Feed additive and application thereof

Abstract

The invention discloses a feed additive and application thereof, ovomucin is prepared from egg white, and the ovomucin is used as a core component to prepare a functional feed for preventing and treating rodent diarrhea and improving weaning survival rate. Experiments prove that ovomucin nutritional composition intervention can significantly improve mouse diarrhea and death caused by weaning stress, improve inflammatory damage caused by bacterial toxins to intestinal mucosa tissues, promote secretion of intestinal immune factors sIgA, and improve oxidative stress imbalance caused by bacterial toxins; the ovomucin nutritional composition has no obvious acute toxicity, and has the potential of being used as a functional feed additive and a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of animal nutrition and disease prevention and treatment, and particularly relates to a feed additive and application thereof. Background Art

[0002] Rodents are important models for biomedical research, and their health during the weaning period directly affects the reliability of experimental data. By improving survival rate and intestinal health, experimental errors caused by diarrhea or growth retardation can be reduced. The weaning period is a critical stage in the life cycle of rodents (such as experimental mice and rats). Due to factors such as imperfect intestinal development, fragile immune system and environmental stress, this stage is often accompanied by diarrhea, growth retardation and increased mortality. Existing technologies mainly intervene in the following ways: (1) Regulating intestinal flora through exogenous probiotics, but there are problems such as low survival rate of live bacteria and unstable colonization effect. (2) Using antibiotic substitutes, such as antimicrobial peptides, plant essential oils, etc., but there are strong dose dependence, poor palatability or potential allergic risks. (3) Functional proteins, such as yolk antibodies (IgY) or recombinant proteins, although they can neutralize pathogens, are expensive and lack direct repair effects on the intestinal mucosa. Ovomucin, a mucin found only in natural egg white, possesses numerous biological activities, including antibacterial and antiviral activities. However, its application in rodents is understudied, particularly regarding its role in regulating weaning-induced diarrhea and survival. Current research on intervention strategies for weaning stress has primarily focused on large commercial species, such as pigs, while weaning intervention programs for rodents remain elusive. Rodents have unique intestinal microenvironments and immune response mechanisms, necessitating the development of functional factors tailored to their physiological characteristics. Summary of the Invention

[0003] Technical problem to be solved: In response to the above technical problems, the purpose of the present invention is to provide a feed additive and its application. First, ovomucin is prepared from poultry eggs, and then a nutritional composition is prepared with ovomucin as the core substance. Experimental results show that ovomucin enhances the intestinal mechanical barrier function of weaned rodents, promotes the production of intestinal epithelial immune proteins, reduces the incidence of diarrhea, increases the survival rate of weaned animals, and improves their growth performance. Ovomucin has no obvious acute toxicity. Ovomucin has the potential to be used as a functional feed additive and has good prospects for the preparation of special medicines, food compositions, food supplements and food additives for rodents to improve weaning survival rate and prevent and treat diarrhea.

