Feed additive for replacing zinc oxide and antibiotics and application thereof

Abstract

The invention provides a feed additive for replacing zinc oxide and antibiotics and application thereof. The weaned piglet feed rich in IPA and IAA with proper concentrations can improve the growth performance and intestinal health of piglets, relieve intestinal flora disorder caused by weaning stress, enhance the intestinal barrier function and recover the intestinal flora structure and functional diversity, and IPA and IAA have a synergistic effect. The invention provides a theoretical basis for application of IPA and IAA in livestock and poultry production, and also provides product reserve for feed resistance forbidding, breeding resistance reduction and reduction of zinc oxide use in China.

Description

Technical Field

[0001] The present invention relates to the field of feed, and in particular to a feed additive that replaces zinc oxide and antibiotics and its application (i.e., a feed that completely replaces zinc oxide and antibiotics, alleviates animal weaning stress, and improves intestinal barrier and flora function and its application). Background Art

[0002] In modern pig production, weaning stress syndrome (WSS) is a key challenge hindering the efficient development of the livestock industry. Due to multiple factors, including feed form changes, environmental stress, maternal antibody decline, and psychological maladjustment, weaned piglets often experience impaired intestinal barrier function, decreased immune systems, and intestinal microbial imbalances. These factors lead to bacterial diarrhea, growth retardation, and high mortality, resulting in billions of dollars in annual economic losses for the global pig industry. Traditional antibiotics are gradually being withdrawn from the market due to the risk of drug resistance and regulatory restrictions. Probiotics and prebiotics, as alternatives, can alleviate weaning stress by modulating the intestinal microbiome or enhancing digestive function, but their targeted effects are limited and their stability is limited. Zinc oxide, due to its advantages in astringency, immune regulation, and pathogen suppression, is widely used in weaned pig feeds. However, the low bioavailability of pharmacologically dosed zinc oxide leads to zinc excretion and environmental contamination, metabolic imbalances, intestinal microbial disturbances, and target organ accumulation, inducing oxidative damage, highlighting the unsustainable use of zinc oxide. The European Union has completely banned high-dose ZnO by 2022, and my country also restricts the use of 1600mg / kg of zinc oxide or basic zinc chloride to the first two weeks after weaning. Therefore, there is an urgent need to develop new, growth-promoting, environmentally friendly, and anti-zinc alternatives to promote the sustainable development of my country's feed industry. Developing a feed additive that can regulate intestinal health through multiple targets, reduce pathogen infection, and mitigate the risks of toxic residues and drug resistance is a key technological breakthrough for improving weaning stress syndrome and enhancing growth performance in piglets. Summary of the Invention

[0003] The purpose of the present invention is to provide a feed additive that replaces zinc oxide and antibiotics and its application (i.e., an antibiotic-free and zinc oxide-free feed that relieves animal weaning stress and maintains intestinal barrier function and microflora homeostasis and its application).

[0004] The present invention is based on the following concept: In the post-antibiotic era, zinc oxide offers significant advantages in promoting growth and combating diarrhea in weaned piglet diets. However, it presents numerous challenges, including environmental pollution, residual effects in vivo, and bacterial resistance. As naturally occurring small-molecule organic acids, IPA and / or IAA may offer the potential to replace antibiotics and zinc oxide in livestock and poultry production. This study aims to investigate the effects of dietary supplementation with IPA and an equal-dose mixture of IPA and IAA on growth performance, diarrhea rates, and intestinal health in weaned piglets.

[0005] To achieve the purpose of the present invention, in a first aspect, the present invention provides a feed additive that replaces zinc oxide and antibiotics, the feed additive comprising component (a) indole-3-propionic acid (IPA); and component (b) indole-3-acetic acid (IAA); The weight ratio of components a and b in the feed additive is 1:4 to 4:1 (preferably 1:1).

[0006] In a second aspect, the present invention provides a use of the feed additive as a feed additive for alleviating animal weaning stress and improving intestinal flora function (achieving zero addition of zinc oxide and antibiotics).

[0007] The animals include, but are not limited to, pigs.

[0008] In a third aspect, the present invention provides a feed for alleviating weaning stress in animals and improving intestinal barrier and flora function, wherein the feed contains the composition and is a low-protein diet feed without zinc oxide, antibiotics and other growth-promoting regulators.

