Application of orange peel hydrolat in preparation of feed, feed additive and medicine

Abstract

The invention discloses application of orange peel hydrolat in preparation of feed, feed additives and drugs. Relates to the technical field of plant extracts. The influence of the orange peel hydrolat on the growth performance, diarrhea rate, organ index, blood routine, serum antioxidant index and liver and jejunum structures and functions of the broiler chickens is researched, and the result shows that the growth performance of the broiler chickens can be remarkably improved and the incidence rate of diarrhea can be reduced by adding a certain dosage of orange peel hydrolat into the feed; meanwhile, the level of inflammatory factors is reduced, the level of immune factors and the activity of antioxidant enzymes are improved, inflammatory response and oxidative stress are reduced, and liver tissue and intestinal mucosa are protected, so that the integrity and functions of the liver and the intestinal tract are recovered. The results show that the broiler bacterial diarrhea can be effectively improved by adding a certain dose of orange peel hydrolat into the feed, and the mechanism of the orange peel hydrolat may be mainly related to the activation of the intestinal immune system and the reduction of inflammatory response.

Description

Technical Field

[0001] The invention relates to the technical field of plant extracts, and more specifically to the application of orange peel hydrosol in the preparation of feed, feed additives and medicines. Background Art

[0002] The broiler industry is an important part of the livestock industry. It has become one of the industries with the best development potential in the global livestock industry due to its short breeding cycle, high feed conversion rate and good economic benefits. However, as intensive and large-scale broiler farming has become a modern farming model, the incidence and transmission risks of various infectious diseases are high. In order to prevent and control the occurrence of these diseases, promote the growth of broilers and improve the production efficiency of broilers, a large number of antibiotics are used in broiler production.

[0003] There are many types of antibiotics, which are mainly divided into synthetic antimicrobial agents such as β-lactam antibiotics, polymyxins and gramicidin antibiotics, tetracycline antibiotics, macrolide antibiotics, aminoglycoside antibiotics, chloramphenicol and quinolones according to their mechanism of action. In animal husbandry, the main functions of antibiotics are to promote growth, enhance immune function, regulate intestinal health, and prevent and treat animal diseases. However, the excessive use of antibiotics in animal husbandry has led to bacterial resistance and antibiotic residues, endangering food, life and environmental safety. "Antibiotic ban" on the feed side, "antibiotic reduction" on the breeding side, and "antibiotic-free" on the product side have become the inevitable choice for green production and high-quality development of animal husbandry.

[0004] Feed antibiotic substitutes are mainly divided into two types: direct substitute products (substitute antibacterial function) and indirect substitute products (substitute growth promotion function). At present, feed antibiotic substitutes mainly include acidifiers, enzyme preparations and plant extracts. Among them, plant extracts have gradually become the focus of the breeding industry because of their advantages of being natural, low toxicity, low residue, no drug resistance, and their physiological activities of anti-inflammatory, antioxidant, and improving intestinal health.

[0005] Plant hydrosol is a fragrant aqueous solution formed by evaporating and condensing the aromatic components and other water-soluble components of plants with steam during the preparation of essential oils. Plant hydrosol contains both a small amount of essential oil components and water-soluble components of plants. Studies have found that tea tree, osmanthus, and camphor tree leaf hydrosols have significant antibacterial activity; galangal hydrosol has significant antioxidant activity and tyrosinase inhibitory activity; lavender hydrosol can not only significantly improve the immunity of Xinjiang brown cattle, but also reduce the diarrhea rate and mortality rate of weaned piglets, improve the growth performance and feed utilization efficiency of weaned piglets, and enhance the ability of weaned piglets to synthesize protein, metabolize fat, scavenge free radicals and resist diseases; adding tea tree hydrosol to the drinking water of laying hens can improve the Haugh unit, yolk color, egg white weight and other indicators of eggs.

[0006] As a Chinese medicine for both medicine and food, orange peel has a strong aromatic smell and has the effects of promoting appetite, regulating qi and strengthening the spleen, drying dampness and removing phlegm. It can improve the animal's own immunity and effectively improve the intestinal health of animals. Essential oil, as one of its main active ingredients, has biological activities such as antibacterial, antiviral, anti-inflammatory, and regulating gastrointestinal motility. However, the production cost of orange peel essential oil is high. For example, 1 kg of orange peel can only obtain about 20 mL of essential oil, and its cost does not meet the development needs of the animal husbandry industry. In the process of producing orange peel essential oil, a large amount of pure dew will be produced. Each kilogram of orange peel can produce more than 3L of pure dew, and the cost of each liter of pure dew is only 2.5 to 3.5 yuan, which is in line with the needs of animal husbandry development. However, the application of orange peel pure dew in animal husbandry production has not been reported.

