Preparation method of natural plant acid and protein compound for animal feed

Abstract

The invention discloses a preparation method of a natural plant acid and protein compound for animal feed, which comprises the following steps: grinding protein and natural plant acid into powder, and combining the natural plant acid with the protein by using a twin-screw extruder to prepare a composite material by adopting a solid-phase co-extrusion preparation technology. The protein-natural plant acid compound prepared by the invention not only has excellent bacteriostatic activity, but also can effectively solve the problem of oxidative deterioration of protein and improve the stability of the protein, and also can enable the natural plant acid to better exert efficacy under the action of a protein carrier and enhance the nutritional value and functional characteristics of the feed; meanwhile, the raw materials are simple and easy to obtain, the preparation method is simple, operability is high, popularization is easy, and a new resource and a new thought are provided for the antibiotic-replacing feed additive and application of the antibiotic-replacing feed additive.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal feed additives. More specifically, the present invention relates to a preparation method of a natural plant acid and protein complex for animal feed. Background Art

[0003] In modern animal husbandry, in order to effectively prevent animal diseases and promote the healthy growth of animals, feed additives are widely used. Antibiotic feed additives once played an important role. They can effectively prevent and treat bacterial infections in animals, and improve the growth rate and feed conversion rate of animals. However, with the long-term and large-scale use of antibiotics in feed, the drug resistance of bacteria to antibiotics has been continuously increasing, resulting in a gradual decline in the efficacy of antibiotics and an increase in the difficulty of disease prevention and treatment. At the same time, antibiotic residues are transmitted through the food chain, may accumulate in animals, and ultimately enter the human body, posing a potential threat to the health of consumers, such as causing human flora imbalance, allergic reactions, and even inducing the generation of drug-resistant bacteria in the human body.

[0004] Therefore, it has become an urgent task to develop new, safe, and efficient feed additives that can replace antibiotics.

[0005] As a green and environmentally friendly feed additive, natural plant acids have received extensive attention in recent years. It mainly regulates the pH value in the animal intestine, inhibits the growth and reproduction of harmful bacteria, and at the same time promotes the proliferation of beneficial bacteria, maintaining the intestinal microecological balance. In addition, some natural plant acids also have multiple functions such as antioxidant and immune enhancement, which helps to improve the health level and production performance of animals.

[0006] Protein is an important nutrient component in animal feed. Common feed proteins include soybean protein, cottonseed protein, rapeseed protein, peanut protein, etc. These protein sources are rich and have high nutritional value, but they are easily affected by oxidation during feed storage and animal digestion, resulting in the loss of nutritional components and a decline in feed quality. Combining natural plant acids with these proteins to prepare composite materials can not only effectively solve the problem of protein oxidation and deterioration, improve its stability, but also enable natural plant acids to better play their functions under the action of protein carriers, enhancing the nutritional value and functional characteristics of feed. Summary of the Invention

[0007] An object of the present invention is to solve at least the above problems and / or deficiencies, and provide at least the advantages described hereinafter.

[0008] To achieve these objects and other advantages of the present invention, there is provided a preparation method of a natural plant acid and protein complex for animal feed, including the following steps: Step 1: Grind the protein and natural plant acid into fine powders respectively, and sieve them for later use. Step 2: Mix the protein powder and natural plant acid powder in proportion and stir evenly, then add them into a rotary twin-screw extruder for extrusion. Dry the extrudate and grind it into powder to obtain the protein-natural plant acid complex.

[0009] Preferably, in Step 1, the protein is one of cottonseed protein, soybean protein, rapeseed protein, and peanut protein.

[0010] Preferably, in Step 1, the natural plant acid is one of shikimic acid, citric acid, lactic acid, and malic acid.

[0011] Preferably, in Step 2, the mass fraction ratio of the protein powder to the natural plant acid powder is 85-96.25%: 3.75-15%.

[0012] Preferably, in Step 2, the parameter settings of the twin-screw extruder are as follows: the feeding speed is 5-15 kg / h, the screw rotation speed is 100-300 rpm, the feeding moisture content is 15-25%, the temperatures of the five individual temperature control zones are 80-100 °C, 90-110 °C, 100-120 °C, 110-130 °C, 120-140 °C respectively, and the die temperature is 120-140 °C.

[0013] Preferably, in Step 2, before mixing the plant protein powder and natural plant acid powder, pre-treat the natural plant acid powder and protein powder respectively. The specific method is as follows: S1: Dissolve the natural plant acid powder in water, stir evenly, then add lecithin and ascorbic acid, disperse evenly by ultrasonic, and heat and evaporate to remove the solvent to obtain the co-blended and modified natural plant acid powder. S2: Disperse the protein powder in water, then add a small amount of fructose and chitosan, heat and stir at low temperature, and then dry under low temperature and vacuum to obtain the pre-treated protein powder.

