied to animal feed.

9. The application according to claim 8, characterized in that, The animal feed also includes vegetable oils; The vegetable oils include one or more of soybean oil, rapeseed oil, corn oil, and palm oil.

10. The application according to claim 8, characterized in that, The bio-emulsifier composition is added to the animal feed at a rate of 50–500 g / t.

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