Sweet orange extract and application thereof in preparation of feed additive

Abstract

The invention relates to the technical field of feed additives, in particular to a sweet orange extract and application thereof in preparation of a feed additive. The sweet orange extract provided by the invention has the total flavone content of 70-90%, can be used for large-scale and low-cost production in factories, and can increase the average daily gain of animals to different degrees, reduce the feed conversion ratio and effectively improve the production performance of the animals when being used as a breeding input for aquatic products, livestock and poultry and other animals.

Description

Technical Field

[0001] The invention relates to the technical field of feed additives, and in particular to a sweet orange extract and application thereof in preparing the feed additive. Background Art

[0002] After growth-promoting drug feed additives (except traditional Chinese medicine) were banned, a variety of nutritional strategies have been developed to replace their use. The main types of alternative additives are: plant extracts, probiotics, enzyme preparations, antimicrobial peptides, acidifiers, etc.

[0003] Citrus aurantium is the dried young fruit of the sour orange (Citrus aurantium L.) and its cultivars or sweet orange (Citrus sinensis Osbeck), which belong to the Rutaceae family. The active ingredients in sweet orange mainly include flavonoids, alkaloids, volatile oils, etc. Flavonoids contain puerarin, naringin, hesperidin, naringenin, hesperidin, nobiletin, etc., which have pharmacological effects such as promoting digestion, anti-oxidation, lowering blood sugar, and anti-inflammatory.

[0004] This patent uses dried young sweet orange fruits as raw materials, and obtains sweet orange total flavonoids extract through process treatment, which is developed and applied as a breeding input. Patent CN118203534A uses natural low eutectic solvents to extract total flavonoids from oranges. The low eutectic solvents used are not suitable for large-scale factory production and have high production costs. This patented process can be used for large-scale factory production at a lower cost. Patent CN201811480875.0 uses a macroporous adsorption method to extract total flavonoids from Citrus aurantium or Citrus aurantium, but the total flavonoids content in the extract obtained is only 73.13% at most.

[0005] Technical problems existing in feed additives and sweet orange processing technology at this stage:

[0006] (1) There is a huge market demand for low-cost, safe and efficient substitutes for feed antibiotics, but the number of products such as Chinese herbal medicine extracts is small.

[0007] (2) The current extraction process of sweet orange total flavonoids extract is relatively complex, with large amounts of wastewater discharged and high costs, and can only be used for small-scale production.

[0008] (3) Currently, there are few research reports on the use of sweet orange total flavonoid extract as a breeding input to improve the production performance and health of target animals such as livestock, poultry and aquatic products. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0010] A sweet orange extract, the preparation method of the sweet orange extract comprising the following steps:

[0011] (1) Extraction: crush the sweet orange raw material, add alkaline solution for extraction, extract 2-3 times, 1h / time-1.5h / time, solid-liquid ratio 1:6-10, extraction temperature 70°C-90°C, filter, collect the extract, and mix;

[0012] (2) concentrating the extract at a concentration temperature of 55° C. to 75° C., concentrating, filtering, and collecting the clear liquid;

[0013] (3) passing the clear solution through an anion exchange resin and eluting with water, alkaline water, and alkaline ethanol in sequence;

[0014] (4) collecting the alkaline ethanol portion, concentrating it until there is no alcohol taste, adding acid to adjust the pH, filtering, and collecting the residue;

[0015] (5) drying the filter residue at a temperature of 60° C.-70° C. to obtain a sweet orange extract.

[0016] Preferably, the alkaline solution is a calcium hydroxide solution.

[0017] Preferably, the alkaline water is sodium hydroxide solution.

[0018] Preferably, the pH of the calcium hydroxide solution is 10-11.

[0019] Preferably, the anion exchange resin is D941 resin.

[0020] Preferably, the preparation method of the sweet orange extract is:

[0021] Preferably, the preparation method of the sweet orange extract comprises the following steps: crushing the sweet orange dried fruit, adding 10 times of calcium hydroxide solution with pH=10, extracting twice, 1h / time, extracting at 70°C, filtering after extraction, mixing and collecting all the extracts, concentrating at 65°C, concentrating to a solid concentration of 10%, filtering, collecting the clear liquid, passing the clear liquid through D941 resin, eluting with 2BV of water, 2BV of sodium hydroxide solution with pH=8, and 2BV of 60% ethanol with pH=11 in sequence, collecting the 60% ethanol segment with pH=11, concentrating until there is no alcohol taste, adding hydrochloric acid to adjust pH=5, filtering, collecting the filter residue, and drying the filter residue at 60°C to obtain the sweet orange extract.