[0009] Preferably, each kilogram of feed contains 100-400 mg of indole-3-propionic acid and 100-400 mg of indole-3-acetic acid (preferably 400 mg of indole-3-propionic acid and 400 mg of indole-3-acetic acid).

[0010] Furthermore, the crude protein CP level of the low-protein diet is 16%-17.5%.

[0011] In a fourth aspect, the present invention provides any one of the following applications of the feed: 1) Used to prepare biological products that can relieve weaning stress in piglets, improve growth performance of weaned piglets, reduce feed-to-weight ratio, increase average body weight and reduce diarrhea rate; 2) Used to prepare biological products that improve the antioxidant capacity and immune function of weaned piglets; 3) Used to promote the proliferation of pig intestinal epithelial cells, increase the diversity of intestinal flora, and promote the growth and reproduction of beneficial intestinal flora in weaned piglets; 4) Used to prepare biological products for improving oxidative stress and intestinal damage in piglets caused by weaning stress.

[0012] Furthermore, under in vitro culture conditions, the addition amounts of IAA and IPA in the culture medium used to promote the proliferation of porcine intestinal epithelial cells were 100~200 μmol / L and 100~200 μmol / L, respectively.

[0013] Furthermore, the beneficial bacteria include but are not limited to microorganisms of the genera Bacteroides, Eubacterium, Acidaminococcus, Anaerofustis, Peptococcus, Alloprevotella, Pseudoramibacter, Lachnospiracea_incertae_sedis, and Oscillibacter.

[0014] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention discovered that a weaned piglet feed enriched with appropriate concentrations of IPA and IAA can improve piglet growth performance and intestinal health, alleviate intestinal flora disturbances caused by weaning stress, improve intestinal barrier function, enhance epithelial cell proliferation activity, and restore intestinal flora structure, function, and diversity. Furthermore, IPA and IAA exhibit a synergistic effect. Experimental results showed that supplementing the diet with 400 mg / kg IAA and 400 mg / kg IPA significantly increased the average daily weight gain of weaned piglets, significantly reduced diarrhea rates, and tended to reduce feed-to-weight ratios. Compared with supplementing the diet with 800 mg / kg IPA or 800 mg / kg IAA alone, a diet supplemented with a combination of 400 mg / kg IAA and 400 mg / kg IPA resulted in a higher percentage of weight gain in piglets during the same period, and exhibited superior growth-promoting and anti-diarrhea effects. Furthermore, under in vitro culture conditions, supplementing the culture medium with 100 μmol / L IAA and 100 μmol / L IPA increased the proliferation activity of pig intestinal epithelial cells. The present invention provides a theoretical basis for the application of IPA and IAA in livestock and poultry production, and also provides product reserves for my country's feed antibiotic ban, breeding antibiotic reduction and reduced use of zinc oxide. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The figure shows the effect of different concentrations of IAA and IPA on the proliferation activity of porcine intestinal epithelial cells IPEC-J2 after 24 h of treatment in a preferred embodiment of the present invention.

[0016] Figure 2 The figure shows the effects of different concentrations of IPA treatment for 24 h on the proliferation activity of IPEC-J2 and the cell survival rate under oxidative stress in a preferred embodiment of the present invention.

[0017] Figure 3 This is the effect of adding IPA to the diet on the α and β diversity of the rectal fecal flora in piglets in a preferred embodiment of the present invention.

[0018] Figure 4This is the effect of adding IPA to the diet in a preferred embodiment of the present invention on the species composition of the rectal fecal flora at the genus level in piglets during the 14th and 28th day of the experimental period. Figure 5 This is a heat map of the differences in genus levels of rectal fecal flora in piglets during the 14-day experimental period in a preferred embodiment of the present invention.

[0019] Figure 6 This is a heat map of the differences in genus levels of rectal fecal flora in piglets during the 28-day experimental period in a preferred embodiment of the present invention.

[0020] Figure 7 This is a KO volcano plot of the differences in intestinal flora function regulated by 200, 400 and 800 mg / kg IPA during the 28-day test period in a preferred embodiment of the present invention.

