Vitamin-containing silkworm feed, and preparation method and application thereof

Abstract

This invention discloses a vitamin-containing silkworm feed, its preparation method, and its application. By weight, the raw materials of this silkworm feed include: 10-25 parts vitamins, 30-50 parts O-carboxymethyl-N-quaternized chitosan, 20-30 parts calcium silicate nano-precursor, 10-15 parts sodium alginate, and 5-15 parts nutritional support matrix; wherein the mass ratio of O-carboxymethyl-N-quaternized chitosan to calcium silicate nano-precursor is (1.2-1.8):1. By constructing an inorganic-organic hybrid microcapsule wall material based on a dynamic charge response mechanism, combined with an aqueous co-precipitation encapsulation process without organic solvents, the surface charge density of the microcapsules is adaptively adjusted according to the intestinal ionic strength, and the active ingredients are precisely controlled for release at specific physiological sites.

Description

Technical Field

[0001] This invention relates to the field of animal nutrition regulation technology, specifically to a vitamin-containing silkworm feed, its preparation method, and its application. Background Technology

[0002] In the fields of fine chemicals and nutrition for livestock, poultry, and economically important insects, microencapsulation technology is often used to encapsulate active components such as vitamins, thereby improving the stability of active substances and achieving targeted release. For example, existing technologies disclose a microcapsule system loaded with water-soluble and fat-soluble active substances. This system uses biodegradable polysaccharides as the wall material and releases the active substances from the core material through enzymatic hydrolysis and polymer chain breakage in the animal's digestive tract.

[0003] While this approach utilizes biopolymers for microencapsulation and provides basic sustained-release effects, it still has significant limitations. Firstly, the preparation process requires the use of organic solvents to dissolve the polymer raw materials, which can easily lead to solvent residues and fail to meet the safety requirements of the food chain and aquaculture products, making it unsuitable for the farming of sensitive economic insects like silkworms. Secondly, the chemical structure and surface charge of the microcapsule wall material are static, lacking the ability to dynamically respond to the ionic strength of the digestive tract, and the release process relies entirely on the action of intestinal microorganisms and enzymes.

[0004] The ionic strength of the midgut physiological environment of silkworms is about 120 mM. Traditional microcapsules have a fixed surface charge and cannot adjust the charge density in real time according to changes in the ionic strength of the environment. This not only easily leads to problems such as particle aggregation and being carried away by the intestinal mucus layer, but also causes active nutrients to leak prematurely in non-target areas or have low adsorption efficiency at the absorption target site. Ultimately, this results in poor bioavailability of nutrients and difficulty in accurately controlling the release behavior.

[0005] Therefore, existing technologies still have problems such as the inability of the surface charge of microcapsules to dynamically adapt to the physiological environment and the poor safety of the preparation process, which restrict the further development and application of efficient and safe silkworm microcapsule feed.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a vitamin-containing silkworm feed, its preparation method, and its application, thereby improving the aforementioned technical problems.

[0008] This invention is implemented as follows: In a first aspect, the present invention provides a vitamin-containing silkworm feed, the raw materials of which, by weight, include: 10-25 parts of vitamins, 30-50 parts of O-carboxymethyl-N-quaternized chitosan, 20-30 parts of calcium silicate nano-precursor, 10-15 parts of sodium alginate, and 5-15 parts of nutritional auxiliary matrix. The mass ratio of the O-carboxymethyl-N-quaternized chitosan to the calcium silicate nano-precursor is (1.2~1.8):1.

[0009] In an optional embodiment, the vitamins include fat-soluble vitamins and water-soluble vitamins, wherein the mass ratio of the fat-soluble vitamins to the water-soluble vitamins is 1:1 to 1:3.

[0010] In an optional embodiment, the fat-soluble vitamin is selected from one or more of vitamin A acetate, vitamin D3, and vitamin E; the water-soluble vitamin is selected from one or more of vitamin B1, vitamin B2, vitamin B6, and vitamin C.

[0011] In an optional embodiment, the calcium silicate nano precursor is amorphous calcium silicate particles with a median particle size of 50–80 nm.

[0012] In an optional embodiment, the nutritional supplement matrix includes mulberry leaf powder, soybean protein powder and mineral premix, wherein the mass ratio of the mulberry leaf powder, the soybean protein powder and the mineral premix is ​​(4-6):(3-5):1.

[0013] In an optional embodiment, the raw materials further include a food-grade emulsifier, wherein the amount of the food-grade emulsifier added is 1% to 5% of the mass of the vitamin.

[0014] Secondly, the present invention also provides a method for preparing the vitamin-containing silkworm feed described in the foregoing embodiments, comprising: The O-carboxymethyl-N-quaternized chitosan and sodium alginate were dissolved in deionized water, the pH of the system was adjusted to 4.5-6.0, and the mixture was stirred until homogeneous to obtain a biomacromolecule solution. The calcium silicate nano precursor was dispersed in deionized water and then ultrasonically dispersed to obtain an inorganic nano dispersion. The vitamins were emulsified and added to the biomacromolecule solution. The inorganic nano-dispersion was then added dropwise under stirring conditions. The ionic strength of the system was gradually increased to 80–150 mM to induce the formation of nascent microcapsules. The nascent microcapsule suspension was heated to 40-60°C and kept warm for maturation. The matured microcapsule suspension is dried, pulverized and sieved, then mixed with the nutrient-assisted matrix, moistened with water, extruded and granulated, and dried to obtain the finished product.

[0015] In an optional embodiment, the stirring temperature for obtaining the biomacromolecule solution is 40–50°C, and the stirring time is 30–45 minutes.

[0016] In an optional embodiment, the ultrasonic treatment has a power of 300–500 W and a frequency of 20–25 kHz.

[0017] In an optional embodiment, the emulsification speed is 8000–12000 rpm and the time is 3–5 minutes; In an optional embodiment, the inorganic nano-dispersion is added at a rate of 2 to 5 mL / min.

[0018] In an optional embodiment, the stirring speed during the heat preservation and maturation period is 400-600 rpm, and the heat preservation and maturation time is 1-2.5 hours.