[0004] Technical solution: A feed additive containing ovomucin. Furthermore, the feed additive is used to improve the weaning survival rate of rodents. Furthermore, the feed additive is used in preparing feed for preventing and treating diarrhea in rodents. Furthermore, the rodent is a mouse. Furthermore, the ovomucin component includes an ovomucin solution, an ovomucin sol, an ovomucin-containing powder and an enzymatic hydrolysis product of ovomucin. Furthermore, the preparation method of the ovomucin comprises the following steps: S1. Wash the egg shells with water, separate the egg whites into a clean container, add 0.1-0.3 mol / L NaCl solution to an amount 3-5 times the volume of the egg white, stir at 400-800 rpm for 30-40 minutes, adjust the pH to 6.0-6.5, and let stand at 4°C for 6-8 hours. S2. The above solution was centrifuged at 8000-12000 rpm for 15-20 min, the supernatant was discarded, 0.1-0.5 mol / L NaCl solution was added to the precipitate, stirred for 30-40 min, and allowed to stand for 2-4 hours. After that, the solution was centrifuged at 11000-12000 rpm for 15-20 min, the supernatant was discarded, and the precipitate was retained; S3. The obtained precipitate was dissolved in 0.01 mol / L edible sodium carbonate solution at a mass ratio of 1:(3-5) and stirred evenly. The precipitate was purified and desalted using a tangential flow ultrafiltration membrane with a molecular weight cutoff of 100 kDa in a flat membrane package system using a concentration-diafiltration combined mode. The retentate was collected and freeze-dried to obtain ovomucin. Furthermore, the concentration-diafiltration combined mode in S3 is specifically to concentrate the sample volume to 1 / 3 of the initial value, adjust the transmembrane pressure and tangential flow rate in stages, use 3-5 times the sample volume for dynamic diafiltration, and combine it with pulse backwash contamination control. Furthermore, the feed additive can be used to prepare oral compositions and food supplements. Furthermore, the dosage forms of the oral composition include tablets, powders, granules and capsules. Furthermore, the oral composition contains 0.1%-5% ovomucin. Beneficial effects: 1. The ovomucin nutritional composition prepared by the present invention can significantly absorb toxins in weaned mice, reduce the rate of soft stool diarrhea and the ammonia content in feces, and improve immune activity. The diarrhea rate of weaned mice fed with the ovomucin nutritional composition is reduced by 40%-50%, and the survival rate 7 days after weaning is increased by 25%-30%. The average daily weight gain of the animals is increased by 10%-15%, and the feed conversion rate is optimized, which can be further developed to promote intestinal health and immunity. 2. The egg white ovomucin prepared by the present invention has a wide range of sources and a mature extraction process, and its cost is only 1 / 3 to 1 / 2 of that of recombinant protein. It has no risk of drug resistance and no gastrointestinal side effects. It can be used in various forms and with various ingredients. Therefore, it can be used with other nutritional foods and health-care ingredients to further achieve synergistic effects, or be used in specially formulated compositions with specific effects. It will have a wide range of applications in ordinary foods, nutritional supplements, health foods, and medicines, and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 This is the HPLC elution curve of the ovomucin prepared in Example 1; Figure 2 Effects of the ovomucin-containing nutritional compositions prepared in Examples 2-3 and Comparative Examples 1-2 on the weight growth rate of normal mice during weaning; Figure 3 The disease activity index (DAI) score of weaned mice induced by bacterial endotoxin (LPS) was obtained by ovomucin intervention described in Examples 6-7 and Comparative Examples 5-7. DETAILED DESCRIPTION The present invention provides a feed additive and its application. In order to make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention will be further described in detail with reference to the following examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Example 1 The preparation of ovomucin comprises the following steps: S1. Wash the egg shells with water, separate the egg whites into a clean container, add 0.1 mol / L NaCl solution to 5 times the volume of the egg white, stir at 600 rpm using a magnetic stirrer for 30 min, adjust the pH to 6.0, and let it stand for 8 h. S2. The above solution was centrifuged at 12000 rpm for 15 min, the supernatant was discarded, 0.5 mol / L NaCl solution was added to the precipitate, stirred for 30 min, allowed to stand for 4 hours, and then centrifuged at 12000 rpm for 15 min. The supernatant was discarded and the precipitate was retained; S3. The obtained precipitate was added into 0.01 mol / L edible sodium carbonate solution for dissolution, the mass ratio of precipitate to sodium carbonate solution was 1:5, and after stirring evenly, a tangential flow ultrafiltration membrane with a molecular weight cutoff of 100 kDa was used in a flat membrane package system, and a concentration-diafiltration combined mode was adopted for purification and desalination. The gradient transmembrane pressure was 1.5 bar, and the tangential flow rate was 2.0 m / s. After the sample volume was concentrated to 1 / 3 of the initial value, the gradient