[0021] Figure 8 This is the KEGG pathway for regulating the functional differences of intestinal flora in the preferred embodiment of the present invention when 200, 400 and 800 mg / kg IPA were used for the 28-day test period. DETAILED DESCRIPTION

[0022] The present invention mainly investigates the effects of adding a mixture of indole-3-propionic acid (IPA) and / or indole-3-acetic acid (IAA) to the diet on the growth performance, diarrhea rate, and intestinal flora structure and functional composition of weaned piglets.

[0023] The present invention adopts the following technical solutions: Experiment 1: Porcine small intestinal epithelial cells IPEC-J2 were cultured in DMEM / F12 complete medium (containing 10% FBS and 1% penicillin-streptomycin-neomycin antibiotic mixture) containing different concentrations of IAA (0 ~ 200 μmol / L) and / or IPA (0 ~ 200 μmol / L). The experimental results showed that the combined treatment of IPEC-J2 cells with IAA and IPA significantly increased the cell proliferation activity compared with the control group and the group with either IAA or IPA alone ( P <0.05), and there was a synergistic effect; higher concentrations of IPA (250 ~ 1,000 μmol / L) co-cultured with porcine small intestinal epithelial cells IPEC-J2 did not cause a decrease in cell viability and cell membrane damage, but significantly improved cell proliferation activity ( P <0.05, with good safety; appropriate concentration of IPA significantly improved the decreased proliferation activity of intestinal epithelial cells caused by oxidative stress ( P <0.05), increased cell survival rate, and improved intestinal barrier function.

[0024] Experiment 2: 270 healthy Durbin × Chang × Da hybrid weaned piglets aged 28 days were selected and randomly divided into 6 dietary treatment groups: control group (basic diet), positive control group supplemented with 2000 mg / kg zinc oxide in the basal diet, experimental group supplemented with 200, 400 and 800 mg / kg IPA or 800 mg / kg IAA in the basal diet, with 5 replicates per group and 9 pigs in each replicate. The experiment lasted for 28 days, and piglet feces were collected on the 14th and 28th days of the experiment. The results showed that IPA can significantly reduce the feed-to-weight ratio on the 15th to 28th days and the 0th to 28th days of the experiment, and significantly reduce the diarrhea rate; IPA can regulate the community structure and species composition of the intestinal microorganisms of piglets, and regulate the functional composition of the intestinal microorganisms of piglets.

[0025] Experiment 3: 144 healthy, 28-day-old weaned piglets of a Duroc×Chang×Dalmatian hybrid were randomly divided into three dietary treatment groups: a control group (basal diet), a positive control group supplemented with 2000 mg / kg zinc oxide in the basal diet, and an experimental group supplemented with 400 mg / kg IAA and 400 mg / kg IPA in the basal diet. Each group had six replicates, with eight pigs per replicate, and the experiment lasted 14 days. Results showed that the addition of 400 mg / kg IAA and 400 mg / kg IPA to the diet significantly increased the average daily gain of weaned piglets, significantly reduced the rate of diarrhea, and showed a trend toward lower feed-to-gain ratio. Compared with the diet supplemented with 800 mg / kg IPA or 800 mg / kg IAA alone, the diet supplemented with a combination of 400 mg / kg IAA and 400 mg / kg IPA resulted in a higher percentage of weight gain in piglets during the same period, and demonstrated superior growth-promoting and anti-diarrhea effects.

[0026] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0027] Example 1 1. Materials and Methods Experiment 1: Porcine small intestinal epithelial cells IPEC-J2 were co-cultured with different concentrations of IAA (0-200 μmol / L) and / or IPA (0-200 μmol / L). Cell proliferation activity was detected using the CCK8 assay, and the interaction effect was calculated using the isodose effect and King's formula.

[0028] Experiment 2: Different concentrations of IPA (250 ~ 1,000 μmol / L) were used to co-culture with porcine small intestinal epithelial cells IPEC-J2. H2O2 was added to induce intestinal epithelial damage, and the cell proliferation activity was detected using the CCK8 method.