[0022] A use of a sweet orange extract in any one or more of the following, wherein the use is:

[0023] (1) Promote the growth of livestock, poultry and aquatic animals and improve production performance;

[0024] (2) Prepare products that promote the growth of livestock, poultry and aquatic animals and improve production performance.

[0025] Preferably, the product is a feed additive.

[0026] Preferably, the livestock, poultry and aquatic animals are bullfrog, chicken, pig, grass carp and Chinese soft-shelled turtle.

[0027] Preferably, when the animal is a bullfrog, the amount of sweet orange extract added to the basic feed is 0.5-4.0 g / kg; when the animal is a chicken, the amount of sweet orange extract added to the basic feed is 1.0-3.0 g / kg; when the animal is a pig, the amount of sweet orange extract added to the basic feed is 1.0-3.0 g / kg; when the animal is a grass carp, the amount of sweet orange extract added to the basic feed is 0.1-2.0 g / kg; when the animal is a Chinese soft-shelled turtle, the amount of sweet orange extract added to the basic feed is 1.5-.2.5 g / kg.

[0028] Preferably, when the animal is a bullfrog, the amount of sweet orange extract added to the basic feed is 1.0 g / kg; when the animal is a chicken, the amount of sweet orange extract added to the basic feed is 2.0 g / kg; when the animal is a pig, the amount of sweet orange extract added to the basic feed is 3.0 g / kg; when the animal is a grass carp, the amount of sweet orange extract added to the basic feed is 1.0 g / kg; when the animal is a Chinese soft-shelled turtle, the amount of sweet orange extract added to the basic feed is 1.5 g / kg.

[0029] Beneficial effects of the present invention:

[0030] (1) Provide a process for extracting total flavonoids from Citrus aurantium (sweet orange), wherein the total flavonoid content is 70%-90%, and can be used for large-scale, low-cost production in factories;

[0031] (2) The total flavonoids extract of sweet orange is used as a breeding input for animals to effectively improve their growth performance and develop a low-cost, safe and efficient plant extract feed additive. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are 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.

[0033] Record experimental plan + experimental results

[0034] Experiment 1: Extraction process of sweet orange extract

[0035] This study aimed to extract flavonoids from sweet orange.

[0036] 1. Extraction: Crush the orange raw material (dried fruit), add alkaline solution for extraction, extract 2-3 times, 1h / time-1.5h / time

[0037] times, the solid-liquid ratio is 1:6-10, the extraction temperature is 70°C-90°C, filter, collect the extract, and mix.

[0038] 2. Concentrate the extract at a temperature of 55°C-75°C, concentrate, filter, and collect the clear liquid.

[0039] 3. The clear liquid is passed through anion exchange resin and eluted with water, alkaline water and alkaline ethanol in sequence.

[0040] 4. Collect the alkaline ethanol part, concentrate it until there is no alcohol taste, add acid to adjust the pH, filter, and collect the residue.

[0041] 5. Dry the filter residue at a temperature of 60°C-70°C to obtain sweet orange extract.

[0042] Embodiment 1:

[0043] The dried sweet orange fruit is crushed, and 10 times of calcium hydroxide solution with pH=10 is added, and the extraction is performed twice, 1h / time, and the extraction temperature is 70°C. After extraction, the extraction is filtered, and all the extracts are mixed and collected, and concentrated at a concentration temperature of 65°C, and the solid concentration is concentrated to 10%, filtered, and the clear liquid is collected. The clear liquid is passed through D941 resin, and 2BV of water, 2BV of sodium hydroxide solution with pH=8, and 2BV of 60% ethanol with pH=11 are used in sequence, and the 60% ethanol section with pH=11 is collected, and concentrated until there is no alcohol taste, hydrochloric acid is added to adjust pH=5, filtered, and the filter residue is collected. The filter residue is dried at 60°C to obtain the sweet orange extract. According to this process, the total flavonoids of the sweet orange extract obtained reaches 90.87%.