transmembrane pressure was increased to 3.0 bar, and the tangential flow rate was increased to 2.5 m / s in stages. Five times the sample volume was used for dynamic diafiltration, and the retained liquid was collected for freeze-drying to obtain ovomucin. Performance Testing (1) HPLC monitoring of ovomucin purity Take 1 mg of freeze-dried protein powder and add 10 mL of distilled water, mix thoroughly, and filter using a 0.45 μm filter membrane. TM Liquid chromatography analysis was performed using a 3 μm SEC-3000LC column. The chromatographic conditions were: 20 μL sample load, 0.05 M phosphate buffer, pH 6.8 mobile phase, column temperature 30°C, flow rate 0.65 mL / min, and diode detection wavelength 280 nm. The HPLC results of the prepared ovomucin are shown in Figure 1. Figure 1 As shown, the peak corresponding to the retention time of about 7 minutes is the ovomucin signal. The area integrals corresponding to the four peaks are 1795983, 13955, 3020, and 12342, respectively. The purity of ovomucin is calculated using the peak area integral calculation formula: Example 2 The preparation of the nutritional composition containing low-dose ovomucin specifically comprises the following steps: S1. 15.7 g casein hydrolysate, 0.3 g L-cystine, 3.0 g whey powder, 30 g wheat flour, 30 g glucose, 4.75 g fructooligosaccharide, 7.88 g coconut oil, 2.62 g soybean oil, 3.53 g mineral mixture, and 1.0 g ovomucin were mixed and then 15 g ethanol was added. The mixture was granulated in a wet granulation apparatus and dried at 50°C for 12 h. S2. The dried substance is mixed with a vitamin mixture and a sucrose fatty acid ester in a mass ratio of 10:1:1, and a drying device is used to prepare a nutritional composition granule containing a low dose of ovomucoid. Example 3 The preparation of the nutritional composition containing a high dose of ovomucin specifically comprises the following steps: S1. 15.7 g casein hydrolysate, 0.3 g L-cystine, 2.0 g whey powder, 30 g wheat flour, 30 g glucose, 4.75 g fructooligosaccharide, 7.88 g coconut oil, 2.62 g soybean oil, 3.53 g mineral mixture, and 2.0 g ovomucin were mixed and then 15 g ethanol was added. The mixture was granulated in a wet granulation apparatus and dried at 50°C for 12 h. S2. The dried substance is mixed with a vitamin mixture and a sucrose fatty acid ester in a mass ratio of 10:1:1, and a drying device is used to prepare a nutritional composition granule containing a high dose of ovomucoid protein. Comparative Example 1 (blank control) The difference between this comparative example and Example 2 is that ovomucin is not added. The preparation of the nutritional composition without ovomucin specifically comprises the following steps: S1. 15.7 g casein hydrolysate, 0.3 g L-cystine, 4.0 g whey powder, 30 g wheat flour, 30 g glucose, 4.75 g fructooligosaccharide, 7.88 g coconut oil, 2.62 g soybean oil, and 3.53 g mineral mixture were mixed and then 15 g ethanol was added. The mixture was granulated in a wet granulation apparatus and dried at 50°C for 12 h. S2. The dried substance is mixed with a vitamin mixture and a sucrose fatty acid ester in a mass ratio of 10:1:1, and a drying device is used to prepare ovomucoid-free nutritional composition particles. Comparative Example 2 (positive control) The difference between this comparative example and Example 2 is that ovomucin is not added, and live bacteria granules of Bacillus subtilis are used instead of ovomucin. Example 4 An experiment investigating the effect of a low-dose ovomucin-containing nutritional composition on the survival and diarrhea of weaned mice involved the following steps: Twenty 21-day-old weaned BALB / c mice were purchased from Hunan SJA Experimental Animal Co., Ltd. and fed the nutritional composition containing ovomucin prepared in Example 2 at a dose of 50 mg / kg BW / d. The mice were maintained in an SPF-grade barrier environment with a relative humidity of 55-70%, an ambient temperature of 24-25°C, and a 12-hour day / night cycle. The mice were fed this composition continuously for seven days, with free access to food and water. The mice's weight change, food intake, mortality, and diarrhea symptoms were recorded daily. After the end of the feeding period, the survival rate, diarrhea rate, and weight gain rate of each group were calculated based on the number of mice that died and the number of mice that experienced diarrhea. Example 5 An experiment investigating the effect of a high-dose ovomucin-containing nutritional composition on the survival and diarrhea of weaned mice involved the following steps: Twenty 21-day-old weaned BALB / c mice were purchased from Hunan SJA Experimental Animal Co., Ltd. and fed the nutritional composition containing ovomucin prepared in Example 3 at a dose of 150 mg / kg BW / d. The mice were maintained in an SPF-grade barrier environment with a relative humidity of 55-70%, an ambient temperature of 24-25°C, and a 12-hour day / night cycle. The mice were fed this composition continuously for seven days, with free access to food and water. The mice's weight change, food intake, mortality, and diarrhea symptoms were recorded daily. After the end of the feeding period, the survival rate, diarrhea rate, and weight gain rate of each group were calculated based on the number of mice that died and the number of mice that experienced diarrhea. Comparative Example 3 The difference between this