[0019] In an optional embodiment, the microcapsule suspension is dried by spray drying or freeze drying; if spray drying is used, the inlet air temperature is controlled at 100-140°C; if freeze drying is used, the pre-freezing temperature is controlled at -40°C to -60°C.

[0020] In an optional embodiment, the pulverized and sieved material has a mesh size of 100 to 150.

[0021] In an optional implementation, the moisture content after wetting with water is controlled at 15-20%.

[0022] In an optional embodiment, the particle diameter obtained by extrusion granulation is 1.0 to 2.0 mm; the drying temperature after granulation is 50 to 60°C, and the drying is carried out until the moisture content is less than 10%.

[0023] Thirdly, the present invention also provides the application of the above-mentioned vitamin-containing silkworm feed in silkworm rearing.

[0024] This invention has the following beneficial effects: The technical solution of this invention breaks through the traditional research and development approach of single polymer encapsulation and enzymatic release in silkworm microencapsulated feed. It is the first to use specific amphoteric electrolyte biomacromolecules and inorganic nano-precursors with opposite charges as core raw materials. In a pure water system without organic solvents, co-precipitation is induced by precise control of pH and ionic strength to construct a dense organic-inorganic hybrid microcapsule wall with dynamic response characteristics. The charged groups on the surface of the microcapsule wall can fine-tune the degree of ionization and rearrange the molecular conformation according to the ionic strength of the medium. This maintains a dense and stable structure in the low ionic strength environment of the silkworm foregut, effectively locking in vitamin nutrients. This invention eliminates premature leakage from non-targeted areas. Under stimulation by specific ion intensity signals in the midgut, a sudden change in charge density occurs, rapidly altering the permeability of the cell wall material and even triggering local disintegration. This enables pulsed, explosive, targeted release of vitamins. Relying on the specific ratio and synergistic effect of each component, the inorganic nano-precursor simultaneously achieves cell wall toughening and charge sensing functions. Combined with flexible biomolecular segments that can adapt to environmental deformation, it effectively adapts to the complex intestinal physiological environment of silkworms, forming a unique stable-burst release dual-modal effect. This significantly improves the adsorption efficiency and bioavailability of nutrient targets—superior performance that cannot be achieved through simple component mixing. Furthermore, this invention uses an aqueous preparation process throughout, completely eliminating the risk of organic solvent residues and greatly improving breeding safety. It effectively solves the dual industry problems of nutrient leakage and inefficient targeted absorption in traditional microcapsules, while also reducing nutrient loss, strengthening the silkworm's constitution, and significantly improving silk quality and overall breeding economic benefits. It possesses strong practicality and application prospects. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0026] The following is a detailed description of a vitamin-containing silkworm feed provided by the present invention, its preparation method, and its application.

[0027] The ionic strength of the midgut fluid in silkworms typically fluctuates over a wide range (e.g., around 120 mM). Traditional fixed-charge microcapsules are prone to particle aggregation due to electrostatic shielding effects in this environment, or are non-specifically adsorbed and removed by the mucus layer due to charge mismatch. This leads to premature leakage of active substances such as vitamins in non-target areas, or insufficient release momentum upon reaching the target site. Conventional improvements in the industry often focus on optimizing polymer molecular weight or adjusting crosslinking degree, attempting to delay release by changing the physical thickness of the coating. However, this only linearly alters the diffusion rate and cannot fundamentally solve the core problem of "poor environmental adaptability." Furthermore, it often requires the introduction of more organic solvents to dissolve the polymer, further exacerbating the safety risks associated with solvent residue. Therefore, based on extensive research and practice, the following technical solution is proposed.

[0028] Some embodiments of the present invention provide a vitamin-containing silkworm feed, the raw materials of which, by weight, include: 10-25 parts of vitamins, 30-50 parts of O-carboxymethyl-N-quaternized chitosan, 20-30 parts of calcium silicate nano-precursor, 10-15 parts of sodium alginate, and 5-15 parts of nutritional auxiliary matrix. The mass ratio of O-carboxymethyl-N-quaternized chitosan to calcium silicate nano-precursor is (1.2~1.8):1.

[0029] The design employs a three-tiered functional formulation of "core-wall-matrix," using low-content, high-activity vitamins as the core functional phase, a high-proportion organic-inorganic hybrid system as the intelligent responsive wall phase, and a small amount of nutrient matrix as a carrier and palatability modifier. The wall component dominates the overall composition, effectively encapsulating the vitamin core and providing ample structural foundation and charge-active sites for aqueous self-assembly into capsules and the formation of dynamic charge-responsive structures. This avoids problems such as incomplete encapsulation, numerous microcapsule defects, and severe leakage caused by an excessively high core proportion. Therefore, this compositional design ensures complete microcapsule formation, dense wall layers, and high encapsulation efficiency, enabling long-term stable storage of vitamins in vitro and in the foregut environment. Simultaneously, it retains sufficient charge-responsive structural units to ensure efficient triggering of charge flipping and release behavior under midgut ion stimulation, balancing formability, stability, and intelligent responsive release performance.

[0030] O-Carboxymethyl-N-quaternized chitosan is an amphoteric polyelectrolyte that is predominantly net positively charged under low ionic strength, providing a flexible polymer framework and ionizable sensitive groups. Calcium silicate nano-precursors are nanoscale negatively charged amorphous particles that serve as a rigid reinforcing phase and a charge-response switch. A ratio of (1.2~1.8):1 ensures a slight excess of positive charge in the system, enabling strong electrostatic assembly under low ionic strength to form a dense and compact hybrid wall material. Simultaneously, sufficient carboxyl ionization response sites are reserved, allowing for significant charge neutralization, increased ionization, and molecular conformational rearrangement when the environmental ionic strength rises to the 80-150 mM range in the silkworm midgut, achieving a controllable charge density mutation. A ratio below 1.2:1 leads to excessive negatively charged particles, loose assembly, and premature material leakage; a ratio above 1.8:1 results in an excess of positive charge, incomplete charge reversal, and ineffective triggering of explosive release in the midgut. This allows for precise matching of the physiological ion thresholds in the silkworm's intestines, achieving a dual-modal effect of highly stable storage in the foregut and step-like explosive release in the midgut.