[0007] Therefore, whether the application of orange peel hydrosol in the preparation of feed, feed additives and medicines can be realized is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0008] In view of this, the present invention provides the application of orange peel hydrosol in the preparation of feed, feed additives, and medicines. The effects of adding orange peel hydrosol, orange peel, and orange seeds to feed on the growth performance, anti-inflammatory ability, and liver and intestinal health of broilers challenged with E. coli were studied, aiming to provide a scientific basis for the development and application of orange peel hydrosol as a new green and safe feed alternative antibiotic additive. Specifically, a high-concentration orange peel hydrosol was extracted by steam distillation, and the components in the orange peel hydrosol were qualitatively and quantitatively analyzed using an electronic nose and GC-MS; orange peel and orange seeds were extracted by decoction, and the components in the orange peel and orange seeds were qualitatively and quantitatively analyzed using an LC-MS analyzer, and these components were used as indicators to ensure the stability of the quality of orange peel hydrosol. Then, a diarrhea model was established using broilers challenged with E. coli, and the effects of adding orange peel hydrosol, orange peel, and orange seeds to feed on the growth performance, anti-inflammatory ability, and liver and intestinal health of broilers were evaluated by examining various physiological and biochemical indicators of broilers.

[0009] In order to achieve the above object, the present invention adopts the following technical solution:

[0010] Application of orange peel hydrosol in the preparation of feed, feed additives and medicines.

[0011] Preferred: Preparation method of orange peel hydrosol: dry the orange peel, cut it into pieces, add water, heat it to 100°C for distillation, and collect the distilled aromatic aqueous solution.

[0012] Preferably: the mass volume ratio of orange peel to water is 1kg:10-15L; the heating distillation time is 6h.

[0013] Preferred: feed, feed additives or drugs that improve the growth performance of broilers, reduce the incidence of diarrhea; reduce the level of inflammatory factors, increase the level of immune factors and antioxidant enzyme activity, reduce inflammatory response and oxidative stress, protect liver tissue and intestinal mucosa, and restore the integrity and function of the liver and intestines.

[0014] Preferably: improving the growth performance of broilers includes improving the average daily weight gain, average daily feed intake and feed-to-meat ratio of broilers;

[0015] Reducing the inflammatory response includes reducing the diarrhea rate and restoring the absolute value of granulocytes, mean hemoglobin content, and mean hemoglobin concentration to normal levels.

[0016] Antioxidant enzyme activities included improving total antioxidant capacity, SOD activity, GPx activity, and CAT activity;

[0017] Protecting liver tissue includes reducing the activities of ALT and AST, and reducing the mRNA levels of IL-6, IL-1β, TNF-α, and IFN-γ, TLR4, NF-κB, and MyD88 in liver tissue;

[0018] Protecting the intestinal mucosa includes reducing the mRNA levels of IL-6, IL-1β, TNF-α, MyD88, IFN-γ, NF-κB, and TLR4 in jejunal tissue, and increasing the mRNA levels of tight junction proteins in jejunal tissue.

[0019] The present invention also provides feed, feed additive or medicine containing orange peel hydrosol prepared based on any of the above applications.

[0020] Preferred: feed additives or drugs, the main active ingredients of orange peel hydrosol include 3-furfural, D-limonene, and 2-hydroxy-1-(hydroxymethyl)ethyl hexadecanoic acid.

[0021] Preferred: the feed includes a basic feed and orange peel hydrosol, and the content of orange peel hydrosol is 1% of the weight of the basic feed.

[0022] It can be known from the above technical scheme that, compared with the prior art, the present invention discloses the use of tangerine peel hydrosol in the preparation of feed, feed additives, and medicines, and the technical effect achieved is that the present invention studies the effects of tangerine peel hydrosol, tangerine peel, and tangerine seeds on the growth performance, diarrhea rate, organ index, blood routine, serum antioxidant index, and liver and jejunal structure and function of broiler chickens. The results show that adding a certain dose of tangerine peel hydrosol to the feed can significantly improve the growth performance of broiler chickens and reduce the incidence of diarrhea; at the same time, it reduces the level of inflammatory factors, increases the level of immune factors and antioxidant enzyme activity, reduces inflammatory response and oxidative stress, protects liver tissue and intestinal mucosa, and thus restores the integrity and function of the liver and intestines. The above results show that adding a certain dose of tangerine peel hydrosol to the feed can effectively improve bacterial diarrhea in broiler chickens, and its mechanism may be mainly related to activating the intestinal immune system and reducing inflammatory response.