[0014] Preferably, in S1, the mass ratio of the natural plant acid powder, water, lecithin, and ascorbic acid is 1: 10-12: 0.1-0.2: 0.01-0.05, the ultrasonic dispersion frequency is 20-40 kHz, the dispersion time is 15-20 min, and the heating and evaporation temperature is 60-75 °C.

[0015] Preferably, in S2, the mass ratio of the protein powder, water, fructose, and chitosan is 1: 15-20: 0.01-0.03: 0.05-0.1, the low-temperature heating temperature is 35-45 °C, the heating time is 4-8 h, and the vacuum drying is carried out at 10-15 °C for 3-6 h.

[0016] The present invention has at least the following beneficial effects: The protein-natural plant acid complex prepared by the present invention not only has excellent antibacterial activity, but also can effectively solve the problem of oxidative deterioration of proteins, improve their stability, and enable natural plant acids to better exert their effects under the action of protein carriers, enhancing the nutritional value and functional characteristics of feeds; The present invention also pre-treats plant protein powder and natural plant acid powder, adds a small amount of fructose and chitosan to the plant protein powder and heats it at low temperature to improve the stability and antioxidant capacity of plant proteins; At the same time, the lecithin and ascorbic acid added to natural plant acids can also enhance the antioxidant capacity to a certain extent, and since the added chitosan has good antibacterial ability, the antibacterial ability of the product is also improved; All the substances added above are edible substances with certain nutritional value and can additionally supplement nutrition for animals. In addition, the protein raw material used in the present invention is derived from plants and has the characteristics of high nutritional value, good palatability, high stability, diverse functions, good cost-effectiveness, sustainability, replaceability, promoting intestinal health, improving feed utilization rate, and reducing environmental pollution, and can be effectively applied to the field of animal feeds; The natural plant acid-protein complex for animal feeds prepared by the present invention can be used as an antibiotic-free feed additive for animal feeds. The raw materials are simple and easy to obtain, the preparation method is simple, the operability is strong, and it is easy to promote, providing a new resource and new idea for antibiotic-free feed additives and their applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a scanning electron microscope image (SEM) of the cottonseed protein-shikimic acid complex and cottonseed protein prepared in Example 1 of the present invention; Figure 2 It is an infrared spectrum diagram (FTIR) of the cottonseed protein-shikimic acid complex and cottonseed protein prepared in Example 1 of the present invention; Figure 3 It is an ultraviolet spectrum diagram (UV) of the cottonseed protein-shikimic acid complex and cottonseed protein prepared in Example 1 of the present invention; Figure 4 It is an endogenous fluorescence spectrum diagram of the cottonseed protein-shikimic acid complex and cottonseed protein prepared in Example 1 of the present invention; Figure 5 It is an antibacterial experiment diagram of the cottonseed protein-shikimic acid complex and cottonseed protein prepared in Example 1 of the present invention against Escherichia coli; Figure 6 It is an antibacterial experiment diagram of the cottonseed protein-shikimic acid complex and cottonseed protein prepared in Example 1 of the present invention against Staphylococcus aureus; Figure 7 It is a DPPH· free radical scavenging experiment diagram of the cottonseed protein-shikimic acid complex and cottonseed protein prepared in Example 1 of the present invention; Figure 8Experimental diagram of the scavenging of ABTS free radicals by the cottonseed protein-shikimic acid complex and cottonseed protein prepared in Example 1 of the present invention; Figure 9 Experimental diagram of the scavenging of hydroxyl free radicals by the cottonseed protein-shikimic acid complex and cottonseed protein prepared in Example 1 of the present invention; Figure 10 Experimental diagram of the scavenging of superoxide anion free radicals by the cottonseed protein-shikimic acid complex and cottonseed protein prepared in Example 1 of the present invention. Detailed implementation mode

[0018] The following further describes the present invention in detail with reference to the drawings and examples, so that those skilled in the art can implement it according to the description in the specification.

[0019] It should be understood that the terms such as "having", "comprising" and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0020] Example 1 A preparation method of a natural plant acid and protein complex for animal feed, comprising the following steps: Step 1: Grind cottonseed protein and shikimic acid into fine powders respectively, and sieve for later use; Step 2: Mix 6 kg of 85% by mass fraction of cottonseed protein powder and 15% by mass fraction of shikimic acid powder in proportion, stir evenly, then add them into a rotary twin-screw extruder for extrusion. Set the parameters of the rotary twin-screw extruder as follows: the feeding speed is 10 kg / h, the screw rotation speed is 200 rpm, the feeding moisture content is 20%, the temperatures of five separate temperature control zones are 90 °C, 100 °C, 110 °C, 120 °C, 130 °C respectively, the die temperature is 130 °C, and the extrusion time is 60 min. Dry the extrudate and grind it into powder to obtain the cottonseed protein-shikimic acid complex (CPC@S15).

