A cottonseed protein-based antibacterial compound for animal feed and a preparation method thereof

Abstract

The present invention discloses a cottonseed protein-based antibacterial composite for animal feed and a preparation method thereof, comprising: in a nitrogen atmosphere, mixing cottonseed protein and deionized water at a mass volume ratio of 1g:50-300mL for 0.5-3h, then adding hydrogen peroxide and ascorbic acid, stirring for 0.5-3h, and obtaining a cottonseed protein mixed solution; mixing the cottonseed protein mixed solution and shikimic acid at a mass molar ratio of 1g:5-80mmol of cottonseed protein to shikimic acid, stirring at 15-35°C for 15-30h, and dialyzing and freeze-drying to obtain a cottonseed protein-based antibacterial composite. The present invention selects high-quality protein cottonseed protein and combines it with shikimic acid with good antibacterial properties. The prepared cottonseed protein-based antibacterial composite has excellent antibacterial activity and can be used as an alternative antibacterial feed additive for preparing animal antibacterial feed. At the same time, the raw materials of the present invention are simple and easy to obtain, the preparation method is simple, the operability is strong, and it is easy to promote, which provides a new resource and new idea for alternative antibacterial feed additives and their applications.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal feed, and more specifically, relates to a cottonseed protein-based antibacterial compound for animal feed and a preparation method thereof. Background Art

[0002] Antibiotics are widely used in feed as feed additives, called "feed antibiotics" to prevent animal diseases, protect animal health, promote livestock and poultry growth and production, and improve feed utilization. When antibiotics are used for drug treatment, while killing pathogens, a certain number of sensitive microorganisms will also be eliminated, resulting in large-scale reproduction of insensitive microorganisms, breaking the pattern of mutual restraint between microorganisms, disrupting the original microecological balance in the intestine, and even inducing new infections. The use of antibiotic additives in animal feed can lead to bacterial resistance, which can be transmitted to humans through the food chain, which has a significant impact on food safety and human health. After consuming excessive antibiotics, the immunity of livestock and poultry decreases, and the flora in the body is imbalanced, which will increase the number of chronic diseases in livestock and poultry. Long-term excessive use of feed antibiotics will cause residues in livestock and poultry bodies. Even if livestock and poultry products are sterilized by high-temperature heating during the cooking process at home, the residual antibiotics cannot be completely eliminated. Over time, antibiotics slowly accumulate in the human body, causing changes in the colony structure of the human body, reducing the body's immunity, and causing lesions in the organs of the body. At the same time, the abuse of antibiotics in feed will increase the probability of infectious diseases in humans and animals and increase the number of zoonotic cases. Therefore, it is of great significance to develop an alternative antibiotic additive suitable for animal feed. Summary of the invention

[0003] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0004] In order to achieve these objects and other advantages of the present invention, a cottonseed protein-based antibacterial compound for animal feed is provided. The cottonseed protein-based antibacterial compound is used as an antibiotic-substitute feed additive for preparing animal antibacterial feed.

[0005] Preferably, the antibacterial rates of the cottonseed protein-based antibacterial compound against Escherichia coli and Staphylococcus aureus are both greater than 90%.

[0006] Preferably, the specific method for preparing the antibacterial feed for animals is: adding the cottonseed protein-based antibacterial compound to the complete feed currently used by the animals, with the added amount being 0.5-2 kg / t.

[0007] A method for preparing the cottonseed protein-based antimicrobial composite for animal feed as described above comprises the following steps:

[0008] Step 1: Under a nitrogen atmosphere, cottonseed protein and deionized water are mixed and stirred at a mass volume ratio of 1 g: 50-300 mL for 0.5-3 h, and then hydrogen peroxide and ascorbic acid are added and stirred for 0.5-3 h to obtain a cottonseed protein mixed solution;

[0009] Step 2: Mix the cottonseed protein mixture and shikimic acid at a mass molar ratio of 1 g:5-80 mmol, stir at 15-35° C. for 15-30 h, dialyze, and freeze-dry to obtain a cottonseed protein-based antibacterial complex for animal feed.

[0010] Preferably, in step 1, the mass volume ratio of cottonseed protein to deionized water is 1 g:80-150 mL.

[0011] Preferably, in step 1, the mass volume ratio of cottonseed protein, hydrogen peroxide and ascorbic acid is 1 g:1-3 mL:0.3-0.8 g.

[0012] Preferably, in step 1, the cottonseed protein and deionized water are mixed and stirred for 1 to 2 hours.

[0013] Preferably, in the step 1, after adding hydrogen peroxide and ascorbic acid, stirring is carried out for 1 to 2 hours.

[0014] Preferably, in the step 2, the mass molar ratio of cottonseed protein to shikimic acid is 1 g:10-60 mmol.