[0023] Specific experimental results:

[0024] (1) The active ingredients of orange peel hydrosol include 3-furfural, D-limonene, 2-hydroxy-1-(hydroxymethyl)ethyl hexadecanoic acid, etc., which are mainly volatile components. The active ingredients of orange peel include naringenin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and nobiletin, etc.; the active ingredients of orange seeds include hesperidin, hesperidin, naringenin, etc., which mainly contain non-volatile components.

[0025] (2) Escherichia coli challenge caused diarrhea in broiler chickens. Compared with orange peel and orange seeds, orange peel hydrosol could significantly increase the ADFI of broiler chickens (P<0.05) and significantly reduce their diarrhea rate (P<0.05).

[0026] (3) Compared with orange peel and orange seeds, the addition of orange peel hydrosol to feed significantly reduced the absolute value of granulocytes (GRA) and increased the mean hemoglobin content (MCH) and mean hemoglobin concentration (MCHC) to normal levels (P<0.05).

[0027] (4) Compared with orange peel and orange seed, orange peel hydrosol can significantly improve the total antioxidant capacity and the enzyme activities of SOD and GPx (P<0.05).

[0028] (5) Compared with orange peel and orange seeds, orange peel hydrosol can significantly reduce the activities of ALT and AST in broiler serum (P<0.05).

[0029] (6) Compared with orange peel and orange seeds, orange peel hydrosol can reduce the mRNA levels of IL-6, IL-1β, TNF-α, IFN-γ, TLR4, NF-κB, and MyD88 in broiler liver tissue, among which IL-6, IL-1β, TNF-α, IFN-γ, TLR4, and MyD88 showed significant differences (P<0.05).

[0030] (7) Compared with orange peel and orange seeds, orange peel hydrosol can significantly reduce the mRNA levels of IL-6, IL-1β, TNF-α, as well as MyD88, IFN-γ, NF-κB, and TLR4 in the jejunal tissue of broiler chickens (P<0.05).

[0031] (8) Compared with orange peel and orange seeds, adding a certain dose of orange peel hydrosol to the feed significantly increased the mRNA level of tight junction protein in the jejunum tissue of broiler chickens (P<0.05).

[0032] (9) Compared with orange peel and orange seeds, orange peel hydrosol can effectively alleviate the damage to liver and jejunal tissues caused by Escherichia coli infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0034] Figure 1 The accompanying drawing is a radar chart of the response value of the electronic nose of orange peel hydrosol provided by the present invention.

[0035] Figure 2 The accompanying drawing is a GC-MS total ion current diagram of the orange peel hydrosol provided by the present invention.

[0036] Figure 3 The attached figure is a diagram showing the effects of tangerine peel hydrosol, tangerine peel and tangerine seeds provided by the present invention on the antioxidant indicators of broilers, wherein A. total antioxidant capacity; B. SOD activity; C. GPx enzyme activity; D. CAT activity.

[0037] Figure 4 The attached drawing is a diagram showing the effects of tangerine peel hydrosol, tangerine peel and tangerine seeds provided by the present invention on serum liver function indicators, wherein A. ALT; B. AST.

[0038] Figure 5 The accompanying drawing is a diagram showing the effects of tangerine peel hydrosol, tangerine peel and tangerine seeds provided by the present invention on liver immune indicators.

[0039] Figure 6 The accompanying drawing is a diagram showing the effects of tangerine peel hydrosol, tangerine peel and tangerine seeds provided by the present invention on liver tissue morphology.

[0040] Figure 7 The attached drawing is a diagram showing the effects of tangerine peel hydrosol, tangerine peel and tangerine seeds provided by the present invention on jejunal immune indicators.

[0041] Figure 8The attached figure is a diagram showing the effects of orange peel hydrosol, orange peel and orange seeds provided by the present invention on the jejunal intestinal barrier.

[0042] Fig. 9 The attached figure is a diagram showing the effects of tangerine peel hydrosol, tangerine peel and tangerine seeds provided by the present invention on jejunal tissue of broiler chickens. DETAILED DESCRIPTION

[0043] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] The present invention discloses the use of orange peel hydrosol in the preparation of feed, feed additives, and medicines. Example 1

[0045] Preparation and component detection of orange peel hydrosol

[0046] Preparation of orange peel hydrosol: Dry and chop fresh orange peel, weigh 3kg of dry orange peel and put it into the extraction tank, add 15 times the water (45L), heat to 100℃ and distill for 6h, and collect the distilled aromatic aqueous solution.