[0021] Example 2 In Step 2 of this example, the mass fraction ratio of cottonseed protein to shikimic acid is 88.75%:11.25%. Other steps are the same as those in Example 1 to obtain the cottonseed protein-shikimic acid complex (CPC@S11.25).

[0022] Example 3 In Step 2 of this example, the mass fraction ratio of cottonseed protein to shikimic acid is 92.5%:7.5%. Other steps are the same as those in Example 1 to obtain the cottonseed protein-shikimic acid complex (CPC@S7.5).

[0023] Example 4 In step two of this embodiment, the mass fraction ratio of cottonseed protein to shikimic acid is 96.25%: 3.75%. The other steps are the same as those in Embodiment 1, and the cottonseed protein - shikimic acid complex (CPC@S3.75) is obtained.

[0024] Embodiment 5 A preparation method of a natural plant acid - protein complex for animal feed, comprising the following steps: Step 1: Grind soy protein and shikimic acid into fine powders respectively, and sieve them for later use; Step 2: Mix 6 kg of 85% mass fraction soy protein powder and 15% mass fraction shikimic acid powder proportionally and stir evenly, then add them into a rotating twin - screw extruder for extrusion. Set the parameters of the rotating twin - screw extruder as follows: the feeding speed is 10 kg / h, the screw speed is 200 rpm, the feeding moisture content is 20%, the temperatures of five separate temperature - controlled zones are 90 °C, 100 °C, 110 °C, 120 °C, 130 °C respectively, the die temperature is 130 °C, and the extrusion time is 60 min. Dry the extrudate and grind it into powder to obtain the soy protein - shikimic acid complex (SPC@S15).

[0025] Embodiment 6 In step two of this embodiment, the mass fraction ratio of soy protein to shikimic acid is 88.75%: 11.25%. The other steps are the same as those in Embodiment 5, and the soy protein - shikimic acid complex (SPC@S11.25) is obtained.

[0026] Embodiment 7 In step two of this embodiment, the mass fraction ratio of soy protein to shikimic acid is 92.5%: 7.5%. The other steps are the same as those in Embodiment 5, and the soy protein - shikimic acid complex (SPC@S7.5) is obtained.

[0027] Embodiment 8 In step two of this embodiment, the mass fraction ratio of soy protein to shikimic acid is 96.25%: 3.75%. The other steps are the same as those in Embodiment 5, and the soy protein - shikimic acid complex (SPC@S3.75) is obtained.

[0028] Embodiment 9 A preparation method of a natural plant acid - protein complex for animal feed, comprising the following steps: Step 1: Grind rapeseed protein and citric acid into fine powders respectively, and sieve them for later use; Step 2: Mix 6 kg of rapeseed protein powder with 85% mass fraction and citric acid powder with 15% mass fraction proportionally and stir evenly, then add them into a rotary twin-screw extruder for extrusion. Set the parameters of the rotary twin-screw extruder as follows: the feeding speed is 10 kg / h, the screw rotation speed is 200 rpm, the feeding moisture content is 20%, the temperatures of five separate temperature control zones are 90 °C, 100 °C, 110 °C, 120 °C, and 130 °C respectively, the die temperature is 130 °C, and the extrusion time is 60 min. Dry and grind the extrudate into powder to obtain rapeseed protein-citric acid complex (RP@C15).

[0029] Example 10 In Step 2 of this example, the mass fraction ratio of rapeseed protein to citric acid is 88.75%:11.25%. Other steps are the same as those in Example 9 to obtain rapeseed protein-citric acid complex (RP@C11.25).

[0030] Example 11 In Step 2 of this example, the mass fraction ratio of rapeseed protein to citric acid is 92.5%:7.5%. Other steps are the same as those in Example 9 to obtain rapeseed protein-citric acid complex (RP@C7.5).

[0031] Example 12 In Step 2 of this example, the mass fraction ratio of rapeseed protein to citric acid is 96.25%:3.75%. Other steps are the same as those in Example 9 to obtain rapeseed protein-citric acid complex (RP@C3.75).

[0032] Example 13 A preparation method of a natural plant acid and protein complex for animal feed, comprising the following steps: Step 1: Grind soy protein and malic acid into fine powder respectively and sieve for later use; Step 2: Mix 6 kg of soy protein powder with 85% mass fraction and malic acid powder with 15% mass fraction proportionally and stir evenly, then add them into a rotary twin-screw extruder for extrusion. Set the parameters of the rotary twin-screw extruder as follows: the feeding speed is 10 kg / h, the screw rotation speed is 200 rpm, the feeding moisture content is 20%, the temperatures of five separate temperature control zones are 90 °C, 100 °C, 110 °C, 120 °C, and 130 °C respectively, the die temperature is 130 °C, and the extrusion time is 60 min. Dry and grind the extrudate into powder to obtain soy protein-malic acid complex (SPC@M15).