comparative example and Example 4 is that the mice were fed with the nutritional composition prepared in Comparative Example 1 that did not contain ovomucin. Comparative Example 4 The difference between this comparative example and Example 4 is that the nutritional composition prepared in Comparative Example 2 was fed to the mice. Performance Testing (1) sIgA content After feeding, the target intestinal tissue was quickly separated, cut open longitudinally, and rinsed with pre-cooled phosphate buffered saline (PBS, pH 7.4) to remove the contents. The mucosal layer tissue was gently scraped with a glass slide and weighed. A solution containing protease inhibitors (1× cOmplete 500) was added at a ratio of 1:9 (mass to volume). TM The cells were homogenized in a lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, pH 7.4) prepared by Roche (Roche). The homogenate was homogenized three times on ice at 10,000 rpm for 10 seconds each, with 30-second intervals. The homogenate was centrifuged at 12,000 × g for 15 minutes at 4°C, and the supernatant was collected. The sIgA content in the supernatant was directly read on a microplate reader using the commercial mouse sIgA detection kit (CUSABIO CSB-E08336m) in strict accordance with the manufacturer's instructions. The sIgA content in intestinal tissue was calculated according to the following formula: Table 1 Statistics of survival rate and diarrhea rate of weaned mice Depend on Figure 2It can be clearly seen from the statistical data in Table 1 that in the experimental group of 3-week-old weaned mice, because the young mice are of low age and weight, their tolerance to environmental stress is weak, and their weight decreases after weaning stress occurs. After ovomucin intervention, the weight loss caused by weaning stress can be significantly alleviated; the sIgA content of Example 5 is 2.0 times that of Comparative Example 1, indicating that ovomucin intervention promotes the secretion of intestinal immune factor sIgA; since weaning can cause stress reactions in mice, leading to intestinal dysfunction, thereby causing diarrhea, and severe cases can cause death. The incidence of diarrhea in Comparative Example 3 was 46%, and the survival rate was 65%. During the weaning process, the ovomucin intervention described in Examples 4 and 5 can reduce the incidence of diarrhea to 10% or less, and the survival rate is increased to more than 90%, indicating that the intervention of the ovomucin nutritional composition can significantly improve the diarrhea and death of mice caused by weaning stress, and the intervention effect is significantly better than the intervention effect of the commercial probiotics shown in Comparative Example 4. Example 6 An intervention experiment on the effect of low-dose ovomucin on the survival and diarrhea of weaned mice induced by bacterial toxins specifically included the following steps: 20 21-day-old weaned BALB / c mice were purchased from Hunan SJA Experimental Animal Co., Ltd. and housed at the Experimental Animal Center of Northwest Agriculture and Forestry University. All mice were fed a basal diet containing Escherichia coli lipopolysaccharide (LPS) toxin, wherein the LPS content was 25 mg / kg. The ovomucin prepared in Example 1 was orally administered at a fixed time daily at a dose of 50 mg / kg body weight per day in an SPF-grade barrier environment with a relative humidity of 55-70%, an ambient temperature of 24-25°C, and a 12-hour day-night cycle for 14 consecutive days, with free access to food and water. Example 7 The intervention experiment of high-dose ovomucin on the survival and diarrhea of weaned mice induced by bacterial toxins was different from Example 6 in that the intervention dose of ovomucin prepared in Example 1 was 150 mg / kg body weight and administered orally at a fixed time every day. Other procedures were the same as in Example 6. Comparative Example 5 (Model Control) The difference between this comparative example and Example 6 and Example 7 is that no ovomucin gavage intervention was performed, and the mice ate and drank normally. Other operations were exactly the same as in Example 7. Comparative Example 6 (positive intervention control) The difference between this comparative example and Example 7 is that ovomucin gavage intervention is not performed, and 10% neomycin sulfate (antibiotic) is gavaged daily instead of ovomucin intervention. Other operations are exactly the same as those in Example 7. Comparative Example 7 (blank control) The difference between this comparative example and comparative example 5 is that the mice were fed a basic feed without LPS, and had a normal diet and water intake without any intervention. Other operations were exactly the same as in Example 5. Performance Testing (1) Disease Activity Index (DAI) The disease activity index (DAI) is a comprehensive score based on the percentage of weight loss, stool consistency, and bleeding in the diseased animals. The total score of the three results is divided by 3 to obtain the DAI value. During the experiment, the weight, stool status, and bloody stool of the mice were recorded daily, and the DAI index of each mouse was calculated according to the standards shown in Table 2. Table 2 Scoring criteria for various DAI indicators The DAI index results of each mouse are as follows Figure 3 As shown, the blank control shown in Comparative Example 7 showed that the DAI value