[0031] In some implementations, the vitamins include fat-soluble vitamins and water-soluble vitamins, with a mass ratio of 1:1 to 1:3. The silkworm's growth, molting, and silk protein synthesis processes simultaneously rely on both fat-soluble vitamins (participating in cell membrane synthesis, antioxidation, and growth regulation) and water-soluble vitamins (participating in metabolism, enzyme activity regulation, and energy cycling). By limiting the compounding ratio, the compatibility ratio between the oil-phase core material and the aqueous system is balanced, avoiding nutritional deficiencies in a single vitamin system. Simultaneously, it adapts to the emulsification and embedding process, ensuring a stable oil-water interface and facilitating the formation of a complete microcapsule structure. This comprehensively covers the nutritional needs of the silkworm throughout its entire growth cycle, balancing growth and development with improved silk quality; at the same time, it optimizes the emulsification performance of the core material, improving the uniformity and embedding rate of microcapsule embedding, avoiding nutritional deficiencies and molding defects caused by a single vitamin.

[0032] For example, fat-soluble vitamins are selected from one or more of vitamin A acetate, vitamin D3, and vitamin E; water-soluble vitamins are selected from one or more of vitamin B1, vitamin B2, vitamin B6, and vitamin C. The selection of these components can precisely address the vitamin deficiencies in conventional silkworm feed, significantly improving silkworm survival rate, uniformity, stress resistance, and silk yield and quality.

[0033] In some embodiments, the calcium silicate nano-precursor is amorphous calcium silicate particles with a median particle size of 50–80 nm. Compared to crystalline calcium silicate, amorphous calcium silicate possesses higher surface activity, more surface charge sites, and better aqueous dispersibility, enabling stronger electrostatic adsorption and interfacial co-precipitation with modified chitosan. The 50–80 nm nanoparticle size exhibits excellent interfacial filling characteristics, filling the gaps between polymer chains and significantly improving the density of the hybrid wall material. Simultaneously, the nanosize effect endows it with extremely high ion sensitivity, allowing it to function as a precise charge-response switch. Excessively large particle sizes can lead to uneven dispersion and wall material defects; excessively small particle sizes can cause agglomeration, affecting assembly uniformity. This approach significantly improves the mechanical strength and density of the microcapsule wall, reducing non-targeted leakage; and ensures that the microcapsules are highly sensitive to changes in ion intensity, achieving precise, rapid, and thorough pulsed release.

[0034] In some embodiments, the nutritional supplement matrix includes mulberry leaf powder, soybean protein powder, and mineral premix, with a mass ratio of (4-6):(3-5):1. Mulberry leaf powder provides the silkworm with its original flavor and natural active substances, improving palatability and feed intake; soybean protein powder provides high-quality plant protein to meet the silkworm's growth and silk protein synthesis needs; and the mineral premix supplements calcium, phosphorus, and trace elements, balancing osmotic pressure and enzyme activity. This formulation balances flavor, protein nutrition, and minerals, adapting to the growth and metabolic patterns of silkworms at different ages. It improves feed palatability and feed intake efficiency, balances the basic nutritional structure, helps improve the robustness of silkworm growth, and forms a synergistic effect with the slow-release vitamin function, achieving both improved quality and increased yield.

[0035] In some embodiments, the raw materials also include food-grade emulsifiers, with the amount of food-grade emulsifier added being 1% to 5% of the vitamin mass. The difference in oil-water interfacial tension between fat-soluble and water-soluble complex vitamin systems easily leads to uneven dispersion, oil droplet aggregation, and uneven core material distribution. An appropriate amount of food-grade emulsifier can reduce the oil-water interfacial tension, enabling the complex vitamin system to form uniform and stable ultrafine emulsion droplets, providing an excellent interfacial foundation for subsequent oil-water interface self-assembly and uniform encapsulation. Too little emulsification results in incomplete emulsification and uneven core material particle size; too much emulsifier can cause an excessively thick interfacial film, interfering with charge response and assembly effects. Therefore, the addition of a specific amount of emulsifier significantly improves the emulsification uniformity and microcapsule embedding regularity of the vitamin core material, increases the embedding rate, reduces empty and broken capsule defects, and ensures batch stability and consistent release.

[0036] Some embodiments of the present invention also provide a method for preparing the vitamin-containing silkworm feed described in the foregoing embodiments, comprising: S1. Dissolve O-carboxymethyl-N-quaternized chitosan and sodium alginate in deionized water, adjust the pH of the system to 4.5-6.0, and stir until homogeneous to obtain a biomacromolecule solution.

[0037] This step yields a highly homogeneous aqueous solution of biomacromolecules in terms of composition, charge, and concentration, ensuring consistency with subsequent assembly with inorganic precursors and reducing microcapsule structural defects. The pH of the system is adjusted to 4.5–6.0. ​​This weakly acidic environment allows O-carboxymethyl-N-quaternized chitosan to be fully protonated, stably exhibiting a net positive charge, while simultaneously ensuring that sodium alginate ionizes into a negatively charged form. Both are electrically stable and do not easily aggregate, laying the charge foundation for subsequent electrostatic self-assembly. A pH deviation from this range will cause abnormal macromolecular charge and decreased solubility.

[0038] In some embodiments, the stirring temperature for obtaining the biomacromolecule solution is 40–50°C, and the stirring time is 30–45 minutes. Appropriate heating can reduce the solution viscosity, accelerate the dissolution rate of the two macromolecules, ensure that the molecules are fully expanded and uniformly dispersed, and avoid localized colloids and agglomeration; sufficient stirring time ensures system homogeneity and eliminates deviations in concentration and charge distribution.

[0039] S2. The calcium silicate nano precursor is dispersed in deionized water and ultrasonically dispersed to obtain an inorganic nano dispersion.