[0033] Example 14 In the second step of this example, the mass fraction ratio of soy protein to malic acid is 88.75%:11.25%. Other steps are the same as those in Example 13, and a soy protein-malic acid complex (SPC@M11.25) is obtained.

[0034] Example 15 In the second step of this example, the mass fraction ratio of soy protein to malic acid is 92.5%:7.5%. Other steps are the same as those in Example 13, and a soy protein-malic acid complex (SPC@M7.5) is obtained.

[0035] Example 16 In the second step of this example, the mass fraction ratio of soy protein to malic acid is 96.25%:3.75%. Other steps are the same as those in Example 13, and a soy protein-malic acid complex (SPC@M3.75) is obtained.

[0036] Example 17 A method for preparing a natural plant acid and protein complex for animal feed, comprising the following steps: Step 1: Grind peanut protein and shikimic acid into fine powders respectively, and sieve them for later use; Step 2: Mix 6 kg of peanut protein powder with a mass fraction of 85% and shikimic acid powder with a mass fraction of 15% in proportion and stir evenly, then add them into a rotating twin-screw extruder for extrusion. Set the parameters of the rotating twin-screw extruder as follows: the feeding speed is 10 kg / h, the screw rotation speed is 200 rpm, the feeding moisture content is 20%, the temperatures of five separate temperature control zones are 90°C, 100°C, 110°C, 120°C, 130°C respectively, the die temperature is 130°C, and the extrusion time is 60 min. Dry and grind the extrudate into powder to obtain a peanut protein-shikimic acid complex (PP@S15).

[0037] Example 18 In the second step of this example, the mass fraction ratio of peanut protein to shikimic acid is 88.75%:11.25%. Other steps are the same as those in Example 17, and a peanut protein-shikimic acid complex (PP@S11.25) is obtained.

[0038] Example 19 In the second step of this example, the mass fraction ratio of peanut protein to shikimic acid is 92.5%:7.5%. Other steps are the same as those in Example 17, and a peanut protein-shikimic acid complex (PP@S7.5) is obtained.

[0039] Example 20 In step two of this embodiment, the mass fraction ratio of peanut protein to shikimic acid is 96.25%: 3.75%. The other steps are the same as those in Example 17, and the peanut protein-shikimic acid complex (PP@S3.75) is obtained.

[0040] Example 21 A preparation method of a natural plant acid and protein complex for animal feed, comprising the following steps: Step one: Grind cottonseed protein and shikimic acid into fine powders respectively, and sieve them for later use; Step two: Pretreat shikimic acid, specifically: dissolve 1 kg of shikimic acid powder in 10 kg of water, stir evenly, then add 0.1 kg of lecithin and 0.03 kg of ascorbic acid, ultrasonically disperse at 25 kHz for 20 min until evenly dispersed, and then heat and evaporate the solvent at 65 °C to obtain a co-blended and modified natural plant acid powder; Mix 6 kg of 85% mass fraction of cottonseed protein powder and 15% mass fraction of pretreated shikimic acid powder in proportion and stir evenly, then add them into a rotary twin-screw extruder for extrusion. Set the parameters of the rotary twin-screw extruder as follows: the feeding speed is 10 kg / h, the screw speed is 200 rpm, the feeding moisture content is 20%, the temperatures of five separate temperature control zones are 90 °C, 100 °C, 110 °C, 120 °C, 130 °C respectively, the die temperature is 130 °C, and the extrusion time is 60 min. Dry and grind the extrudate into powder to obtain the cottonseed protein-shikimic acid complex (CPC@S15-A).

[0041] Example 22 A preparation method of a natural plant acid and protein complex for animal feed, comprising the following steps: Step one: Grind cottonseed protein and shikimic acid into fine powders respectively, and sieve them for later use; Step two: Pretreat cottonseed protein, specifically: disperse 10 kg of protein powder in 120 kg of water, then add 0.2 kg of fructose and 0.5 kg of chitosan thereto, heat and stir at 35 °C for 6 h, and then vacuum dry at 10 °C to obtain the pretreated protein powder; After mixing 6 kg of pre-treated cottonseed protein powder with 85% mass fraction and shikimic acid powder with 15% mass fraction proportionally and stirring evenly, add them into a rotary twin-screw extruder for extrusion. Set the parameters of the rotary twin-screw extruder as follows: the feeding speed is 10 kg / h, the screw rotation speed is 200 rpm, the feeding moisture content is 20%, the temperatures of five separate temperature control zones are 90 °C, 100 °C, 110 °C, 120 °C, 130 °C respectively, the die temperature is 130 °C, and the extrusion time is 60 min. Dry the extrudate and grind it into powder to obtain cottonseed protein-shikimic acid complex (CPC@S15-B).