[0029] 2. Results and Analysis 2.1 IAA and IPA synergistically enhance the proliferation activity of porcine jejunal epithelial cells IPEC-J2 The experiment investigated the effects of adding 0, 100, and 200 μmol / L IAA and IPA alone or in combination on the proliferation activity of IPEC-J2 cells. Quantitative analysis using the CCK-8 assay showed that compared with the control group, all five treatment groups significantly increased cell proliferation activity ( P <0.05); compared with the combined treatment group of 100 μmol / L IAA and 100 μmol / L IPA, the cell viability of the groups treated with 100 μmol / L IAA or IPA alone and 200 μmol / L IAA alone was significantly lower than that of the combined treatment group ( P <0.05) ( Figure 1 Based on the isodose effect, the cell viability of 100 μmol / L IAA + 100 μmol / L IPA was significantly higher than that of 200 μmol / L IAA, indicating a synergistic effect between IAA and IPA. According to King's formula, the cell viability of 100 μmol / L IAA + 100 μmol / L IPA was significantly higher than that of either 100 μmol / L IAA or 100 μmol / L IPA, indicating a synergistic effect calculated using King's formula. In summary, IAA and IPA have a synergistic effect on the proliferation and viability of porcine jejunal epithelial cells within an appropriate concentration range (Table 1).

[0030] Table 1 Effects of IPA, IAA, or a combination of IPA and IAA in the culture medium on the proliferation activity of porcine jejunal epithelial cells IPEC-J2 2.2 IPA alleviates oxidative stress injury in porcine jejunal epithelial cells IPEC-J2 The experiment investigated the effects of IPA on the proliferation activity and oxidative stress damage of porcine intestinal epithelial cells. The CCK-8 assay was used to determine the cell proliferation activity of IPEC-J2 cells after treatment with different concentrations of IPA for 24 hours. The effect of adding 1000 μmol / L hydrogen peroxide for 4 hours after treatment with different concentrations of IPA for 24 hours on cell survival was also determined. The results showed that compared with the control group, 500, 750, and 1000 μmol / L IPA significantly increased cell proliferation activity ( P <0.05), maintain and promote the growth of IPEC-J2 cells ( Figure 2 Compared with the oxidative stress model group, 500 and 750 μmol / L IPA significantly increased cell survival rate ( P <0.05), relieve cellular oxidative stress, and improve intestinal epithelial barrier function ( Figure 3 ).

[0031] Example 2 1. Materials and Methods 1.1 Experimental Design and Experimental Animals Experiment 1: Weaned piglet trial (addition of IAA or IPA alone) A total of 270 healthy, 28-day-old weaned piglets (Durham × Changhe × Dahua hybrids) were randomly assigned to six dietary treatments using a randomized complete block design, split equally between males and females, and based on sex and body weight: a control diet (basal diet); a positive control diet supplemented with 2000 mg / kg zinc oxide; and experimental diets supplemented with 200, 400, and 800 mg / kg IPA or 800 mg / kg IAA, respectively. Each group had five replicates, with nine pigs per replicate, for a 28-day experiment. Serum and feces were collected on days 14 and 28.

[0032] Experiment 2: Weaned piglet trial (IAA and IPA added together) A total of 144 healthy, 28-day-old weaned piglets of a Duroc × Chang × Da hybrid were randomly assigned to three dietary treatments using a randomized complete block design, split equally between males and females, based on sex and body weight: a control group (basal diet), a positive control group supplemented with 2000 mg / kg zinc oxide, and an experimental group supplemented with 400 mg / kg IAA and 400 mg / kg IPA. Each group had six replicates, with eight pigs per replicate, and the experiment lasted 14 days. IPA and IAA were purchased from Hubei Jiufenglong Chemical Co., Ltd.

[0033] 1.2 Experimental diet and feeding management This experiment was conducted at the Fengning Animal Experimental Base of the Ministry of Agriculture and Rural Affairs' Feed Industry Center. A full-in, full-out housing model was adopted. The piggery temperature was maintained at 22 ± 3°C, the humidity was 50%–70%, and the floors within the pens were slatted. Piglets had free access to food and water and were fed 3–4 times daily. The pens were cleaned daily, and the drinking fountains and feed troughs within were maintained clean and hygienic. Piglet immunizations were performed according to the farm's routine procedures. During the experiment, weaned piglets' feed intake and health were observed and recorded daily. Feed intake was recorded for each pen, and diarrhea was recorded daily in the morning and afternoon. The experimental diets were all mash, formulated according to the national standard (China Pig Nutrient Requirements GB / T 39235-2020, 8–25 kg Piglets), to meet the nutritional needs of the piglets. No antibiotics or zinc oxide were added to the basal diet. The composition and chemical composition of the experimental basal diet are shown in Table 2.