[0044] Embodiment 2:

[0045] The dried sweet orange fruit is crushed, 6 times of calcium hydroxide solution with pH=11 is added, extracted twice, 1.5h / time, extraction temperature is 90℃, filtered after extraction, all extracts are mixed and collected, concentrated, the concentration temperature is 55℃, concentrated to a solid concentration of 10%, filtered, the clear liquid is collected, the clear liquid is passed through D941 resin, 2BV of water, 2BV of sodium hydroxide solution with pH=8, 2BV of 65% ethanol with pH=10 are used in sequence, the 65% ethanol segment with pH=10 is collected, concentrated to no alcohol taste, hydrochloric acid is added to adjust pH=3, filtered, the filter residue is collected, and the filter residue is dried at 60℃ to obtain the sweet orange extract. According to this process, the total flavonoids of the sweet orange extract obtained reaches 84.39%.

[0046] Embodiment three:

[0047] The dried sweet orange fruit is crushed, 6 times of calcium hydroxide solution with pH=11 is added, extracted 3 times, 1h / time, extraction temperature is 80℃, filtered after extraction, all extracts are mixed and collected, concentrated, the concentration temperature is 75℃, concentrated to a solid concentration of 10%, filtered, the clear liquid is collected, the clear liquid is passed through D941 resin, 2BV of water, 2BV of sodium hydroxide solution with pH=9, 2BV of 70% ethanol with pH=10 are used in sequence, the 70% ethanol segment with pH=10 is collected, concentrated to no alcohol taste, hydrochloric acid is added to adjust pH=4, filtered, the filter residue is collected, and the filter residue is dried at 60℃ to obtain the sweet orange extract. According to this process, the total flavonoids of the sweet orange extract obtained reaches 70.15%.

[0048] Experiment 2: Identification and content detection of sweet orange extract

[0049] 1. Test solution and solution preparation

[0050] Sodium hydroxide test solution: Take 4.3g of sodium hydroxide and add water to dissolve it into 100ml.

[0051] Dilute ethanol test solution: Measure 529 ml of ethanol, add water to 1000 ml, mix well, and you have the solution.

[0052] 5% sodium nitrite solution: weigh 2.0g of sodium nitrite, add 100ml of pure water to dissolve it, mix well, and you have it.

[0053] 10% aluminum nitrate solution: Weigh 4.0 g of aluminum nitrate, add 100 ml of pure water to dissolve, mix well, and you have it.

[0054] 2. Preparation of standard curve solution

[0055] Preparation of reference solution

[0056] Take an appropriate amount of rutin reference substance, weigh it accurately, add ethanol to make a solution containing 0.20 mg of rutin per 1 ml (ultrasonic treatment to dissolve it if necessary).

[0057] Preparation of standard curve

[0058] Accurately measure 1ml, 2ml, 3ml, 4ml, 5ml, and 6ml of the reference solution, place them in 25ml volumetric bottles, add water to 6ml each, add 1ml of 5% sodium nitrite solution, mix well, let stand for 6 minutes, add 1ml of 10% aluminum nitrate solution, shake well, let stand for 6 minutes, add 10ml of sodium hydroxide test solution, add water to the scale, shake well, let stand for 15 minutes, use the corresponding reagent as blank, measure the absorbance at a wavelength of 500nm according to the UV-visible spectrophotometry, use absorbance A as the ordinate and concentration as the abscissa, and draw a standard curve. Conc.=83.0729×A+0.2980, R=0.9996.

[0059] 3. Preparation of test samples

[0060] Take 0.5g of this product, weigh accurately, place in a stoppered conical flask, accurately add 25ml of dilute ethanol, seal, shake well, ultrasonically treat for 5 minutes, leave for more than 3 hours, filter, accurately measure 3ml of the filtrate, place in a 25ml volumetric flask, dilute with water to the scale, shake well, as the test solution. Accurately measure 4ml of the test solution, place in a 25ml volumetric flask, and measure the absorbance according to the method under "2 Preparation of standard curve", starting from "add water to 6ml", and at the same time accurately measure 2ml of the test solution, place in a 25ml volumetric flask, add water to the scale, shake well, as a blank solution. Read the amount of rutin in the test solution from the standard curve and calculate.

[0061] Experiment 3 Effects of sweet orange extract on growth performance of bullfrog

[0062] 1 Materials and methods

[0063] 1.1 Test materials

[0064] Sweet orange extract (flavonoid content 70.15%) was provided by Hunan Jiuanhe Biotechnology Co., Ltd.