[0042] Example 23 A preparation method of a natural plant acid and protein complex for animal feed, comprising the following steps: Step 1: Grind cottonseed protein and shikimic acid into fine powder respectively, and sieve them for standby; Step 2: Pretreat cottonseed protein and shikimic acid respectively, specifically: S1: Dissolve 1 kg of shikimic acid powder in 10 kg of water, stir evenly, then add 0.1 kg of lecithin and 0.03 kg of ascorbic acid, disperse ultrasonically at 25 kHz for 20 min until evenly dispersed, and then heat and evaporate the solvent at 65 °C to obtain a blend-modified natural plant acid powder; S2: Disperse 10 kg of protein powder in 120 kg of water, then add 0.2 kg of fructose and 0.5 kg of chitosan thereto, heat and stir at low temperature at 35 °C for 6 h, and then dry in vacuum at 10 °C to obtain pre-treated protein powder; After mixing 6 kg of pre-treated cottonseed protein powder with 85% mass fraction and shikimic acid powder with 15% mass fraction proportionally and stirring evenly, add them into a rotary twin-screw extruder for extrusion. Set the parameters of the rotary twin-screw extruder as follows: the feeding speed is 10 kg / h, the screw rotation speed is 200 rpm, the feeding moisture content is 20%, the temperatures of five separate temperature control zones are 90 °C, 100 °C, 110 °C, 120 °C, 130 °C respectively, the die temperature is 130 °C, and the extrusion time is 60 min. Dry the extrudate and grind it into powder to obtain cottonseed protein-shikimic acid complex (CPC@S15-C).

[0043] Comparative Example 1 The difference between this comparative example and Example 1 is that no natural plant acid is added to obtain CPC@S0.

[0044] Comparative Example 2 The difference between this comparative example and Example 1 is that no natural plant acid is added, and no twin-screw extruder is used for extrusion. Only grind cottonseed protein into powder to obtain CPC.

[0045] Figure 1 Scanning electron microscopy (SEM) images of the products of Example 1 and Comparative Examples 1-2 of the present invention; Figure 1 As can be seen, cottonseed protein (CPC) presents a rough surface state without passing through a rotary twin-screw extruder. After cottonseed protein is extruded through a rotary twin-screw extruder (CPC@S0), the surface presents a porous foamed state. After cottonseed protein is mixed with shikimic acid and then further extruded through a rotary twin-screw extruder (CPC@S15), it presents a further smooth and porous state, and at the same time, the number of white particles increases, because solid-phase co-extrusion makes shikimic acid uniformly incorporated into cottonseed protein.

[0046] Figure 2 Fourier transform infrared spectroscopy (FTIR) images of the products of Example 1 and Comparative Examples 1-2 of the present invention; Changes in the secondary structure of proteins are usually explained by the stretching vibration of C=O in amide I (1700 - 1600 cm -1 ), and the stretching vibration of C-N and bending vibration of N-H in amide II (1600 - 1500 cm -1 ). As can be seen from Figure 2 , the broad peak near 3292 cm -1 of cottonseed protein (CPC) belongs to the stretching vibration peak of -OH, and the peak at 2970 cm -1 indicates the presence of C-H. The peaks at 1631 cm -1 , 1540 cm -1 and 1399 cm -1 are the characteristic absorption peaks of amide I, II, and III bands of proteins respectively; after cottonseed protein is extruded through a rotary twin-screw extruder (CPC@S0), the peak at 3292 cm -1 redshifts to 3398 cm -1 , indicating that the intramolecular structure of the protein is damaged, forming new hydrogen bonds or enhancing the strength of existing hydrogen bonds; after cottonseed protein and shikimic acid are mixed and then extruded through a rotary twin-screw extruder (CPC@S15), there are no obvious changes in amide I band, II band, and III band. It can be seen that the stretching vibration peak of -OH becomes significantly wider, which is mainly due to the introduction of a large number of hydroxyl groups by shikimic acid. The change in the peak intensity at 1162 cm -1 is attributed to the vibration of hydroxyl groups, indicating that shikimic acid and cottonseed protein in CPC@S15 prepared in Example 1 are mainly combined through non-covalent interactions such as hydrogen bonds and van der Waals forces.