[0015] Preferably, in step 2, the stirring is performed at 20 to 30° C. for 20 to 25 hours.

[0016] Preferably, in step 2, the freeze-drying temperature is -60 to -100°C.

[0017] Preferably, in step 2, the freeze-drying time is 40 to 50 hours.

[0018] Preferably, modified shikimic acid is used to replace the shikimic acid in step 2. The preparation method of the modified shikimic acid is as follows: shikimic acid is added to water, stirred for 20 to 40 minutes, xanthan gum and tannic acid are added, and ultrasonic oscillation mixing treatment is performed. After each ultrasonic treatment for 5 to 10 minutes, the ultrasonic treatment is continued for 5 to 10 minutes, and the cycle is repeated 4 to 6 times. During the ultrasonic treatment, the mixed solution is irradiated with an ultrashort pulse laser, and then freeze-dried to obtain modified shikimic acid.

[0019] Preferably, the mass volume ratio of the shikimic acid to water is 1 g:10-50 mL; the mass ratio of the shikimic acid, xanthan gum and tannic acid is 1:0.1-0.5:0.1-0.5.

[0020] Preferably, the ultrasonic power is 300-500 W, and the frequency is 50-70 kHz; the ultrashort pulse laser has a power of 1-4 W, a pulse width of 100-200 fs, a wavelength of 355-1064 nm, and a frequency of 100-1000 Hz.

[0021] The present invention includes at least the following beneficial effects: the present invention selects high-quality protein cottonseed protein (CPI) and combines it with shikimic acid with good antibacterial properties. The prepared cottonseed protein-based antibacterial complex has excellent antibacterial activity and can be used as an alternative antibiotic feed additive for preparing animal antibacterial feed. At the same time, the raw materials of the present invention 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 alternative antibiotic feed additives and their applications.

[0022] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The infrared spectra of the cottonseed protein-based antibacterial composite and cottonseed protein prepared in Examples 1 to 3 of the present invention;

[0024] Figure 2 The fluorescence spectra of the cottonseed protein-based antibacterial composite and cottonseed protein prepared in Examples 1 to 3 of the present invention;

[0025] Figure 3 The ultraviolet spectra of the cottonseed protein-based antibacterial composite, cottonseed protein and shikimic acid prepared in Examples 1 to 3 of the present invention;

[0026] Figure 4 CD spectra of cottonseed protein-based antibacterial composites and cottonseed protein prepared in Examples 1 to 3 of the present invention;

[0027] Figure 5 This is a graph showing the antibacterial effect of the cottonseed protein-based antibacterial composite and cottonseed protein prepared in Examples 1 to 3 of the present invention on Escherichia coli. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0029] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0030] Example 1

[0031] A method for preparing a cottonseed protein-based antibacterial composite for animal feed comprises the following steps:

[0032] Step 1: Under a nitrogen atmosphere, 1 g of cottonseed protein and 100 mL of deionized water were mixed and stirred for 2 h, and then 2 mL of hydrogen peroxide and 0.5 g of ascorbic acid were added and stirred for 2 h to obtain a cottonseed protein mixture;

[0033] Step 2: Add 10 mmol of shikimic acid to the cottonseed protein mixture obtained in step 1, stir at 25°C for 24 hours, dialyze to remove unreacted shikimic acid, and freeze-dry at -80°C for 48 hours to obtain a cottonseed protein-based antibacterial complex for animal feed, recorded as CPI-S10.

[0034] Example 2

[0035] In this example, the amount of shikimic acid used is 30 mmol, and the remaining steps are the same as those in Example 1 to obtain a cottonseed protein-based antibacterial composite, which is recorded as CPI-S30.

[0036] Example 3

[0037] In this example, the amount of shikimic acid used is 60 mmol, and the remaining steps are the same as those in Example 1 to obtain a cottonseed protein-based antibacterial composite, which is denoted as CPI-S60.

[0038] Figure 1 The infrared spectra of the cottonseed protein-based antibacterial composite and cottonseed protein prepared in Examples 1 to 3 of the present invention are shown in Figure 1. -1 The broad peak near 2960 cm-1 is attributed to the stretching vibration peak of -OH. -1 The peak at 1655 cm indicates the presence of CH. -1 、1542cm -1 and 1239cm -1 The peaks at 3409cm-1 and 3410cm-2 are the characteristic absorption peaks of amide I, II and III of proteins respectively. In the infrared spectrum of CPI-S (S10, S30, S60), it can be seen that the -OH stretching vibration peak is significantly broadened and increases from 3409cm-1 to 3410cm-2. -1 Blue shift to 3301cm -1 This is mainly due to the introduction of a large number of hydroxyl groups by shikimic acid; at the same time, -1 and 626cm -1 New infrared absorption peaks appeared at , which were attributed to the stretching vibration of CO and the in-plane stretching vibration of CH on the benzene ring, respectively. In summary, it can be confirmed that shikimic acid has been successfully fixed to cottonseed protein. At the same time, the movement of amide Ⅰ, Ⅱ and Ⅲ band peaks indicates that the interaction between shikimic acid and cottonseed protein may be related to C=O (amide Ⅰ band) and -NH- (amide Ⅱ band).