[0065] 1.2 Bullfrog breeding experiment

[0066] 1.2.1 Experimental design and feeding management

[0067] 600 bullfrogs with healthy physique and uniform size were selected. The initial body weight of the experimental bullfrog was (23±0.02) g. They were randomly divided into 5 groups, with 3 replicates in each group and 40 bullfrogs in each replicate. The basic diet was prepared with chicken powder as the protein source. Five experimental feeds with equal nitrogen and energy were prepared by adding 0, 0.5g / kg, 0.1g / kg, 2g / kg, and 4g / kg of sweet orange extract to the basic diet (labeled as CON group, CA-0.5 group, CA-1 group, CA-2 group, and CA-4 group). The composition and nutritional level of the basic diet are shown in Table 1. According to the feed formula, the raw materials were crushed, mixed, and passed through a 60-mesh sieve, and then made into floating feed with a diameter of 2.5 mm using a feed extruder. The experimental period was 50 days, and the feeding time was 08:00 and 18:00. During the breeding period, the water level was 60-80cm and the water temperature was (25±3)℃.

[0068] Table 1 Basic bait composition and nutritional level (air-dried basis) Unit: %

[0069] 1) Each kilogram of premix contains: VB1 10mg, riboflavin 8mg, pyridoxine hydrochloride 10mg, VB12 0.2mg, VK310mg, inositol 100mg, calcium pantothenate 20mg, niacin 50mg, folic acid 2mg, biotin 2mg, VA 400mg, VD 5mg, VE100mg, ethoxyquinoline 150mg, dapoxetine powder 132.8mg; potassium chloride 200mg, potassium iodide 60mg, cobalt sulfate (1%) 100mg, copper sulfate 24mg, ferrous sulfate 400mg, zinc sulfate 174mg, manganese sulfate 78mg, magnesium sulfate 800mg, sodium selenite (1%) 50mg, zeolite powder 3 114mg;

[0070] 2) Nutritional levels are all measured values.

[0071] 2 Growth performance determination

[0072] At the end of the experiment, the bullfrogs in each cage were counted and weighed after starvation for 24 h, and the final weight and number of live frogs were reported to calculate the weight gain rate (WGR) and feed conversion rate (FCR).

[0073] Weight gain rate (%) = 100 × [final body weight (g) - initial body weight (g)] / initial body weight (g);

[0074] Feed coefficient = total feeding amount (g) / [final body weight (g)-initial body weight (g)];

[0075] 3 Results

[0076] The effect of sweet orange extract on the growth performance of bullfrogs is shown in Table 2. The results show that sweet orange extract can not only improve the growth performance of bullfrogs, but also reduce the mortality of bullfrogs. Adding 0.5g / kg to 2g / kg of sweet orange extract to the feed can improve the final average weight, weight gain rate and feed conversion rate of bullfrogs (p<0.05), and adding 0.5g / kg to 4g / kg of sweet orange extract to the feed can significantly increase the survival rate of bullfrogs (p<0.05).

[0077] In summary, sweet orange extract can reduce the mortality rate of bullfrogs and improve their growth performance, among which adding 1g / kg sweet orange extract is the best.

[0078] Table 2 Effect of sweet orange extract on growth performance of bullfrog

[0079]

[0080] Note: Data in the same row without letters or with the same letters indicate no significant difference (P>0.05), while data with different letters indicate significant difference (P<0.05)

[0081] Experiment 3 Effects of sweet orange extract on growth performance of broiler chickens

[0082] 1. Test methods

[0083] 1.1 Test materials

[0084] Sweet orange extract (flavonoid content 70.15%) was provided by Hunan Jiuanhe Biotechnology Co., Ltd.

[0085] 1.1.2 Experimental animals and diet

[0086] In this experiment, 240 one-day-old AA white-feathered broilers (Arbor Acres, Arbor Acres) were purchased from Hunan Shuncheng Industrial Co., Ltd. The experimental chicken feed was prepared by itself with reference to the agricultural industry standard "Chicken Feeding Standard" (NY / T33-2004) and did not contain any drug additives. The daily diet ratio and nutritional level of the feed are shown in Table 3.

[0087] Table 3 Daily rations and nutritional levels of experimental broiler feed (air-dried basis)

[0088]

[0089]

[0090] Note: 1) The premix provides per kilogram of feed: VA 12000IU, VD 35000IU, VE 80IU, VK3 3.2mg, VB1 3.2mg, VB265mg, VB6 4.3mg, VB12 0.017mg, pantothenic acid 20mg, biotin 0.22mg, folic acid 2.2mg, I1.25mg, Fe 20mg, Mn120, Se 0.3mg, Zn 110mg.