[0047] Figure 3UV spectra of the products of Example 1 and Comparative Examples 1-2 of the present invention; the absorption of molecules by ultraviolet light is related to their molecular structure. Therefore, by examining the characteristics of the ultraviolet spectrum and the changes in absorption intensity at specific wavelengths, qualitative or quantitative analysis of molecules can be performed. For organic compounds, the most commonly used absorption spectra are generated based on n-π* and π-π* transitions. As can be seen from Figure 3, the absorbance of cottonseed protein (CPC) at 210 nm is significantly stronger than that at 260 nm, and the molar absorption coefficient (ε) of the amide is lower than that of the benzene ring. It can be easily concluded that the two characteristic peaks of CPC are mainly caused by the π-π* transition of the benzene rings of aromatic amino acids (tryptophan, tyrosine, and phenylalanine). After cottonseed protein is extruded through a rotating twin-screw extruder (CPC@S0), the wavelength at 210 nm becomes larger and undergoes a red shift, and the peak at 280 nm is obvious, because the protein structure is damaged and a conjugated system is formed on the benzene rings of aromatic amino acids. When cottonseed protein and shikimic acid are mixed and then extruded through a rotating twin-screw extruder (CPC@S15), the wavelength at 210 nm further becomes larger and undergoes a red shift, and the characteristic peak at 280 nm decreases, indicating that the conjugated system has changed significantly, because shikimic acid binds to the hydrophobic amino acids exposed after co-extrusion.

[0048] Figure 4 Endogenous fluorescence spectra of the products of Example 1-4 and Comparative Examples 1-2 of the present invention; in protein fluorescence analysis, 280 nm is selected as the fixed excitation wavelength, and at this wavelength, fluorescence is mainly generated by tryptophan, tyrosine, and phenylalanine. As can be seen from Figure 4, cottonseed protein (CPC) exhibits strong fluorescence intensity at 350 nm. After being extruded through a rotating twin-screw extruder (CPC@S0), the fluorescence intensity becomes lower because the hydrophobic amino acid residues are exposed after solid-phase co-extrusion of the protein. When cottonseed protein and shikimic acid are mixed and then extruded through a rotating twin-screw extruder (CPC@S15), further fluorescence quenching occurs because shikimic acid increases the environmental polarity.

[0049] Figure 5 Antibacterial experiment graphs of the products of Example 1-4 and Comparative Examples 1-2 of the present invention against Escherichia coli. As can be seen from Figure 5 it, cottonseed protein (CPC) has no inhibitory effect on Escherichia coli, and after being extruded through a rotating twin-screw extruder (CPC@S0), it also has no inhibitory effect on Escherichia coli, indicating that cottonseed protein or puffing treatment does not confer antibacterial activity. When cottonseed protein and shikimic acid are mixed and then extruded through a rotating twin-screw extruder (CPC@S15), an obvious antibacterial zone is formed. This is attributed to the fact that shikimic acid (SA) confers significant antibacterial activity to cottonseed protein through mechanisms such as reducing pH, destroying cell membranes, and chelating metal ions, indicating that shikimic acid (SA) is the key synergistic factor, providing a scientific basis for the development of functional feed additives.

[0050] Figure 6This is the antibacterial experiment diagram of the products of Examples 1-4 and Comparative Examples 1-2 of the present invention against Staphylococcus aureus. From Figure 6 it can be seen that cottonseed protein (CPC) has no inhibitory effect on Staphylococcus aureus, and there is also no inhibitory effect on Staphylococcus aureus after extrusion by a rotary twin-screw extruder (CPC@S0). After mixing cottonseed protein and shikimic acid and then extruding through a rotary twin-screw extruder (CPC@S15), an obvious antibacterial zone is formed. It shows that shikimic acid can endow cottonseed protein with antibacterial activity.