[0040] The above operations can obtain a monodisperse inorganic nano-dispersion liquid, which fully exposes the interfacial activity and charge sites of the particles, resulting in more uniform subsequent binding with macromolecules, and batch-to-batch stability of the microcapsule wall structure and charge response performance.

[0041] Specifically, the power of the ultrasonic treatment is 300–500 W, and the frequency is 20–25 kHz.

[0042] Calcium silicate nano precursors have a large specific surface area and are prone to particle aggregation under normal conditions. Ultrasound with selected power and frequency can break up aggregated particles through cavitation effect and mechanical shear force, so that 50-80 nm amorphous particles are stably dispersed in water as single particles. If the frequency is too low, the dispersion will be incomplete and the particles will clump together; if the frequency is too high, it will easily cause particle breakage, abnormal particle size, and damage to the nano-response characteristics.

[0043] S3. After emulsifying the vitamins, add them to the biological macromolecule solution. Under stirring conditions, add the inorganic nano-dispersion dropwise, and control the ionic strength of the system to gradually increase to 80-150 mM to induce the formation of nascent microcapsules.

[0044] Specifically, the emulsification speed is 8000–12000 rpm, and the time is 3–5 minutes; the rate of adding the inorganic nano-dispersion is 2–5 mL / min.

[0045] High-speed, high-shearing can shear fat-soluble and water-soluble complex vitamins into fine, uniform emulsion droplets, forming a stable oil-water interface that serves as the microcapsule core. Insufficient speed and duration can lead to larger droplets, uneven distribution, and incomplete encapsulation.

[0046] The slow droplet addition of inorganic nano-dispersion allows inorganic nanoparticles to gradually enter the system, with a steady gradient increase in ionic strength, avoiding sudden changes in local ion concentration that could lead to instantaneous aggregation and large-scale precipitation; it also ensures that the wall material components undergo orderly electrostatic adsorption, co-precipitation, and dynamic self-assembly at the vitamin droplet interface.

[0047] The ionic strength of the control system is 80–150 mM, which matches the physiological ionic environment threshold of the silkworm midgut. This is not only the critical condition for the in-situ generation of dense hybrid microcapsule walls, but also allows the wall material to pre-construct a complete ion-charge response structure.

[0048] By controlling the above parameters, the vitamin core material can be made uniform in particle size, with regular microcapsule core-shell structure and high encapsulation rate; the nascent microcapsules are fully formed without large flocs, and a hybrid interface with ion-responsive capability is pre-constructed.

[0049] S4. Heat the nascent microcapsule suspension to 40-60℃ and keep it warm to mature.

[0050] Specifically, the stirring speed during the heat preservation and maturation period is 400-600 rpm, and the heat preservation and maturation time is 1-2.5 hours.

[0051] Gentle heating promotes further entanglement and cross-linking of macromolecular segments, strengthens the organic-inorganic interface bonding, and allows the amorphous calcium silicate precursor to be transformed in situ into stable nanoparticles, gradually densifying the capsule walls. Excessive temperature can destroy vitamin activity, while insufficient temperature results in slow cross-linking reactions and porous wall materials. Low-speed stirring prevents microcapsules from settling and sticking together, ensuring uniform heat and mass transfer. Sufficient maturation time is crucial to ensure the complete directional alignment of charge-responsive groups and lock in dynamic charge regulation performance; insufficient time leads to unstable structure and response performance, while excessive time increases energy consumption and reduces production efficiency.

[0052] S5. The matured microcapsule suspension is dried, pulverized and sieved, then mixed with the nutrient-assisted matrix, moistened with water and extruded into granules, and dried to obtain the finished product.

[0053] In some embodiments, the microcapsule suspension is dried using spray drying or freeze drying; if spray drying is used, the inlet air temperature is controlled at 100–140°C; if freeze drying is used, the pre-freezing temperature is controlled at -40°C to -60°C. The pulverized and sieved material is 100–150 mesh. The moisture content after wetting is controlled at 15–20%. The particle diameter obtained by extrusion granulation is 1.0–2.0 mm; the drying temperature after granulation is 50–60°C, and drying is carried out until the moisture content is below 10%.

[0054] Spray drying utilizes high-temperature hot air for rapid dehydration, resulting in high production efficiency and suitability for large-scale production. The 100–140℃ temperature range minimizes thermal degradation of heat-sensitive vitamins while achieving rapid drying. Freeze-drying, on the other hand, operates in a consistently low-temperature environment, providing optimal protection for the activity of various vitamins and making it suitable for products with high activity requirements. Temperatures of -40 to -60℃ ensure complete freezing of the system, preventing material melting and structural collapse. Both methods yield loose microcapsule powders, fully preserving the original structure and charge response characteristics of the microcapsules.

[0055] The screen mesh size limits the powder particle size, which can remove lumps and large particles and ensure that the microcapsule powder has a uniform particle size. This facilitates uniform mixing with the subsequent nutritional supplement matrix and ensures that the final feed pellets dissolve and release consistently.

[0056] Choosing the appropriate moisture content after wetting can give the powder suitable plasticity, making it easy to form during extrusion granulation and preventing the particles from breaking easily; if the moisture content is too low, the material will be dry and difficult to form, while if the moisture content is too high, the particles will be soft and easy to stick together, increasing the subsequent drying load.

[0057] A particle diameter of 1.0–2.0 mm is suitable for the feeding characteristics of silkworm mouthparts, ensuring good palatability and smooth feeding; if the particle size is too large, feeding becomes difficult, and if it is too small, dust is generated and losses increase.

[0058] Low-temperature drying after granulation avoids high-temperature damage to vitamins and microcapsule structures; the final moisture content is controlled below 10%, which can prevent mold, clumping, and microbial growth during feed storage, extend shelf life, and ensure product storage stability.

[0059] Some embodiments of the present invention also provide the application of the above-mentioned vitamin-containing silkworm feed in silkworm rearing.