[0047] Component detection of orange peel hydrosol: An electronic nose was used to qualitatively analyze the components of orange peel hydrosol; Agilent's gas phase separation and mass spectrometry detection method was established using Agilent's gas chromatography-mass spectrometry to detect the chemical components in orange peel hydrosol. Mass spectrometry data was identified using MassHunter data analysis software in combination with NIST, mainlib, replib standard spectral libraries and traditional Chinese medicine databases to reveal the material basis of orange peel hydrosol.

[0048] Identification of the ingredients of orange peel hydrosol: The qualitative analysis of the ingredients of orange peel hydrosol using an electronic nose revealed that orange peel hydrosol is mainly composed of methyls, alcohols and some aromatic compounds, organic sulfides, inorganic sulfides and terpenes ( Figure 1 , Table 1). By establishing a GC-MS method to detect the chemical components of orange peel hydrosol, a total of 20 chemical components were screened in combination with multiple databases, among which the main active ingredients were 3-furfural, D-limonene, 2-hydroxy-1-(hydroxymethyl)ethyl hexadecanoic acid, etc. ( Figure 2 , Table 2).

[0049] Table 1 Electronic nose response values ​​of orange peel hydrosol

[0050] Serial number Sensor Name Response material type Response value Proportion (%) 1 W1C Sensitive to aromatic compounds 1.635 4.98 2 W5S Sensitive to nitrogen oxides 2.35 7.15 3 W3C Sensitive to ammonia and aromatic compounds 1.118 3.40 4 W6S Sensitive to hydrogen 1.281 3.90 5 W5C Sensitive to alkanes and aromatic compounds 1.105 3.36 6 W1S Sensitive to methyl 8.182 24.91 7 W1W Sensitive to inorganic sulfides and terpenes 3.885 11.83 8 W2S Sensitive to alcohols and some aromatic compounds 6.748 20.54 9 W2W Sensitive to aromatic compounds and organic sulfides 5.405 16.45 10 W3S Sensitive to alkanes 1.139 3.47

[0051] Table 2 Chemical composition of orange peel hydrosol

[0052]

[0053] Comparative experiment

[0054] Comparative Example 1

[0055] Preparation and component detection of tangerine peel decoction

[0056] Preparation of orange peel decoction: Dry and chop fresh orange peel, weigh 3 kg of dry orange peel and put it into an extraction tank, add 15 times of water (45L), boil for 30 minutes, and collect the orange peel decoction.

[0057] Component detection of tangerine peel decoction: Agilent's liquid chromatography-mass spectrometry instrument was used to establish a liquid phase separation and mass spectrometry detection method to detect the chemical components in tangerine peel hydrosol. The mass spectrometry data was identified through data analysis software combined with multiple traditional Chinese medicine databases to reveal the material basis of tangerine peel decoction.

[0058] Identification of components of tangerine peel decoction: An LC-MS method was established to detect the chemical components of tangerine peel decoction, and 31 chemical components were obtained by screening multiple databases, mainly including naringenin, 3,5,6,7,8,3',4'-heptamethoxyflavone, nobiletin, etc. (Table 3).

[0059] Table 3 Chemical composition of orange peel

[0060]

[0061]

[0062] Comparative Example 2

[0063] Preparation and component detection of orange seed decoction

[0064] Preparation of orange seed decoction: Dry fresh orange seeds, weigh 3 kg of dry seeds and put them into an extraction tank, add 15 times of water (45 L), boil for 30 minutes, and collect the orange seed decoction.

[0065] Component detection of tangerine seed decoction: Agilent's LC / MS instrument was used to establish a liquid phase separation and mass spectrometry detection method to detect the chemical components in tangerine peel hydrosol. The mass spectrometry data was identified through data analysis software combined with multiple traditional Chinese medicine databases to reveal the material basis of tangerine peel decoction.

[0066] Identification of components of orange seed decoction: The chemical components of orange seed decoction were detected by establishing an LC-MS method. A total of 31 chemical components were obtained by screening in combination with multiple databases, mainly including hesperidin, hesperidin, naringenin, etc. (Table 4).

[0067] Table 4 Chemical composition of orange seeds

[0068]

[0069]

[0070] The results show that:

[0071] The active ingredients of orange peel hydrosol include 3-furfural, D-limonene, 2-hydroxy-1-(hydroxymethyl)ethyl hexadecanoic acid, etc., which are mainly volatile components.