[0034] 1.3 Test sample collection On days 14 and 28 of the experiment, two piglets were selected from each replicate, and rectal feces were collected by sampling swabs. The samples were immediately placed in liquid nitrogen and then stored in a -80°C refrigerator for fecal microbial sequencing analysis.

[0035] 1.4 Detection indicators and measurement methods (1) Growth performance Piglets were weighed on replicate days 0, 14, and 28 to calculate average daily gain (ADG) for each replicate and for the entire experiment. Piglets were fasted for 12 hours before weighing. Feed consumption was recorded for each replicate to calculate average daily feed intake (ADFI). Feed-to-gain ratio (FGR) was calculated based on ADG and ADFI.

[0036] Average daily gain (ADG) = (final weight - initial weight) / number of days of experiment Average daily feed intake (ADFI) = feed consumption / (number of piglets per group × number of experimental days) Feed to Gain Ratio (F / G) = Average Daily Feed Intake / Average Daily Gain (2) Diarrhea rate Diarrhea was assessed in piglets from days 0 to 28: The health and diarrhea status of each piglet was observed and recorded daily at 9:00 AM and 3:00 PM during the experimental period. Piglet feces were blindly evaluated and scored according to the following fecal scoring criteria: 0 for normal cylindrical solid feces, 1 for soft but solid feces, 2 for sticky, unformed feces with a high moisture content, and 3 for liquid, unformed feces with a high moisture content. Piglets with fecal scores of 2 or 3 for two consecutive days were considered to have diarrhea.

[0037] Diarrhea rate (%) = Number of piglets with diarrhea during the experimental period / (number of piglets in each group × number of experimental days) × 100 (3) Fecal microbiome DNA extraction and 16S rRNA gene sequencing The experimental operation and data processing were completed by the sequencing company.

[0038] 1.5 Data Statistical Analysis The data obtained from the experiment were organized using Microsoft Excel 2019, and all data were tested for normal distribution and homogeneity of variance. Cell data were analyzed by one-way analysis of variance using GraphPad Prism 7.0 software and plotted. Piglet results were analyzed by one-way analysis of variance ANOVA. If there were significant differences between treatments, Duncan's method was used for multiple comparisons. GraphPad Prism 7.0 was used for plotting, and the results were expressed as mean ± standard error of the mean (SEM). The significance threshold for all data was set to P <0.05 indicates significant difference. P <0.01 indicates extremely significant difference, 0.05< P <0.10 indicated a statistical trend in the difference.

[0039] Table 2 Basic diet composition and nutrient levels (%, feeding basis) Note: Vitamin and mineral premix provides the following nutrients per kg of diet: Vitamin A, 12,000 IU; Vitamin D3, 2,500 IU; Vitamin E, ≥ 30 IU; Vitamin K3, ≥ 2.5 mg; Vitamin B1, ≥ 2.5 mg; Vitamin B2, ≥ 4 mg; Vitamin B6, ≥ 3 mg; Vitamin B 12 , ≥ 0.02 mg; niacinamide, ≥ 40 mg; pantothenic acid, ≥ 12.5 mg; folic acid, ≥ 0.7 mg; biotin, ≥ 0.14 mg; iron, 0.3 g; copper, 0.09 g; zinc, 0.09 g; manganese, 0.05 g; iodine, 0.5 mg; selenium, 0.33 mg. Ratios are expressed in weight percentage.

[0040] 2. Results and Analysis 2.1 Adding IPA and IAA to feed has a synergistic effect and improves the growth performance of piglets The effects of adding IPA, IAA, or a combination of IPA and IAA to the diet on the weight of weaned piglets are shown in Table 3. The results of the synergistic effect calculated by the equal-dose verification method are shown in Table 4. Compared with the addition of 800 mg / kg IPA (Comparative Example 1) or 800 mg / kg IAA (Comparative Example 2) alone, the diet supplemented with a mixture of 400 mg / kg IAA and 400 mg / kg IPA (Example 1) resulted in a higher percentage of weight gain in piglets during the same period, and a better growth-promoting effect.