[0060] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0061] Example 1 This embodiment provides a vitamin-containing silkworm feed, the raw materials of which, by weight, include the following components: 18 parts vitamins, 36 parts O-carboxymethyl-N-quaternized chitosan, 24 parts calcium silicate nano-precursor, 12 parts sodium alginate, and 10 parts nutritional supplement matrix.

[0062] The vitamins are composed of fat-soluble vitamins (equal masses of vitamin A acetate, vitamin D3, and vitamin E) and water-soluble vitamins (equal masses of vitamin B1, vitamin B2, vitamin B6, and vitamin C) in a 1:2 mass ratio. The nutritional supplement matrix includes 6 parts mulberry leaf powder, 3 parts soybean protein powder, and 1 part mineral premix.

[0063] The calcium silicate nano-precursor consisted of amorphous calcium silicate particles with an average particle size of 60 nm. The mineral premix was Sangbaojing-Silkworm Composite Mineral Premix (model: SY-M01) purchased from Jiangsu Haian Xinyuan Biopharmaceutical Co., Ltd. O-Carboxymethyl-N-quaternized chitosan was prepared by the following steps: Chitosan with a degree of deacetylation of 85% and a molecular weight of 150 kDa was placed in a 40% sodium hydroxide solution and alkalized at 40°C for 2 h to complete the activation of hydroxyl and amino groups. The mixture was then filtered and squeezed dry. The alkalized chitosan was dispersed in a 70% ethanol solution, and chloroacetic acid was added at a molar ratio of chitosan structural units to chloroacetic acid of 1:1.2. The mixture was heated to 55°C and reacted at this temperature for 3 h. After the reaction, the pH of the system was neutralized with glacial acetic acid. The mixture was dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 10,000 to remove small molecule impurities and salts. The resulting O-carboxymethyl chitosan was then freeze-dried. The obtained O-carboxymethyl chitosan was dissolved in deionized water, and the pH of the system was adjusted to 8.0. The mixture was then added at a molar ratio of O-carboxymethyl chitosan to 3-chloro-2-hydroxypropyltrimethylammonium chloride of 1:0.3. The quaternizing reagent was slowly added dropwise, and the reaction was stirred at 60°C for 2 h. After the reaction was completed, the mixture was dialyzed again for 24 h and then freeze-dried to finally obtain O-carboxymethyl-N-quaternized chitosan.

[0064] The preparation method of this embodiment includes the following steps: S1: Dissolve 36 parts of O-carboxymethyl-N-quaternized chitosan and 12 parts of sodium alginate in 900 parts of deionized water. Adjust the pH of the system to 5.0 using dilute hydrochloric acid or sodium hydroxide solution. Stir at 45°C for 35 minutes until completely dissolved to obtain a homogeneous and transparent biomacromolecule solution.

[0065] S2: Disperse 24 parts of calcium silicate nano precursor in 250 parts of deionized water, turn on an ultrasonic cell disruptor, and sonicate for 20 minutes at a power of 400 W and a frequency of 23 kHz to ensure that it is fully dispersed and free from agglomeration, thus obtaining a stable inorganic nano dispersion.

[0066] S3: 18 parts of vitamins were added to an appropriate amount of food-grade Tween-80 (2% of the vitamin mass), and emulsified for 4 minutes at 10,000 rpm in a high-speed shear emulsifier to form an oil-phase emulsion. This emulsion was slowly added to the biomacromolecule solution prepared in S1, while maintaining vigorous stirring (750 rpm). Subsequently, the inorganic nano-dispersion prepared in S2 was slowly added dropwise at a rate of 3 mL / min. During this process, the ionic strength of the system was monitored in real time, and the ionic strength was gradually increased to 115 mM by controlling the dropping rate and adding a small amount of sodium chloride solution. This ionic strength environment induced positively charged modified chitosan and negatively charged calcium silicate and sodium alginate to undergo dynamic self-assembly and co-precipitation at the oil-water interface, rapidly forming a dense primary microcapsule structure.

[0067] S4: The nascent microcapsule suspension obtained in S3 was transferred to a constant-temperature reactor, heated to 50°C, and aged for 1.8 hours. During this period, a gentle stirring state of 500 rpm was maintained to promote further densification of the inorganic-organic interface inside the wall material and to complete the directional arrangement of charge-responsive groups, thereby enhancing the structural stability of the microcapsules.

[0068] S5: The solidified microcapsule suspension is fed into a spray drying tower, with the inlet air temperature controlled at 120℃ and the outlet air temperature at 78℃ for instant drying; the dried material is then pulverized by airflow and passed through a 120-mesh sieve to obtain microcapsule powder with good flowability.

[0069] S6: Mix the obtained microcapsule powder with 10 parts of nutrient supplement matrix in a three-dimensional mixer for 25 minutes. Add an appropriate amount of deionized water to the mixture to moisten it (moisture content controlled at 18%), and granulate it using a twin-screw extruder. After extruding the strips, cut them into granules with a diameter of 1.5 mm using a pelletizer. Finally, place the wet granules in a fluidized bed dryer and dry them at a low temperature of 55℃ until the moisture content is less than 10%, thus obtaining the finished silkworm microcapsule feed.

[0070] Example 2 The only difference between this embodiment and Example 1 is that the raw materials, by weight, include the following components: 18 parts of vitamins, 45 parts of O-carboxymethyl-N-quaternized chitosan, 25 parts of calcium silicate nano-precursor, 12 parts of sodium alginate, and 10 parts of nutritional auxiliary matrix.

[0071] For other conditions and preparation methods, please refer to Example 1.

[0072] Example 3 The only difference between this embodiment and Example 1 is that the raw materials, by weight, include the following components: 22 parts of vitamins, 34 parts of O-carboxymethyl-N-quaternized chitosan, 28 parts of calcium silicate nano-precursor, 6 parts of sodium alginate, and 10 parts of nutritional auxiliary matrix.

[0073] For other conditions and preparation process, please refer to Example 1.

[0074] Example 4 The raw materials used in this embodiment are the same as those in Example 1. The only difference between this embodiment and Example 1 is that the preparation process is slightly different, specifically: In step S4, the nascent microcapsule suspension is heated to 58°C, and the curing time is shortened to 1.0 hour.