[0051] Figure 7 This is the DPPH· free radical scavenging experiment diagram of the products of Examples 1-4 and Comparative Examples 1-2 of the present invention. The DPPH· (1,1-diphenyl-2-picrylhydrazine) free radical scavenging experiment is a classic method for evaluating the antioxidant ability of compounds. The DPPH· free radical simulates the free radical chain reaction in lipid peroxidation, and a high scavenging rate indicates that the sample can effectively inhibit lipid peroxidation and protect the integrity of cell membranes. From Figure 7It can be seen that the clearance rate of only the puffed treatment group CPC@S0 (denoted as C@S0 in the figure) is slightly higher than that of untreated CPC, but the difference is not significant, indicating that the improvement of the DPPH clearance rate by puffing depends on the synergistic effect of SA. At all concentrations, the DPPH clearance rate of CPC@S15 (denoted as C@S15 in the figure) is significantly higher than that of CPC and CPC@S0. Especially at low concentrations, i.e., 0.13 mg / mL, its clearance rate is 27.32%, which is 4 times that of CPC, indicating that SA still has high antioxidant activity at low doses. This is attributed to the fact that on the one hand, the hydroxyl group of SA directly provides hydrogen atoms (H·) to DPPH free radicals, terminating the free radical chain reaction. On the other hand, SA can bind to the hydrophobic region of puffed cottonseed protein, enhancing its dispersibility in the lipophilic system (DPPH / ethanol model) and improving the contact efficiency. CPC@S15 significantly improves the DPPH free radical clearance rate through the hydrogen atom supply and hydrophobic synergistic effect of shikimic acid, especially showing excellent performance at low concentrations (0.13–0.5 mg / mL), providing a key basis for its application in lipid peroxidation protection, anti-aging, and feed preservation. In addition, DPPH· free radical scavenging experiments and antibacterial experiments against Escherichia coli were carried out on Examples 21-23. It was found that compared with Example 1, the antibacterial property of Example 21 did not change much, but after pretreatment with shikimic acid, the clearance rate of the prepared product against DPPH· free radicals reached 30.21% at a low concentration (0.13 mg / mL). This is because the lecithin and ascorbic acid added to the shikimic acid powder played a role in enhancing the antioxidant ability. Compared with Example 1, the antibacterial property of Example 22 was significantly improved, and after pretreatment of cottonseed protein powder, the clearance rate of the product against DPPH· free radicals reached 31.66% at a low concentration (0.13 mg / mL). This is because after adding fructose and chitosan to cottonseed protein and reacting, it can also provide hydrogen atoms to DPPH· free radicals to a certain extent, thereby increasing the clearance rate of the product against DPPH· free radicals. Compared with Example 1, in Example 23, after pretreatment of both cottonseed protein and shikimic acid, the antibacterial property was significantly improved, and the clearance rate of the product against DPPH· free radicals reached 36.62% at a low concentration (0.13 mg / mL), and its antioxidant ability was also greatly improved. In addition to the above-mentioned advantages, the substances added in Examples 21-23 are all edible substances with certain nutritional value and can also supplement nutrition for animals.

[0052] Figure 8This is the experimental graph of ABTS radical scavenging for the products of Examples 1-4 and Comparative Examples 1-2 of the present invention. The ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging experiment is a classic method for evaluating the total antioxidant capacity of substances. It reflects the ability of a sample to neutralize water-soluble radicals through hydrogen atom transfer (HAT) and single electron transfer (SET) mechanisms, simulating the antioxidant defense in the aqueous phase environment (such as cytoplasm, blood) in vivo. From Figure 8 It can be seen that the scavenging rate of only the extruded CPC@S0 (denoted as C@S0 in the figure) is slightly higher than that of the untreated CPC, but the difference is small, indicating that extrusion slightly improves the antioxidant activity of CPC. This is attributed to the extrusion destroying the dense structure of cottonseed protein and releasing bound antioxidant components (such as phenolic acids, flavonoids). At the same time, extrusion increases the dispersibility of CPC in water and promotes the contact efficiency with ABTS radicals. The extruded CPC@S15 (denoted as C@S15 in the figure) treated with shikimic acid has a significantly higher scavenging rate than CPC and CPC@S0 at all concentrations, proving that shikimic acid (SA) is the core synergistic component. This is attributed to the hydroxyl group of SA providing a hydrogen atom to neutralize the ABTS⁺ radical. The treatment of CPC@S15 significantly improves the ABTS radical scavenging ability through the direct antioxidant effect of shikimic acid and the synergistic effect of the extrusion process, with the highest scavenging rate of 99.09%, verifying its potential as an efficient natural antioxidant and providing a key scientific basis for the development of functional feed additives.

[0053] Figure 9 This is the experimental graph of hydroxyl radical scavenging for the products of Examples 1-4 and Comparative Examples 1-2 of the present invention. Hydroxyl radicals are the most destructive reactive oxygen species (ROS) in vivo, and the improvement of their scavenging rate is crucial for cell protection. The enhancement of hydroxyl radical scavenging ability can effectively protect cell membranes, DNA, and proteins from oxidative damage and reduce the release of inflammatory factors (such as IL-6, TNF-α). From Figure 9It can be seen that only the extruded CPC@S0 (denoted as C@S0 in the figure) has a slightly higher clearance rate than untreated CPC, indicating that extrusion improves the activity by releasing bound antioxidant components, but the increase is limited and requires the synergistic effect of SA. The hydroxyl radical clearance rate of CPC@S15 (denoted as C@S15 in the figure) is significantly higher than that of CPC and CPC@S0 at all concentrations. Especially at the low concentration of 0.13 mg / mL, its clearance rate is 28.31%, which is 1.96 times that of CPC, indicating that SA can still effectively neutralize highly reactive free radicals at extremely low doses. This is attributed to the fact that the hydroxyl group of SA directly provides hydrogen atoms to terminate the free radical chain reaction. In addition, SA can also bind to Fe²⁺ (a Fenton reaction catalyst) to inhibit the generation of ·OH. In summary, CPC@S15 significantly improves the hydroxyl radical clearance rate through the hydrogen atom supply and metal chelation of shikimic acid, combined with the synergistic effect of the extrusion process, and exhibits high antioxidant activity especially at low concentrations (0.13–0.5 mg / mL), providing key support for its application in preventing DNA damage, protein oxidation, and chronic diseases.