[0039] Figure 2The fluorescence spectra of the cottonseed protein-based antibacterial complex and cottonseed protein prepared in Examples 1 to 3 of the present invention are shown. The main fluorescent substances in cottonseed protein are tryptophan and tyrosine. 280 nm is selected as the fixed excitation wavelength. At this wavelength, it can be considered that the endogenous fluorescence in cottonseed protein comes from tryptophan and tyrosine. It can be seen that compared with CPI, the fluorescence intensity of myoglobin at the emission wavelength of 334 nm is significantly increased, which shows that shikimic acid fully combines with the chromophore tryptophan and tyrosine in cottonseed protein to avoid the damage of tryptophan and tyrosine by active free radicals. After adding shikimic acid, cottonseed protein is obviously red-shifted to 337 nm. This red-shift phenomenon indicates that the tryptophan residues of CPI are exposed to a more hydrophilic environment.

[0040] Figure 3 The UV spectra of cottonseed protein-based antibacterial composites, cottonseed protein and shikimic acid prepared in Examples 1 to 3 of the present invention are shown. It can be seen that with the addition of shikimic acid (S), the peak value decreases and blue shifts, and Example 2 (S30) has the largest shift, indicating that shikimic acid and protein interact with each other, which also indicates that their interaction causes significant changes in the conjugated system.

[0041] Figure 4 CD spectra of cottonseed protein-based antibacterial complexes and cottonseed proteins prepared in Examples 1 to 3 of the present invention. The CD spectra of all samples show a broad negative band at around 190 to 250 nm, indicating that CPI is rich in random coil structures. As shown in Table 1, after CPI is combined with S, the α-helix and β-sheet content decreases, and the β-turn and random coil content increases, which means that the α-helix and β-sheet are converted to β-turns and random coils.

[0042] Table 1

[0043]

[0044] Antibacterial experiment: dilute the cultured bacterial solution (Escherichia coli or Staphylococcus aureus) to 10 6 CFU / mL, and then use physiological saline to prepare 1mg / mL antibacterial solution, add 100μL bacterial solution and 50μL antibacterial solution to each well of a sterile 96-well plate, and culture for 24 hours at 37°C and 90% relative air humidity. After 24 hours, the culture of the 96-well plate is evenly inoculated on the agar plate and cultured again for 24 hours. The control group uses physiological saline instead of antibacterial solution, and the antibacterial rate is calculated using the following formula:

[0045] Bacterial inhibition rate (%) = (1-concentration of bacterial solution in experimental group / concentration of bacterial solution in control group) × 100%

[0046] The antibacterial test of cottonseed protein-based antibacterial composites and cottonseed protein against Escherichia coli prepared in Examples 1 to 3 of the present invention is shown in the figure Figure 5As shown in Table 2, the antibacterial rate can be seen that the number of colonies in the culture dish with the addition of the cottonseed protein-based antibacterial composite is significantly reduced, indicating that the cottonseed protein of the present invention is combined with shikimic acid to effectively improve its antibacterial activity. Among them, when the addition amount of shikimic acid is 60mmol, the antibacterial rate is the highest.

[0047] Table 2

[0048] Antibacterial rate Example 1 Example 2 Example 3 CPI Escherichia coli 76.4％ 75.1％ 92.3％ 18.7％ Staphylococcus aureus 77.2％ 76.7％ 93.8％ 19.3％

[0049] Example 4

[0050] In this embodiment, modified shikimic acid is used to replace the shikimic acid in Example 3, and the remaining steps are the same as those in Example 3; wherein, the preparation method of modified shikimic acid is as follows: 10 g of shikimic acid is added to 300 mL of water, stirred for 30 min, 2 g of xanthan gum and 3 g of tannic acid are added, and mixed and treated with ultrasonic oscillation at 400 W and 55 kHz, and ultrasonic treatment is continued for 5 min after each ultrasonic treatment for 10 min, and the mixture is cycled for 5 times, and then freeze-dried at -80°C for 48 h to obtain modified shikimic acid.

[0051] In this embodiment, xanthan gum and tannic acid are used to modify shikimic acid, and the biochemical interaction between shikimic acid and bacterial membrane reduces the stability of the membrane, thereby improving the antibacterial activity of shikimic acid.