[0075] The operating parameters for the remaining steps S1, S2, S3, S5, and S6 are consistent with those in Example 1.

[0076] Example 5 The raw materials used in this embodiment are the same as those in Example 1. The only difference between this embodiment and Example 1 is that the preparation process is slightly different, specifically: In step S3, the dropping rate is controlled to gradually increase the ionic strength of the system to 150 mM.

[0077] The operating parameters for the remaining steps S1, S2, S4, S5, and S6 are consistent with those in Example 1.

[0078] Example 6 The only difference between this embodiment and Example 1 is that the raw materials, by weight, include the following components: 20 parts vitamins, 35 parts O-carboxymethyl-N-quaternized chitosan, 25 parts calcium silicate nano-precursor, 10 parts sodium alginate, and 10 parts nutritional supplement matrix. The nutritional supplement matrix consists of: 4 parts mulberry leaf powder, 5 parts soybean protein powder, and 1 part mineral premix.

[0079] For other conditions and preparation process, please refer to Example 1.

[0080] Example 7 The raw materials used in this embodiment are the same as those in Example 1. The only difference between this embodiment and Example 1 is that the preparation process is slightly different, specifically: In step S5, freeze-drying was used. The solidified microcapsule suspension was pre-frozen at -50°C for 5 hours, and then sublimated and dried under a vacuum of 15 Pa for 22 hours, with the desorption drying temperature controlled at 25°C.

[0081] The operating parameters for the remaining steps S1, S2, S3, S4, and S6 are consistent with those in Example 1.

[0082] Example 8 The raw materials used in this embodiment are the same as those in Example 1. The only difference between this embodiment and Example 1 is that the preparation process is slightly different, specifically: In step S1, adjust the pH of the solution to 6.0.

[0083] The operating parameters for the remaining steps S2, S3, S4, S5, and S6 are consistent with those in Example 1.

[0084] Example 9 The raw materials used in this embodiment are the same as those in Example 1. The only difference between this embodiment and Example 1 is that the preparation process is slightly different, specifically: In step S2, the ultrasonic treatment time is extended to 30 minutes and the power is increased to 500 W.

[0085] The operating parameters for the remaining steps S1, S3, S4, S5, and S6 are consistent with those in Example 1.

[0086] Comparative Example 1 The only difference between the raw materials in this comparative example and those in Example 1 is that, by weight, it includes the following components: 18 parts vitamins, 36 parts O-carboxymethyl-N-quaternized chitosan, 12 parts sodium alginate, and 10 parts nutritional supplement matrix.

[0087] The preparation process is different from that in Example 1 except that it does not include step S2, and there is no operation of adding inorganic nano-dispersion in step S3.

[0088] Comparative Example 2 The only difference between the raw materials in this comparative example and those in Example 1 is that, by weight, it includes the following components: 18 parts vitamins, 20 parts O-carboxymethyl-N-quaternized chitosan, 40 parts calcium silicate nano-precursor, 12 parts sodium alginate, and 10 parts nutritional support matrix.

[0089] For other conditions and preparation process, please refer to Example 1.

[0090] Comparative Example 3 The raw materials used in this comparative example are the same as those in Example 1. The only difference between this example and Example 1 is that the preparation process is slightly different, specifically: In step S1, the solvent is changed from "deionized water" to "ethanol-water mixed solvent (volume ratio 1:1)" to dissolve part of the O-carboxymethyl-N-quaternized chitosan.

[0091] In step S3, 18 parts of vitamins were added to an appropriate amount of food-grade Tween-80 (2% of the vitamin mass), and then emulsified at 10,000 rpm for 4 minutes in a high-speed shear emulsifier to form an oil phase emulsion. This emulsion was slowly added to the biomacromolecule solution prepared in step S1, while maintaining vigorous stirring (750 rpm), and an inorganic nano-dispersion was added. The system temperature was controlled at 25 ℃, and the stirring speed was maintained at 400 rpm. Food-grade acetone was measured at a 1:1 ratio to the total aqueous phase liquid volume of the system and added to the system all at once within 10 seconds. The mixture was stirred continuously for 6 minutes, allowing O-carboxymethyl-N-quaternized chitosan, sodium alginate, and calcium silicate nano-precursors to precipitate, deposit, and cross-link at the oil-water interface of the vitamin emulsion droplets, resulting in a nascent microcapsule suspension.

[0092] Comparative Example 4 The raw materials used in this comparative example are the same as those in Example 1. The only difference between this example and Example 1 is that the preparation process is slightly different, specifically: In step S4, the heat preservation temperature is reduced to 25°C and the heat preservation time is shortened to 0.3 hours.

[0093] Comparative Example 5 This comparison is based on commercially available products.

[0094] Product information is as follows: Manufacturer: Shanghai Novartis Animal Health Co., Ltd.; Product Name: Multivitamin Premix (General Type); Product Model / Brand: NH-Vit-Mix-2023.

[0095] Instructions for use: Mix directly into mulberry leaf powder and soybean meal matrix according to the recommended dosage in the product instructions to make feed pellets.

[0096] Experimental Example 1: Microcapsule Morphology Characterization and Vitamin Encapsulation Efficiency Determination Experimental equipment: Laser particle size analyzer: Model Mastersizer 3000, Manufacturer Malvern Panalytical; High performance liquid chromatograph (HPLC): Model Agilent 1260 Infinity II, Manufacturer Agilent Technologies.

[0097] The experimental method includes the following steps: S1: Take an appropriate amount of each sample and disperse it in deionized water. Use a laser particle size analyzer to determine its particle size distribution (D10, D50, D90) and polydispersity index (PDI). Each sample is measured in parallel 3 times and the average value is taken.

[0098] S2: Accurately weigh 0.5 g of each sample, add the cell wall-breaking enzyme solution (a mixture of cellulase and pectinase), and thoroughly hydrolyze the wall material in a 37℃ water bath to release all vitamins. Centrifuge and collect the supernatant, filter through a 0.22 μm filter membrane, and determine the total vitamin content (W_total) using HPLC. Separately, take an equal mass of unbroken samples, directly extract the free vitamins from the surface with methanol, and determine the surface vitamin content (W_surface).