[0041] Table 3 Effects of dietary supplementation with IPA, IAA, or a combination of IPA and IAA on body weight of weaned piglets

[0042] Table 4 Synergistic effect of dietary supplementation with IPA, IAA, or a combination of IPA and IAA on body weight and daily gain of weaned piglets 2.2 The addition of IPA and IAA to feed significantly improved the growth performance and diarrhea rate of weaned piglets, and there was no significant difference compared with the positive control group supplemented with zinc oxide. The effects of adding IPA and IAA combination to the diet on the growth performance and diarrhea rate of weaned piglets are shown in Table 5. The results showed that compared with the control group, the IPA and IAA combination significantly increased the average daily weight gain of piglets from day 0 to 14 of the experiment and significantly reduced the diarrhea rate ( P<0.05), and the feed-to-weight ratio showed a decreasing trend. Compared with the positive control group, the positive effects of IPA and IAA combination on growth promotion and anti-diarrhea in piglets were not significantly different from those of zinc oxide supplementation.

[0043] Table 5 Effects of dietary IPA and IAA combination on growth performance and diarrhea rate in weaned piglets 2.3 IPA improved the growth performance and diarrhea rate of weaned piglets, but there was no significant difference compared with the same dose of IAA The effects of dietary IPA on growth performance and diarrhea rates in weaned piglets are shown in Table 6. The results showed that IPA had no significant effect on average daily gain and daily feed intake in weaned piglets, but it significantly reduced feed-to-gain ratios during the experimental periods of days 15-28 and 0-28. Regarding diarrhea rates, dietary IPA reduced them to varying degrees compared to the control group. Doses of 400 and 800 mg / kg reduced diarrhea rates by half, to 3.37 and 4.32, respectively. There was no significant difference between 800 mg / kg IPA and 800 mg / kg IAA.

[0044] Table 6 Effects of dietary IPA on growth performance and diarrhea rate in weaned piglets 2.4 IPA significantly regulates the rectal fecal flora of weaned piglets The study further investigated the effects of dietary IPA supplementation on the diversity, richness, and species composition of rectal fecal microbiota in piglets. Fresh rectal feces were collected from the control group and the 200-800 mg / kg IPA-treated groups on days 14 and 28 of the experiment. A total of 4,637,904 optimized sequences were obtained, with an effective length of 1,926,537,690 bp and an average length of 415 bp. Non-repeated sequences were clustered using the USEARCH11-uparse algorithm at 97% similarity. Chimeras were removed during the clustering process to obtain representative sequences and OTU counts for all rectal fecal microbiota OTUs.

[0045] (1) IPA can regulate the community structure and species composition of intestinal microorganisms in piglets 16S rDNA sequencing was used to detect changes in the structural and functional composition of intestinal microorganisms. Figure 3A and 3B show that compared with the blank control group, IPA had no significant effect on the richness and uniformity of the rectal fecal flora at 14 and 28 days of the experimental period. PCoA analysis based on Bray-Curtis showed that the addition of IPA to the diet could affect the overall structure and composition of the intestinal microbial community at 28 days of the experimental period, and feeding 400 mg / kg IPA had the greatest effect on the intestinal flora ( Figure 3 C). In terms of species composition, the top five genera in terms of relative abundance in intestinal microorganisms are mainly Lactobacillus, Clostridium_sensu_stricto, Prevotella and Blautia ( Figure 4 A and Figure 4 B). Further genus-level difference analysis found that different feeding levels of IPA had different effects on species composition. On day 14 of the experiment, 200 mg / kg IPA increased the relative abundance of Anaerofustis and decreased the abundance of Desulfovibrio and Olsenella; 400 mg / kg IPA significantly increased the relative abundance of Acidaminococcus and Peptococcus and significantly decreased the relative abundance of Streptococcus; interestingly, feeding 800 mg / kg IPA increased the abundance of Escherichia / Shigella and decreased the abundance of Gemmiger ( Figure 5 ). When the experimental period was 28 days, the number of differential genera affected by feeding IPA was also greater ( Figure 6 Compared with the control group, 200 mg / kg IPA increased the relative abundance of Alloprevotella and Pseudoramibacter, and decreased the abundance of Prevotella. 400 mg / kg IPA increased the relative abundance of Bacteroides, Eubacterium, Lachnospiracea_incertae_sedis, and Oscillibacter, and decreased the relative abundance of Actinobacillus, Faecalibacterium, and Roseburia. Compared with the 200 and 400 mg / kg IPA-supplemented groups, 800 mg / kg IPA increased the relative abundance of Lachnospiracea_incertae_sedis and Olsenella, and decreased the relative abundance of Fusicatenibacter.