[0091] 2. Test methods

[0092] 2.1 Experimental design

[0093] In this study, 300 one-day-old AA (Arbor Acres) white-feathered broilers were selected and randomly divided into 4 groups according to body weight and sex, with 60 chickens in each group (6 replicates per group, 10 chickens in each replicate). Among them, 4 dosage groups (CA-1, CA-2, CA-3, CA-4 groups) were fed with feed supplemented with 1g / kg, 2g / kg, 3g / kg and 4g / kg of sweet orange extract, respectively; 1 blank group (CON group) was fed with a basic diet.

[0094] 2.2 Feeding and management

[0095] The experimental animals were caged. The chicken coops and cages were cleaned before the test, and then disinfected with 1% sodium hydroxide solution. After 24 hours, they were washed with clean water, and then the chicken coops, feed trays, feed buckets and drinking water equipment were fumigated with formalin and potassium permanganate (30ml formalin and 15g potassium permanganate per cubic meter) for 48 hours. The test began after the chicken coop was ventilated for five days. The test used a combination of natural and artificial lighting, free access to food and water, and observed the health status, feeding, drinking water and feces of the experimental chickens every morning and evening, and disinfected and immunized according to routine procedures.

[0096] 2.3 Determination of production performance

[0097] With reference to the agricultural industry standard "Poultry Production Performance Ranking Terminology and Measurement Statistical Methods" (NY / T823-2020), the whole empty stomach was weighed at 1 day, 21 days, and 42 days of age, the remaining feed was weighed, and the number of deaths and diarrhea was recorded. The average daily feed intake (ADFI), average daily gain (ADG), and feed-to-meat ratio (F / G) of each group in the early growth period (1-21 days of age), late growth period (21-42 days of age), and full growth period (1-42 days of age) were calculated.

[0098] 3 Results and discussion

[0099] The effects of different amounts of sweet orange extract on the growth performance of broilers are shown in Table 4. The results showed that 1g / kg, 2g / kg and 3g / kg of sweet orange extract could significantly increase the final weight of AA broilers (p<0.01). 4g / kg of sweet orange extract significantly reduced the final weight. Sweet orange extract had no significant effect on body weight at 21 days of age, body weight at 21 days of age, average daily weight gain from 1 to 21 days of age, and average daily feed intake. The high dose (4g / kg) reduced the average daily feed intake by 3.71g, which may be due to the high dose of sweet orange extract, which led to a decrease in feed intake in broilers, further resulting in the main reason that the body weight of broilers in the CA-4 group at 42 days of age was higher than that of other groups.

[0100] In summary, sweet orange extract can improve the growth performance of broiler chickens, and adding 2g / kg sweet orange extract is the best.

[0101] Table 4 Effects of different amounts of sweet orange extract on the growth performance of broiler chickens

[0102]

[0103] Note: Data in the same row without letters or with the same letters indicate no significant difference (P>0.05), while data with different letters indicate significant difference (P<0.05).

[0104] Experiment 4 Effects of sweet orange extract on growth performance of grass carp

[0105] 1 Materials and methods

[0106] 1.1 Test materials

[0107] Sweet orange extract (flavonoid content 70.15%) was provided by Hunan Jiuanhe Biotechnology Co., Ltd.

[0108] 1.2 Grass carp feeding experiment

[0109] 1.2.1 Experimental design

[0110] The experiment selected 360 healthy grass carps with similar body size and weight, and randomly divided them into 4 groups, with 3 replicates in each group and 30 fish in each replicate. The CON group was fed with a basic diet, and the three treatment groups of CA low, medium and high were added with 0.1g / kg (0.01%), 1g / kg (0.1%), 1.5g / kg (0.15%) and 2g / kg (0.2%) sweet orange extract in the basic diet respectively. The experimental period was 52 days, including a pre-feeding period of 10 days and a main test period of 42 days. The experimental diets are shown in Table 5.

[0111] Table 5 Basic bait composition and nutritional level (air-dried basis) Unit: %

[0112]

[0113] 1) Note: 1. Each kilogram of premix provides the following: magnesium 5g, iron 5.50g, copper 200mg, manganese 0.80g, iodine 75mg, selenium 4.50mg, VA 280000IU, VB 2.80g, VB28g, VB120.02g, VC75g, VE45g, VD250000IU, D-pantothenate calcium 7g, folic acid 2.50g, D-biotin 0.06g02. The nutritional levels are all measured values.