[0099] S3: Calculate the encapsulation efficiency (EE%) using the formula: EE% = (W_total - W_surface) / W_total × 100%. Calculate the average encapsulation efficiency for fat-soluble and water-soluble vitamins respectively.

[0100] The experimental results are shown in Table 1.

[0101] Table 1 Regarding particle size, the D50 of Examples 1-9 is concentrated between 2.5-4.5 μm, and the PDI is less than 0.2, indicating good particle size uniformity. In contrast, Comparative Example 3 (organic solvent method) has a wide particle size distribution due to excessively fast precipitation rate, and the PDI is as high as 0.45 or more.

[0102] Encapsulation efficiency tests showed that the average encapsulation efficiency of vitamins in Examples 1-9 was between 88% and 94%, significantly higher than that of the comparative examples. In particular, Comparative Example 5 (a commercially available product) suffered significant loss of active ingredients during granulation due to a lack of effective encapsulation, with the actual effective content being only about 75% of the labeled amount. Example 1, with its optimized inorganic-organic ratio and suitable curing conditions, achieved the highest encapsulation efficiency (93.5%), demonstrating that the dynamic self-assembly process can effectively lock vitamins within the microcapsule core, reducing processing losses. Comparative Example 2, due to charge mismatch, experienced a significant drop in encapsulation efficiency to 65%, indicating that a specific mass ratio is crucial for forming a dense wall material.

[0103] Experimental Example 2: Study on Dynamic Release Behavior in Simulated Silkworm Digestive Tract Environment Experimental equipment: Intelligent drug dissolution apparatus: model RC8MS, manufacturer: Tianjin Tianda Tianfa Technology Co., Ltd.; Ion meter: model FE30-Standard, manufacturer: Mettler-Toledo; Ultraviolet-Visible spectrophotometer: model UV-2600, manufacturer: Shimadzu Corporation.

[0104] Experimental methods: This experiment simulates the foregut (low ionic strength) and midgut (high ionic strength) environments of silkworms to investigate the release characteristics of microcapsules and verify the "dynamic charge response" mechanism.

[0105] The above experimental method includes the following steps: S1: Prepare simulated silkworm foregut solution (SIF-I): pH 6.0, ionic strength 40 mM; prepare simulated silkworm midgut solution (SIF-M): pH 7.2, ionic strength 120 mM. Both solutions were preheated to 28±0.5℃ (silkworm body temperature).

[0106] S2: Take an amount of microcapsule powder equivalent to 10 mg of vitamin from each example and comparative example, and place it in the dissolution apparatus basket. First, soak it in SIF-I for 30 minutes to simulate the foregut passage process, and take samples every 5 minutes to determine the release amount.

[0107] S3: After 30 minutes, quickly replace the medium with SIF-M and continue incubation for 120 minutes to simulate the release process after entering the midgut. Take samples every 10 minutes to measure the release amount and replenish with an equal volume of medium at the same temperature.

[0108] S4: Plot the cumulative release curve, calculate the release rate constants for the two phases, and observe whether there is a "pulse" release characteristic (i.e., whether the release rate increases significantly after switching to SIF-M).

[0109] The experimental results are shown in Table 2.

[0110] Table 2 The experimental results show that Examples 1-9 exhibited extremely low release rates in SIF-I (foregut mimicry solution) (cumulative release <8% within 30 minutes), indicating that the microcapsules have excellent stability in non-target areas and can effectively prevent premature vitamin leakage. Upon transfer to SIF-M (midgut mimicry solution, ionic strength 120 mM), the release curves of Examples 1-9 immediately showed a steep "step" increase, with the cumulative release rapidly reaching 70%-85% within the following 30-40 minutes, exhibiting typical pulsatile release behavior. This confirms that the charge density on the microcapsule surface abruptly increased with increasing ionic strength, triggering a sudden increase in the permeability of the wall material.

[0111] In contrast, Comparative Example 1 (traditional static microcapsules) already exhibited approximately 15% leakage in SIF-I, and the release rate did not significantly improve after transfer to SIF-M, showing a slow, linear release lacking targeted burst power. Comparative Example 2, due to charge mismatch, underwent partial disintegration in SIF-I, resulting in disordered release. Comparative Example 5 (commercially available product) released extremely rapidly in SIF-I, exceeding 60% release within 30 minutes, causing most vitamins to be lost or destroyed before reaching absorption sites.

[0112] The pulse effect in Example 4 (high-temperature short-time curing) was slightly weaker than that in Example 1, indicating that sufficient curing is crucial for constructing a sensitive charge-responsive interface. The experimental data strongly demonstrate the unique advantages of the present invention in achieving a "stable-explosive" dual-mode release, which is impossible with existing static embedding techniques.

[0113] Experimental Example 3 Experiment Title: Silkworm Feeding Experiment and Bioavailability Evaluation Experimental equipment: Electronic balance: Model XS204, manufacturer Mettler-Toledo; Biochemical analyzer: Model AU5800, manufacturer Beckman Coulter; Constant temperature and humidity incubator: Model RXZ-300B, manufacturer Ningbo Jiangnan Instrument Factory.

[0114] The experimental method includes the following steps: S1: 600 healthy, uniformly developed fifth-instar silkworms were selected and randomly divided into 14 groups (corresponding to 9 examples and 5 comparative examples), with 3 replicates in each group and approximately 15 silkworms in each replicate.

[0115] S2: Each group was fed the feed prepared according to the corresponding embodiment or comparative example (using fresh mulberry leaves as a carrier, added in a uniform ratio). The rearing environment was controlled at a temperature of 24-26℃, a relative humidity of 70-75%, and a photoperiod of 12L:12D. Feeding continued for 5 days until the end of the five-year-old's peak feeding period.

[0116] S3: Record the weight gain of silkworms in each group and calculate the weight gain rate. Collect silkworm excrement and blood samples (5 silkworms randomly selected from each group).