did not increase significantly with the extension of the feeding time, indicating that the diarrhea caused by weaning will improve with the development of the animal's intestinal immune system. However, exposure to bacterial toxins during the weaning process will significantly aggravate intestinal diarrhea and bloody stools. As the bacterial toxin LPS intervention time is prolonged, the DAI value of the model group mice shown in Comparative Example 5 is significantly increased, indicating that the bacterial toxin LPS significantly aggravates the clinical symptoms of diarrhea and bloody stools induced by weaned mice. However, after intervention with ovomucin, whether in Example 6 or Example 7, the DAI value changes tended to be gentle and had significant differences from the model group, indicating that after ovomucin intervention, the increase in the disease activity index caused by LPS was improved, and this improvement effect was better than the positive antibiotic intervention shown in Comparative Example 6. After the experiment, the total mortality rate, diarrhea rate, and bloody stool rate for each group were calculated, and the results are shown in Table 3. The experiment found that in the experimental group of mice weaned on day 21, due to their younger age and weight, they had a weaker tolerance to environmental stress and lost weight after weaning stress. In particular, in the model group (Comparative Example 5) exposed to bacterial toxins, mice became listless and had blood in their stools, with deaths occurring on day 4. At the end of the modeling, the survival rate of mice in Comparative Example 5 was only 15%, while the survival rates of the two groups treated with ovomucin (Examples 6 and 7) were 65% and 85%, respectively, and the survival rate was positively correlated with the dose of ovomucin ingested. Table 3 Statistics of survival rate and diarrhea rate of weaned mice induced by bacterial toxins (2) IL10, IL6, and TNF-α levels in mouse serum and intestinal sIgA content After the feeding period, serum samples were collected from each group, and the IL10, IL6, TNF-α levels in the serum and the intestinal sIgA content in the mice of different groups were detected using ELISA kits. The results are shown in Table 4. Table 4 Effects of ovomucin on the concentrations of cytokines related to inflammation and stress in weaned mice induced by bacterial toxin LPS As can be seen in Table 4, compared with Comparative Example 7, the concentrations of the pro-inflammatory factors TNF-α and IL-6 in Comparative Example 5 were significantly increased, while the concentration of the anti-inflammatory factor IL-10 was significantly decreased, further demonstrating that LPS exacerbates the systemic inflammatory stress response in weaned mice. In contrast, after ovomucin intervention as shown in Examples 6 and 7, the levels of TNF-α and IL-6 were significantly suppressed, while the level of IL-10 was the opposite, indicating that ovomucin's effect on diarrhea is related to inhibiting the activation of these stress and inflammatory cytokines. Furthermore, the intestinal sIgA content in Example 7 was 2.4 times that of Comparative Example 5, indicating that ovomucin intervention promoted the secretion of the intestinal immune factor sIgA, ameliorating oxidative stress and intestinal immune imbalance caused by bacterial toxins. Example 8 The toxicity and safety of ovomucin were evaluated with reference to the test method of "GB / T 23179 Feed Toxicological Evaluation - Subacute Toxicity Test", comprising the following steps: 20 SD rats, half male and half female, were orally gavaged with the nutritional composition containing ovomucin prepared in Example 3 at a maximum tolerated dose (MTD) of 20 g / kg BW. The symptoms of poisoning, such as performance, abnormal behavior, and death of the test rats were recorded within 14 days. The test animals were weighed on day 1, day 7, and day 14, and then the animals were sacrificed and grossly dissected to observe changes in organs such as the liver, kidney, spleen, heart, and lungs. Table 5 Results of acute toxicity test of ovomucin on rats As shown in Table 5, both male and female rats gained weight over the 14-day period, with no acute poisoning symptoms or mortality observed. Autopsies revealed that the liver, kidneys, spleen, heart, lungs, stomach, and intestines of the mice were all normal, demonstrating that the ovomucin composition had an acute toxicity (MTD) greater than 20 g / kg BW in rats, placing it at a non-toxic level according to the acute toxicity classification standard. The above examples used a weaned BALB / c mouse model to investigate the effects of oral administration of an ovomucin-containing nutritional composition on the occurrence of diarrhea and mortality caused by weaning stress. The results demonstrated that ovomucin significantly improved intestinal immunity and significantly reduced mortality and diarrhea caused by weaning stress and bacterial toxins, while exhibiting no significant acute toxicity. The composition has the potential to be used as a functional feed additive and holds great promise for future applications. The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, use the methods and technical contents disclosed above to make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A feed additive, characterized in that The feed additive contains ovomucin component.