[0114] 2) Nutritional levels are all measured values.

[0115] 1.2.2 Experimental management

[0116] The experimental grass carp was domesticated for 10 days, during which time it was fed with basic feed, once at 8:30 and 16:00 a day. During the formal test, each replicate of the grass carp was raised in a 1.5m×1.0m×1.0m net cage, and fed once at 6:30, 12:00 and 17:30 a day. The daily feeding amount was 4% of the grass carp's body weight, and the feeding amount was adjusted at any time according to the water temperature, weather and feeding conditions. During the test, oxygen was continuously supplied. The water was changed every 3 days, and the amount of water changed each time was 1 / 3. The pH value of the aquaculture water was about 7.5, the water temperature was controlled at about 28℃, the ammonia nitrogen concentration was less than 0.15mg / L, the nitrite concentration was less than 0.08mg / L, and the dissolved oxygen concentration was greater than 5.50mg / L.

[0117] 2 Growth performance determination

[0118] At the end of the experiment, the experimental fish in each cage were counted and weighed after 24 hours of starvation, and the weight gain rate (WGR) and feed conversion rate (FCR) were calculated.

[0119] Weight gain rate (%) = 100 × [final body weight (g) - initial body weight (g)] / initial body weight (g);

[0120] Feed coefficient = total feeding amount (g) / [final body weight (g)-initial body weight (g)];

[0121] Specific growth rate (% / d) = 100 × [ln final body weight (g) - ln initial body weight (g)] / culture days (d);

[0122] Survival rate (%) = 100 × [final number / initial number].

[0123] 3 Results

[0124] The effect of sweet orange extract on the growth performance of grass carp is shown in Table 6. The results show that adding 0.01% and 1% sweet orange extract to the feed can significantly increase the final average weight, weight gain rate and specific growth rate of grass carp (p<0.05), while 0.15% and 0.2% sweet orange extract have no effect on the final average weight and weight gain rate of grass carp (p>0.05). The feed conversion rate of sweet orange extract increased significantly (p<0.05), but the feed conversion rate of 0.1% sweet orange extract was significantly lower than that of other groups (p<0.05). Sweet orange extract had no significant effect on the survival rate of grass carp, but there was a trend of increasing the survival rate of grass carp (p>0.05).

[0125] In summary, sweet orange extract can improve the growth performance of grass carp, and adding 1g / kg sweet orange extract is the best.

[0126] Table 6 Effects of sweet orange extract on growth performance of grass carp

[0127]

[0128] Note: Data in the same row without letters or with the same letters indicate no significant difference (P>0.05), while data with different letters indicate significant difference (P<0.05).

[0129] Experiment 5 Effects of sweet orange extract on growth performance of Chinese soft-shelled turtle

[0130] 1 Materials and methods

[0131] 1.1 Test materials

[0132] Sweet orange extract (flavonoid content 70.15%) was provided by Hunan Jiuanhe Biotechnology Co., Ltd.

[0133] 1.2 Turtle breeding experiment

[0134] 1.2.1 Experimental design and feeding management

[0135] Before the experiment, a one-week temporary rearing was carried out, and the feed was fed with water and pellets. During the temporary rearing period, the young turtles were fed feed without sweet orange extract. After the temporary rearing, they were fasted for 24 hours and the formal breeding experiment began. 480 test turtles were randomly divided into 4 groups, with 3 replicates in each group and 40 turtles in each replicate; they were placed in 12 cement breeding pools (5m×4m) for breeding; they were fed with test feeds made of sweet orange extract preparations without or with 0.15%, 0.2%, and 0.25% respectively. Feeding was done twice a day (8:00 and 18:00), and the daily feed amount accounted for 3% of body weight. Clean up the bait residue in time, record the number of deaths, water temperature, and food intake, and clean the breeding pool regularly. During the entire experimental period, the average water temperature was maintained at (30±0.25)℃, the dissolved oxygen content in the water was not less than 3mg / L, and the pH was 6.7-7.7. Natural light was used for breeding for 30 days.