[0072] The active ingredients of tangerine peel include naringenin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and nobiletin, etc.; the active ingredients of tangerine seeds include hesperidin, hesperidin, naringenin, etc. Both mainly contain non-volatile components and are highly similar.

[0073] That is, the ingredients of the orange peel hydrosol product prepared by the present invention are significantly different from those of orange peel and orange seeds, and the effect is unpredictable.

[0074] Comparative experiment 2

[0075] Effects of orange peel hydrosol, orange peel and orange seeds on growth performance, anti-inflammatory ability, liver and intestinal health of broiler chickens challenged with Escherichia coli

[0076] Animals and groups

[0077] Animal grouping and treatment: 375 healthy white-feathered broilers at 1 day old were selected and adaptively raised for 7 days. On the 8th day, they were randomly divided into 5 groups, namely, blank group (group C), Escherichia coli challenge group (group M), orange peel hydrosol group (group CL), orange peel group (group JP), and orange seed group (group JZ). Each group had 5 replicates, and each replicate had 15 chickens. The control group and the Escherichia coli challenge group were fed a basal diet, the hydrosol group (group CL) added 1% orange peel hydrosol to the basal diet, the orange peel group (group JP) added 1% orange peel to the basal diet, and the orange seed group (group JZ) added 1% orange seed to the basal diet. The experimental period was 28 days (8-35 days old), and a 7-day challenge experiment was conducted at 29-35 days of age (2 mL of 1.0×10 9 coli K88 liquid (CFU / mL), and group C was given 2 mL of sterile saline).

[0078] Measurement indicators and methods

[0079] Growth performance and diarrhea rates

[0080] The broilers were weighed after fasting for 12 hours (without feed but without water) at 8, 29 and 35 days of age, and the feed intake of each group was recorded every day. The average daily weight gain (ADG), average daily feed intake (ADFI) and feed-to-weight ratio (F / G) were calculated at 8-28 days and 29-35 days of age. During the experiment, the health of the broilers was observed every day. During the E. coli challenge period (29-35 days of age), the feces of normal chickens were columnar spiral cones, light gray-green in color, with white ribbon-like urate attached to the surface of the chicken feces, soft and fluffy; when the chickens had diarrhea, the performance was diverse, with loose stools such as white, red and green. By observing the dirtiness of the broiler anus and the proportion of loose feces in the feces tray, the diarrhea of ​​each group of broilers was recorded every day, the daily diarrhea rate was calculated, and the average diarrhea rate of 7 days was finally taken for statistical analysis. After collecting blood samples, the broilers were bled to death through the carotid artery and then slaughtered for sampling.

[0081] The results show that:

[0082] The broilers were divided into 5 groups, namely, the blank group (group C), the E. coli challenge group (group M), the orange peel hydrosol group (group CL), the orange peel group (group JP), and the orange seed group (group JZ). By testing the growth indicators such as the average daily weight gain (ADG), average daily feed intake (ADFI), feed-to-meat ratio (FCR), and diarrhea rate of the broilers before and after the E. coli challenge, it was found that during the period of 8 to 28 days of age, compared with the blank group, the ADG, ADFI, and FCR of the broilers in the CL group increased significantly (P<0.05); after the E. coli challenge (at 29 to 35 days of age), compared with the blank group, the ADG and ADFI of the challenge group decreased significantly, and the FCR and diarrhea rate increased significantly (P<0.05); compared with the challenge group, the hydrosol group significantly increased ADFI and FCR (P<0.05), and significantly reduced the diarrhea rate (P<0.05); the orange peel group and the orange seed group significantly increased FCR (P<0.05) (Table 5). The results showed that adding a certain dose of orange peel hydrosol to feed can significantly promote the appetite of broilers and reduce the diarrhea rate of broilers, while the effects of orange peel and orange seeds on promoting appetite and reducing diarrhea rate are not obvious.

[0083] Table 5 Effects of orange peel hydrosol, orange peel and orange seeds on growth performance and diarrhea rate of broiler chickens

[0084]

[0085]

[0086] Note: The same letters indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05)

[0087] Blood routine index test

[0088] After the experiment, the routine blood indicators of broilers were tested, including white blood cell count (WBC), red blood cell count (RBC), hemoglobin content (HGB), granulocyte absolute value (GRA), lymphocyte absolute value (LYM), platelet number (PLT), mean corpuscular volume (MCV), mean hemoglobin content (MCH), mean hemoglobin concentration (MCHC), mean platelet volume (MPV), etc.