2. Use of a feed additive according to claim 1 in improving the weaning survival rate of rodents.

3. Use of the feed additive according to claim 1 in preparing feed for preventing and treating diarrhea in rodents.

4. The use according to claim 2 or 3, characterized in that The rodents are mice.

5. A feed additive according to claim 1, characterized in that, The ovomucin component includes ovomucin solution, ovomucin sol, ovomucin-containing powder and ovomucin enzymatic hydrolysis product.

6. A feed additive according to claim 1, characterized in that, The preparation method of the ovomucin comprises the following steps: S1. Wash the egg shells with water, separate the egg whites into a clean container, add 0.1-0.3 mol / L NaCl solution to an amount 3-5 times the volume of the egg white, stir at 400-800 rpm for 30-40 minutes, adjust the pH to 6.0-6.5, and let stand at 4°C for 6-8 hours. S2. The above solution was centrifuged at 8000-12000 rpm for 15-20 min, the supernatant was discarded, 0.1-0.5 mol / L NaCl solution was added to the precipitate, stirred for 30-40 min, and allowed to stand for 2-4 hours. After that, the solution was centrifuged at 11000-12000 rpm for 15-20 min, the supernatant was discarded, and the precipitate was retained; S3. The obtained precipitate was dissolved in 0.01 mol / L edible sodium carbonate solution at a mass ratio of 1:(3-5) and stirred evenly. The precipitate was purified and desalted using a tangential flow ultrafiltration membrane with a molecular weight cutoff of 100 kDa in a flat membrane package system using a concentration-diafiltration combined mode. The retentate was collected and freeze-dried to obtain ovomucin.

7. A feed additive according to claim 6, characterized in that, The concentration-diafiltration combined mode in S3 is specifically to concentrate the sample volume to 1 / 3 of the initial value, and then adjust the transmembrane pressure and tangential flow rate in stages, use 3-5 times the sample volume for dynamic diafiltration, and combine it with pulse backwash contamination control.

8. A feed additive according to claim 1, characterized in that, The feed additive can be used to prepare oral compositions and food supplements.

9. A feed additive according to claim 8, characterized in that, The dosage forms of the oral composition include tablets, powders, granules and capsules.

10. A feed additive according to claim 8, characterized in that: The oral composition contains 0.1%-5% ovomucin.

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