[0117] S4: Determine the concentrations of vitamins A, E, and B vitamins in silkworm hemolymph and calculate bioavailability indicators. Simultaneously, statistically analyze the cocooning rate, cocoon layer ratio, and total cocoon weight for each group of silkworms.

[0118] The experimental results are shown in Table 3.

[0119] Table 3 Feeding results showed that silkworms fed with the feeds of Examples 1-9 had significantly higher weight gain rates during the fifth instar than the comparative groups. Specifically, the average weight gain rate of the Example 1 group reached 450%, significantly better than that of Comparative Example 1 (380%) and Comparative Example 5 (360%). This indicates that the microencapsulated feed of the present invention improves the efficiency of nutrient digestion and absorption.

[0120] Hemolymphatic vitamin concentration detection data showed that vitamin levels in silkworms in Examples 1-9 remained high and stable, especially the concentrations of fat-soluble vitamins A and E, which were 1.8-2.2 times higher than those in Comparative Example 5. This confirmed that the dynamically charged responsive microcapsules successfully achieved targeted release and efficient absorption of vitamins in the midgut. Comparative Examples 1 and 2, due to defects in their release mechanisms, exhibited large fluctuations in vitamin levels and were generally low.

[0121] Regarding cocoon production performance, the cocooning rate of groups 1-9 in Examples 1-9 was all above 96%, with a cocoon layer rate increase of 3-5 percentage points. However, in Comparative Example 4, due to poor microencapsulation stability, some vitamins were degraded before feeding, resulting in poor nutritional status of the silkworms and a cocooning rate of only 88%. While the commercially available product group (Comparative Example 5) could provide basic nutrition, its growth-promoting effect and cocoon quality improvement were significantly less than those of the embodiments of this invention due to the lack of targeted delivery capabilities.

[0122] In summary, this invention, through its unique dynamic charge response mechanism, not only protects vitamin activity but also precisely matches the physiological needs of silkworms, thereby achieving unexpected technological advancements in growth performance and economic benefits.

[0123] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A vitamin-containing silkworm feed, characterized in that, By weight, its raw materials include: 10-25 parts vitamins, 30-50 parts O-carboxymethyl-N-quaternized chitosan, 20-30 parts calcium silicate nano precursor, 10-15 parts sodium alginate, and 5-15 parts nutritional auxiliary matrix. The mass ratio of the O-carboxymethyl-N-quaternized chitosan to the calcium silicate nano-precursor is (1.2~1.8):

1.

2. The vitamin-containing silkworm feed according to claim 1, characterized in that, The vitamins include fat-soluble vitamins and water-soluble vitamins, and the mass ratio of the fat-soluble vitamins to the water-soluble vitamins is 1:1 to 1:

3.

3. The vitamin-containing silkworm feed according to claim 2, characterized in that, The fat-soluble vitamin is selected from one or more of vitamin A acetate, vitamin D3, and vitamin E; the water-soluble vitamin is selected from one or more of vitamin B1, vitamin B2, vitamin B6, and vitamin C.

4. The vitamin-containing silkworm feed according to claim 1, characterized in that, The calcium silicate nano precursor is amorphous calcium silicate particles with a median particle size of 50–80 nm.

5. The vitamin-containing silkworm feed according to claim 1, characterized in that, The nutritional supplement matrix includes mulberry leaf powder, soybean protein powder and mineral premix, and the mass ratio of the mulberry leaf powder, the soybean protein powder and the mineral premix is ​​(4-6):(3-5):

1.

6. The vitamin-containing silkworm feed according to claim 1, characterized in that, The raw materials also include food-grade emulsifiers, and the amount of food-grade emulsifiers added is 1% to 5% of the mass of the vitamins.

7. A method for preparing a vitamin-containing silkworm feed as described in any one of claims 1 to 6, characterized in that, It includes: The O-carboxymethyl-N-quaternized chitosan and sodium alginate were dissolved in deionized water, the pH of the system was adjusted to 4.5-6.0, and the mixture was stirred until homogeneous to obtain a biomacromolecule solution. The calcium silicate nano precursor was dispersed in deionized water and then ultrasonically dispersed to obtain an inorganic nano dispersion. The vitamins were emulsified and added to the biomacromolecule solution. The inorganic nano-dispersion was then added dropwise under stirring conditions. The ionic strength of the system was gradually increased to 80–150 mM to induce the formation of nascent microcapsules. The nascent microcapsule suspension was heated to 40-60°C and kept warm for maturation. The matured microcapsule suspension is dried, pulverized and sieved, then mixed with the nutrient-assisted matrix, moistened with water, extruded and granulated, and dried to obtain the finished product.

8. The method for preparing vitamin-containing silkworm feed according to claim 7, characterized in that, The stirring temperature for obtaining the biomacromolecule solution is 40–50°C, and the stirring time is 30–45 minutes. And / or, the power of the ultrasonic treatment is 300-500 W and the frequency is 20-25 kHz; And / or, the emulsification speed is 8000-12000 rpm, and the time is 3-5 minutes; And / or, the rate at which the inorganic nano-dispersion is added is 2–5 mL / min; And / or, the stirring speed during the heat preservation and maturation period is 400-600 rpm, and the heat preservation and maturation time is 1-2.5 hours.

9. The method for preparing vitamin-containing silkworm feed according to claim 7, characterized in that, The microcapsule suspension is dried by spray drying or freeze drying. If spray drying is used, the inlet air temperature is controlled at 100-140℃. If freeze drying is used, the pre-freezing temperature is controlled at -40℃ to -60℃. And / or, the pulverized and sieved mesh size is 100-150 mesh; And / or, the moisture content after moistening with water should be controlled at 15-20%; And / or, the particle diameter obtained by extrusion granulation is 1.0 to 2.0 mm; the drying temperature after granulation is 50 to 60°C, and the drying is carried out until the moisture content is less than 10%.

10. The application of the vitamin-containing silkworm feed as described in any one of claims 1 to 6 in silkworm rearing.

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