[0089] The results show that:

[0090] Compared with the blank group, the absolute value of granulocytes (GRA) and the absolute value of lymphocytes (LYM) in the challenge group increased significantly (P<0.05); the number of platelets (PLT), the mean hemoglobin content (MCH), and the mean hemoglobin concentration (MCHC) decreased significantly (P<0.05). Compared with the challenge group, the absolute value of granulocytes (GRA) in the pure dew group decreased significantly, and the mean hemoglobin content (MCH) and the mean hemoglobin concentration (MCHC) increased significantly to normal levels (P<0.05); the absolute value of granulocytes (GRA) in the orange peel group decreased significantly, and the mean hemoglobin content (MCH) and the mean hemoglobin concentration (MCHC) increased significantly to normal levels (P<0.05); the mean hemoglobin content (MCH) and the mean hemoglobin concentration (MCHC) in the orange seed group increased significantly (P<0.05) (Table 6). The results showed that adding a certain dose of orange peel hydrosol to the feed can significantly increase the immunity of broilers and reduce symptoms such as inflammation, and its improvement effect is better than that of the orange peel group and orange seed group.

[0091] Table 6 Effects of orange peel hydrosol, orange peel and orange seeds on blood routine indexes of broilers

[0092]

[0093] Note: The same letters indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05)

[0094] Serum antioxidant index

[0095] After broilers were fasted for 12 hours (without feed but without water), one chicken with a body weight close to the average was selected from each replicate, and blood was collected from the subwing vein. The blood was centrifuged at 4°C and 3000r / min for 15 minutes, and the serum was separated and frozen for storage. The total antioxidant capacity detection kit (FRAP method) (Biyuntian Biotechnology Co., Ltd.), the total SOD activity detection kit (WST-8 method) (Biyuntian Biotechnology Co., Ltd.), the total glutathione peroxidase detection kit (Biyuntian Biotechnology Co., Ltd.), and the catalase detection kit (Biyuntian Biotechnology Co., Ltd.) were used to detect the total antioxidant capacity of the serum of each group of broilers and the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) in the serum.

[0096] The results show that:

[0097] Since superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) are all very important antioxidant enzymes that can remove harmful free radicals or peroxides in the body. Therefore, they are often used as important indicators for detecting antioxidant effects. By detecting the total antioxidant capacity and SOD, GPx, and CAT activities in broiler serum, it was found that compared with the blank group, the total antioxidant capacity, SOD, GPx, and CAT activities of the challenge group were reduced, among which the total antioxidant capacity and SOD and GPx enzyme activities were significantly reduced (P<0.05); compared with the challenge group, the tangerine peel hydrosol group can significantly improve the total antioxidant capacity and the enzyme activities of SOD and GPx (P<0.05), and improve CAT activity; the tangerine peel group and the tangerine seed group both have increased total antioxidant capacity and SOD, GPx, and CAT activities, but not significantly (P>0.05) ( Figure 3 ). The results showed that adding a certain dose of orange peel hydrosol to feed can reduce oxidative stress and enhance the body's antioxidant capacity by increasing the activity of antioxidant enzymes in broilers challenged with E. coli, and the effect is better than that of orange peel and orange seeds.

[0098] Liver function indexes and immune-related indexes

[0099] RT-qPCR was used to detect liver function indicators such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) to evaluate the health of broiler livers. RT-qPCR was used to detect the mRNA levels of pro-inflammatory factors such as IL-6, IL-1β, TNF-α, and immune regulatory factors such as Toll-like receptor 4 (TLR4), mucosa-associated molecule glycoprotein 88 (MyD88), interferon-γ (IFN-γ), and nuclear factor κB (NF-κB) in the liver tissues of broilers in each group.

[0100] The results show that:

[0101] ① Effects of orange peel hydrosol, orange peel, and orange seeds on serum liver function indicators

[0102] Liver function indicators such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are a group of biochemical indicators used to evaluate the health of the liver. By testing the serum liver function indicators of broiler chickens, it was found that compared with the blank group, the activities of ALT and AST in the challenge group increased significantly. Adding orange peel hydrosol, orange peel, and orange seeds to the feed can significantly reduce the ALT activity in broiler serum (P<0.05), and the hydrosol can also significantly reduce the activity of AST (P<0.05) ( Figure 4 ).