[0054] Figure 10 This is the experimental graph of the scavenging of superoxide anion radicals by the products of Examples 1-4 and Comparative Examples 1-2 of the present invention. Superoxide anion (O2⁻) is one of the most common reactive oxygen species (ROS) in organisms, and its scavenging experiment directly reflects the neutralization ability of substances to primary free radicals. The O2⁻ scavenging ability is a key indicator for evaluating the protective effect of substances against early oxidative stress. As can be Figure 10 seen, only the extruded CPC@S0 (denoted as C@S0 in the figure) has a slightly higher clearance rate than untreated CPC, but the increase is limited, indicating that extrusion partially improves the activity by releasing bound antioxidant components and requires the synergistic effect of SA. The superoxide anion clearance rate of CPC@S15 (denoted as C@S15 in the figure) is significantly higher than that of CPC and CPC@S0 at all concentrations. This is attributed to the fact that, on the one hand, the hydroxyl group of SA directly donates electrons to the superoxide anion to reduce it to H2O2, which is further decomposed by catalase (CAT) or glutathione peroxidase (GSH-Px) subsequently. On the other hand, SA binds to Fe²⁺ / Cu²⁺ to inhibit the Haber-Weiss reaction (O2⁻+H2O2→·OH + OH⁻+O2) and block the generation of ·OH. In summary, CPC@S15 significantly improves the superoxide anion clearance rate through the electron transfer and metal chelation of shikimic acid, combined with the synergistic effect of the extrusion process, providing key support for improving mitochondrial function, balancing immune responses, and maintaining intestinal health.

[0055] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the examples shown and described herein.

Claims

1. A preparation method of a natural plant acid and protein complex for animal feed, characterized in that, It includes the following steps: Step 1: Grind the protein and natural plant acid into fine powders respectively, and sieve them for later use; Step 2: After mixing and stirring the protein powder and natural plant acid powder evenly in proportion, add them into a rotary twin-screw extruder for extrusion. Dry and grind the extrudate into powder to obtain a protein-natural plant acid complex.

2. The preparation method of the natural plant acid and protein complex for animal feed according to claim 1, characterized in that, In Step 1, the protein is one of cottonseed protein, soybean protein, rapeseed protein, and peanut protein.

3. The preparation method of the natural plant acid and protein complex for animal feed according to claim 1, characterized in that In Step 1, the natural plant acid is one of shikimic acid, citric acid, lactic acid, and malic acid.

4. The preparation method of the natural plant acid and protein complex for animal feed according to claim 1, characterized in that, In Step 2, the mass fraction ratio of the protein powder to the natural plant acid powder is 85~96.25%:3.75~15%.

5. The preparation method of the natural plant acid and protein complex for animal feed according to claim 1, characterized in that, In Step 2, the parameter settings of the twin-screw extruder are as follows: the feeding speed is 5~15 kg / h, the screw rotation speed is 100~300 rpm, the feeding moisture content is 15~25%, the temperatures of the five separate temperature control zones are 80~100 °C, 90~110 °C, 100~120 °C, 110~130 °C, 120~140 °C respectively, and the die temperature is 120~140 °C.

6. The preparation method of the natural plant acid and protein complex for animal feed according to claim 1, characterized in that, In Step 2, before mixing the plant protein powder and natural plant acid powder, pre-treat the natural plant acid powder and protein powder respectively. The specific method is as follows: S1: Dissolve the natural plant acid powder in water, stir evenly, then add lecithin and ascorbic acid, disperse evenly by ultrasonic wave, and heat to evaporate the solvent to obtain a blend-modified natural plant acid powder; S2: Disperse the protein powder in water, then add a small amount of fructose and chitosan, heat and stir at low temperature, and then dry under low-temperature vacuum to obtain a pre-treated protein powder.

7. The preparation method of the natural plant acid and protein complex for animal feed according to claim 6, characterized in that, In S1, the mass ratio of the natural plant acid powder, water, lecithin, and ascorbic acid is 1:10~12:0.1~0.2:0.01~0.05, the ultrasonic dispersion frequency is 20~40 kHz, the dispersion time is 15~20 min, and the heating evaporation temperature is 60~75 °C.

8. The preparation method of the natural plant acid and protein complex for animal feed according to claim 6, characterized in that, In S2, the mass ratio of the protein powder, water, fructose, and chitosan is 1:15~20:0.01~0.03:0.05~0.1, the low-temperature heating temperature is 35~45 °C, the heating time is 4~8 h, and the vacuum drying is carried out at 10~15 °C for 3~6 h.

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