[0046] (2) IPA regulates the functional composition of piglet intestinal microorganisms Picrust2 was used to analyze how dietary IPA affects the functional composition of the intestinal microbiome in piglets. As shown in the volcano plot, high concentrations of IPA had no significant effect on the functional KOs of the microbiome, while 200 mg / kg and 400 mg / kg could affect the KO composition of the microbiome. 200 mg / kg IPA significantly reduced the expression of 25 KOs and increased the expression of 444 KOs; 400 mg / kg IPA significantly reduced the expression of 98 KOs and increased the expression of 151 KOs ( Figure 7 A. Figure 7 B and Figure 7 C). LeFSe was further used to analyze the KEGG pathways regulated by IPA: 200 mg / kg IPA increased the expression of sucrose degradation, butanediol synthesis, and urea cycle pathways, and decreased the expression of PreQ0 biosynthesis, lipid molecule mycolate synthesis, TDP synthesis, and other pathways; 400 mg / kg IPA promoted the expression of pathways such as the urea cycle, pyruvate fermentation, and aspartate synthesis, and inhibited the expression of pathways such as NAD synthesis, polyamine synthesis, and aromatic sugar and rhamnose degradation; 800 mg / kg IPA increased the expression of the urea cycle and purine and adenosine rescue pathways, and decreased the expression of lipid molecule mycolate synthesis, heme synthesis, and aromatic sugar and rhamnose degradation pathways ( Figure 8 A. Figure 8 B and Figure 8 C).

[0047] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A feed additive that replaces zinc oxide and antibiotics, characterized in that: The feed additive comprises component (a) indole-3-propionic acid; and component (b) indole-3-acetic acid; The weight ratio of components a and b in the feed additive is 1:4 to 4:1, preferably 1:

1.

2. Use of the feed additive according to claim 1 as a feed additive for alleviating weaning stress in animals and improving intestinal flora function.

3. The use according to claim 2, characterized in that The animals include pigs.

4. A feed for alleviating weaning stress in animals and improving intestinal barrier and flora function, characterized in that: The feed contains the composition according to claim 1, and is a low-protein diet feed without zinc oxide, antibiotics and other growth-promoting regulators.

5. The feed according to claim 4, characterized in that Each kilogram of feed contains 100-400 mg of indole-3-propionic acid and 100-400 mg of indole-3-acetic acid, preferably 400 mg of indole-3-propionic acid and 400 mg of indole-3-acetic acid.

6. The feed according to claim 4 or 5, characterized in that The crude protein CP level of the low protein diet is 16%-17.5%.

7. Any of the following uses of the feed according to any one of claims 4 to 6: 1) Used to prepare biological products that relieve weaning stress in piglets, improve growth performance of weaned piglets, reduce feed-to-weight ratio, increase average body weight and reduce diarrhea rate; 2) Used to prepare biological products that improve the antioxidant capacity and immune function of weaned piglets; 3) Used to promote the proliferation of pig intestinal epithelial cells, increase the diversity of intestinal flora, and promote the growth and reproduction of beneficial intestinal flora in weaned piglets; 4) Used to prepare biological products for improving oxidative stress and intestinal damage in piglets caused by weaning stress.

8. The use according to claim 7, characterized in that The beneficial bacteria include but are not limited to microorganisms of the genera Bacteroides, Eubacterium, Acidaminococcus, Anaerofustis, Peptococcus, Alloprevotella, Pseudoramibacter, Lachnospiracea_incertae_sedis, and Oscillibacter.

9. The use according to claim 7, characterized in that Under in vitro culture conditions, the addition amounts of IAA and IPA in the culture medium used to promote the proliferation of porcine intestinal epithelial cells were 100~200 μmol / L and 100~200 μmol / L, respectively.

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