[0103] ②The effects of orange peel hydrosol, orange peel and orange seeds on liver immune indicators

[0104] By detecting the mRNA levels of proinflammatory factors such as IL-6, IL-1β, TNF-α and immunomodulatory factors such as Toll-like receptor 4 (TLR4), mucosal-associated glycoprotein 88 (MyD88), interferon-γ (IFN-γ), and nuclear factor κB (NF-κB) in broiler liver tissue, it was found that compared with the blank group, the mRNA levels of proinflammatory factors and immunomodulatory factors in the challenge group were significantly increased (P<0.05). Adding a certain dose of orange peel hydrosol to the feed can significantly reduce the mRNA levels of the broiler liver group. Tangerine peel could significantly reduce the mRNA levels of IL-6, IL-1β, TNF-α, IFN-γ, TLR4, NF-κB, and MyD88 in the tissues (P<0.05); tangerine peel could significantly reduce the mRNA levels of IL-6, IL-1β, IFN-γ, and MyD88 (P<0.05); tangerine seeds could reduce the mRNA levels of IL-6, IL-1β, TNF-α, IFN-γ, TLR4, NF-κB, and MyD88, but the effect was not significant (P>0.05)( Figure 5 ).

[0105] ③The effects of tangerine peel hydrosol, tangerine peel and tangerine seeds on liver tissue morphology

[0106] By observing the pathological changes of broiler liver tissue through staining of liver tissue pathological sections, it was found that compared with the blank group, the liver tissue structure of the challenge group was disordered, the hepatic cords were arranged in a disordered manner, the hepatocytes were degenerated and necrotic, the hepatic sinusoids were significantly dilated, and multiple inflammatory cell infiltration foci were seen in the hepatic sinusoids; the hepatocytes in the tangerine peel pure dew group did not show degeneration and necrosis, the hepatic cords were arranged in a disordered manner, the hepatic sinusoids were slightly dilated, and a small number of inflammatory cell infiltration foci were seen in the hepatic sinusoids; the hepatocytes in the tangerine peel group did not show degeneration and necrosis, the hepatic cords were arranged in a disordered manner, the hepatic sinusoids were dilated, and a small number of inflammatory cell infiltration foci were seen in the hepatic sinusoids; the liver tissue structure of the tangerine seed group was disordered, the hepatic cords were arranged in a disordered manner, the hepatocytes were degenerated and necrotic, the hepatic sinusoids were significantly dilated, and more inflammatory cell infiltration foci were seen in the hepatic sinusoids. ( Figure 6 ). The results showed that orange peel hydrosol and tangerine peel can effectively alleviate liver damage caused by E. coli attack, and orange peel hydrosol is more effective than tangerine peel.

[0107] Jejunal immune indexes and intestinal barrier indexes

[0108] RT-qPCR was used to detect the mRNA levels of proinflammatory factors such as IL-6, IL-1β, TNF-α, and immune regulatory factors such as Toll-like receptor 4 (TLR4), mucosa-associated glycoprotein 88 (MyD88), interferon-γ (IFN-γ), and nuclear factor κB (NF-κB) in the jejunal tissue of broilers. RT-qPCR was used to detect the mRNA levels of tight junction proteins such as ZO-1, MUC2, Occludin, and Claudin-1 in the jejunal tissue of broilers.

[0109] The results show that:

[0110] ① Effects of orange peel hydrosol, orange peel, and orange seeds on jejunal immune indexes

[0111] By detecting the mRNA levels of proinflammatory factors such as IL-6, IL-1β, TNF-α and immunomodulatory factors such as Toll-like receptor 4 (TLR4), mucosal-associated molecule glycoprotein 88 (MyD88), interferon-γ (IFN-γ), and nuclear factor κB (NF-κB) in the jejunal tissue of broilers, it was found that compared with the blank group, the mRNA levels of proinflammatory factors and immunomodulatory factors in the challenge group were significantly increased (P<0.05). Adding a certain dose of orange peel hydrosol to the feed significantly reduced the mRNA levels of IL-6, IL-1β, TNF-α, IFN-γ, TLR4, NF-κB, and MyD88 in the jejunal tissue of broilers (P<0.05); orange peel can significantly reduce the mRNA levels of IFN-γ, TLR4, NF-κB, and MyD88 (P<0.05); orange seeds can significantly reduce the mRNA level of TLR4 (P<0.05) ( Figure 7 ). The results showed that orange peel hydrosol and orange peel can effectively improve the abnormal jejunal immune indicators, and orange peel hydrosol is more effective than orange peel.