[0052] Example 5

[0053] In this embodiment, the preparation method of modified shikimic acid is as follows: 10g of shikimic acid is added to 300mL of water, stirred for 30min, 2g of xanthan gum and 3g of tannic acid are added, and mixed under 400W, 55kHz ultrasonic oscillation. After each ultrasonic treatment for 5min, the ultrasonic treatment is continued after an interval of 5min, and the cycle is repeated 5 times. While the ultrasonic treatment is being carried out, the mixed solution is irradiated with an ultrashort pulse laser, and then freeze-dried at -80°C for 48h to obtain modified shikimic acid; wherein the power of the ultrashort pulse laser is 2W, the pulse width is 120fs, the wavelength is 800nm, and the frequency is 500Hz; the remaining steps are the same as those in Example 4.

[0054] In the modification process of this embodiment, ultrashort pulse laser is used to irradiate the mixed solution, which helps to improve the modification effect and further enhance the antibacterial activity of shikimic acid.

[0055] The antibacterial rates of the cottonseed protein-based antibacterial composites prepared in Examples 3 to 5 are shown in Table 3. It can be seen that compared with Example 3, the antibacterial rates of Examples 4 to 5 are significantly improved, among which Example 5 has the highest antibacterial rate.

[0056] Table 3

[0057] Antibacterial rate Example 3 Example 4 Example 5 Escherichia coli 92.3％ 95.5％ 97.6％ Staphylococcus aureus 93.8％ 96.8％ 98.1％

[0058] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A cottonseed protein-based antimicrobial composite for animal feed, characterized in that: The cottonseed protein-based antibacterial compound is used as an antibiotic-substitute feed additive to prepare antibacterial feed for animals; The method for preparing the cottonseed protein-based antibacterial composite comprises the following steps: Step 1: Under a nitrogen atmosphere, cottonseed protein and deionized water are mixed at a mass volume ratio of 1 g: 50-300 mL and stirred for 0.5-3 h, and then hydrogen peroxide and ascorbic acid are added and stirred for 0.5-3 h to obtain a cottonseed protein mixed solution; Step 2: Mix the cottonseed protein mixture and shikimic acid at a mass molar ratio of 1 g:5-80 mmol, stir at 15-35° C. for 15-30 h, dialyze, and freeze-dry to obtain a cottonseed protein-based antibacterial complex for animal feed.

2. A cottonseed protein-based antimicrobial composite for animal feed according to claim 1, characterized in that: The antibacterial rates of the cottonseed protein-based antibacterial compound on Escherichia coli and Staphylococcus aureus are both greater than 90%.

3. The method for preparing a cottonseed protein-based antibacterial compound for animal feed according to claim 1, characterized in that: The following steps are involved: Step 1: Under a nitrogen atmosphere, cottonseed protein and deionized water are mixed at a mass volume ratio of 1 g: 50-300 mL and stirred for 0.5-3 h, and then hydrogen peroxide and ascorbic acid are added and stirred for 0.5-3 h to obtain a cottonseed protein mixed solution; Step 2: Mix the cottonseed protein mixture and shikimic acid at a mass molar ratio of 1 g:5-80 mmol, stir at 15-35° C. for 15-30 h, dialyze, and freeze-dry to obtain a cottonseed protein-based antibacterial complex for animal feed.

4. The method for preparing a cottonseed protein-based antibacterial compound for animal feed according to claim 3, characterized in that: In the step 1, the mass volume ratio of cottonseed protein to deionized water is 1 g:80-150 mL.

5. The method for preparing a cottonseed protein-based antibacterial compound for animal feed according to claim 3, characterized in that: In the step 1, the mass volume ratio of cottonseed protein, hydrogen peroxide and ascorbic acid is 1 g:1-3 mL:0.3-0.8 g.

6. The method for preparing a cottonseed protein-based antimicrobial compound for animal feed according to claim 3, characterized in that: In the step 1, the cottonseed protein and deionized water are mixed and stirred for 1 to 2 hours.

7. The method for preparing a cottonseed protein-based antimicrobial compound for animal feed according to claim 3, characterized in that: In the step 1, after adding hydrogen peroxide and ascorbic acid, stirring is performed for 1 to 2 hours.

8. The method for preparing a cottonseed protein-based antibacterial compound for animal feed according to claim 3, characterized in that: In the step 2, the mass molar ratio of cottonseed protein to shikimic acid is 1 g:10-60 mmol.

9. The method for preparing a cottonseed protein-based antimicrobial compound for animal feed according to claim 3, characterized in that: In the step 2, the stirring is performed at 20-30° C. for 20-25 hours.

10. The method for preparing a cottonseed protein-based antibacterial compound for animal feed according to claim 3, characterized in that: In the step 2, freeze drying is performed at -60 to -100°C for 40 to 50 hours.

Images