[0112] ② Effects of orange peel hydrosol, orange peel, and orange seeds on the jejunal intestinal barrier

[0113] By detecting the mRNA levels of tight junction proteins such as MUC2, ZO-1, Occludin, and Claudin-1 in the jejunal tissue of broiler chickens, it was found that compared with the blank group, the mRNA levels of tight junction proteins in the challenge group were significantly reduced (P<0.05). Adding a certain dose of orange peel hydrosol to the feed significantly increased the mRNA levels of tight junction proteins such as MUC2, ZO-1, Occludin, and Claudin-1 in the jejunal tissue of broiler chickens (P<0.05); orange peel can significantly increase the mRNA levels of MUC2 and ZO-1 (P<0.05); orange seeds have a tendency to increase the mRNA level of tight junction proteins, but it is not significant (P>0.05) ( Figure 8 ).

[0114] Jejunal tissue morphology

[0115] 1-2 cm of the middle jejunum and liver tissue were taken, rinsed with 0.9% saline, fixed in 4% paraformaldehyde solution, embedded in paraffin, sliced, baked, dewaxed to water, stained with hematoxylin and eosin, and then subjected to pathological examination.

[0116] The results show that:

[0117] By staining the jejunal tissue pathological sections and observing the pathological changes of the jejunal tissue of broiler chickens, it was found that compared with the blank group, the jejunal villi of the challenged group became shorter, the number decreased, and the arrangement was irregular. The mucosal epithelial cells were severely necrotic and fell off into the lumen, and more inflammatory cells were seen in the lamina propria. Compared with the challenged group, the jejunal villi of the tangerine peel pure dew group were arranged more disorderly, the mucosal epithelial cells did not show degeneration or necrosis, and scattered inflammatory cells were seen in the lamina propria; the jejunal villi of the tangerine peel group were arranged more disorderly, the mucosal epithelial cells did not show degeneration or necrosis, and a small amount of inflammatory cells were seen in the lamina propria; the jejunal villi of the tangerine seed group were shortened and arranged disorderly, the intestinal glands were sparse, the mucosal epithelial cells were necrotic and fell off into the lumen, and more inflammatory cells were seen in the lamina propria. ( Fig. 9 ). The above results show that orange peel hydrosol and orange peel can activate the intestinal immune system, reduce inflammatory response in the jejunum, regulate the permeability of intercellular junctions, repair intestinal mucosal damage, maintain the integrity and function of the intestinal barrier, and effectively improve intestinal damage caused by E. coli infection.

[0118] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0119] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of orange peel hydrosol in the preparation of feed, feed additives and medicines.

2. The use according to claim 1, characterized in that The preparation method of the orange peel hydrosol comprises the following steps: drying and chopping the orange peel, adding water, heating to 100° C. for distillation, and collecting the distilled aromatic aqueous solution.

3. The use according to claim 2, characterized in that The mass volume ratio of the orange peel to water is 1kg:10-15L; the heating distillation time is 6h.

4. The use according to claim 3, characterized in that The feed, feed additive or medicine is a feed, feed additive or medicine that improves the growth performance of broilers and reduces the incidence of diarrhea; reduces the level of inflammatory factors, increases the level of immune factors and the activity of antioxidant enzymes, reduces inflammatory reactions and oxidative stress, protects liver tissue and intestinal mucosa, and restores the integrity and function of the liver and intestines.

5. The use according to claim 4, characterized in that Improving the growth performance of broilers includes increasing the average daily weight gain and average daily feed intake of broilers; The reduction of inflammatory response includes reducing the diarrhea rate and restoring the absolute value of granulocytes, average hemoglobin content, and average hemoglobin concentration to normal levels. The antioxidant enzyme activity includes improving the total antioxidant capacity, SOD activity, GPx activity and CAT activity; the liver tissue protection includes reducing the activities of ALT and AST, and reducing the mRNA levels of IL-6, IL-1β, TNF-α, IFN-γ, TLR4, NF-κB and MyD88 in liver tissue; The intestinal mucosa protection includes reducing the mRNA levels of IL-6, IL-1β, TNF-α, MyD88, IFN-γ, NF-κB, and TLR4 in jejunal tissue, and increasing the mRNA level of tight junction protein in jejunal tissue.

6. Feed, feed additive or medicine containing orange peel hydrosol prepared based on any one of claims 1 to 5.

7. The feed, feed additive or medicine according to claim 6, wherein the main active ingredients of the orange peel hydrosol include 3-furfural, D-limonene, and 2-hydroxy-1-(hydroxymethyl)ethyl hexadecanoic acid.

8. The feed, feed additive or medicine according to claim 7, characterized in that: The feed includes a basic feed and orange peel hydrosol, wherein the content of the orange peel hydrosol is 1% of the weight of the basic feed.

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