[0136] Table 7 Basic bait composition and nutritional level (air-dried basis) Unit: %

[0137]

[0138]

[0139] 1) Each kilogram of premix contains: VB1 10mg, riboflavin 8mg, pyridoxine hydrochloride 10mg, VB12 0.2mg, VK310mg, inositol 100mg, calcium pantothenate 20mg, niacin 50mg, folic acid 2mg, biotin 2mg, VA 400mg, VD 5mg, VE100mg, ethoxyquinoline 150mg, dapoxetine powder 132.8mg; potassium chloride 200mg, potassium iodide 60mg, cobalt sulfate (1%) 100mg, copper sulfate 24mg, ferrous sulfate 400mg, zinc sulfate 174mg, manganese sulfate 78mg, magnesium sulfate 800mg, sodium selenite (1%) 50mg, zeolite powder 3 114mg;

[0140] 2) Nutritional levels are all measured values.

[0141] 2 Growth performance determination

[0142] At the end of the experiment, the Chinese soft-shelled turtles in each cage were counted and weighed after starvation for 24 hours, and the final weight and number of live turtles were counted. The final average weight, survival rate, intestine-to-body ratio, liver-to-body ratio, organ-to-body ratio, weight gain rate (WGR) and feed conversion rate (FCR) were calculated.

[0143] Weight gain rate (%) = 100 × [final body weight (g) - initial body weight (g)] / initial body weight (g);

[0144] Feed coefficient = total feeding amount (g) / [final body weight (g)-initial body weight (g)];

[0145] Specific growth rate (% / d) = 100 × [ln final body weight (g) - ln initial body weight (g)] / culture days (d);

[0146] Fatness (g / cm3) = 100 × body weight (g) / body length 3 (cm);

[0147] Viscera to body ratio (%) = 100 × viscera weight (g) / body weight (g);

[0148] Liver-to-body ratio (%) = 100 × liver weight (g) / body weight (g);

[0149] Intestinal fat ratio (%) = 100 × intestinal fat weight (g) / body weight (g).

[0150] 3 Results

[0151] The effect of sweet orange extract on the growth performance of Chinese soft-shelled turtle is shown in Table 8. The results show that adding 1.5g / kg to 2.5g / kg of sweet orange extract to the feed can significantly increase the final average weight and weight gain rate of Chinese soft-shelled turtle (p<0.05). At the same time, sweet orange extract can significantly increase the survival rate of Chinese soft-shelled turtle (p<0.05). Sweet orange extract has no negative impact on the intestine-to-body ratio, liver-to-body ratio, organ-to-body ratio, fatness and feed coefficient of Chinese soft-shelled turtle (p>0.05).

[0152] In summary, adding 1.5g / kg to 2.5g / kg of sweet orange extract to feed can improve the growth performance of Chinese soft-shelled turtle, among which 1.5g / kg is the best.

[0153] Table 8 Effect of sweet orange extract on growth performance of Chinese soft-shelled turtle

[0154]

[0155]

[0156] Note: Data in the same row without letters or with the same letters indicate no significant difference (P>0.05), while data with different letters indicate significant difference (P<0.05).

[0157] Experiment 6 Effect of sweet orange extract on growth performance of fattening pigs

[0158] 1. Test methods

[0159] 1.1 Test materials

[0160] 1.1.1 Preparation of sweet orange extract

[0161] Provided by Hunan Jiuanhe Biotechnology Co., Ltd., the content of total flavonoids in sweet orange extract is 70.15%.

[0162] 1.1.2 Experimental design and diet

[0163] The experiment selected 160 healthy fattening pigs weighing about 60 kg and randomly divided them into 4 groups according to a single-factor design, with 5 replicates in each group and 8 fattening pigs in each replicate. The fattening pigs in the control group were fed with a basic diet without sweet orange extract, and the fattening pigs in the sweet orange extract group were fed with an experimental diet with 1, 2, and 3 g / kg sweet orange extract added, respectively. The basic diet for the experimental fattening pigs was prepared in accordance with the nutritional requirements standard for large pigs in "Nutritional Requirements of Pigs" (GB / T 39235-2020), which is a corn-soybean meal-based basic diet. Its composition and nutritional level are shown in Table 9.

[0164] Table 9 Composition and nutritional level of corn-soybean meal basal diet

[0165]

[0166] 1.1.3 Feeding and management

[0167] Before the experiment, the pig house was disinfected. During the pre-experiment, all fattening pigs were fed with a basic diet. In the formal experiment, fattening pigs had free access to food and water, and deworming and immunization procedures were carried out normally. During the breeding process, the health status of fattening pigs in each group was observed. Relevant immunization procedures were carried out according to the requirements of the pig farm.

[0168] 2. Index determination and methods

[0169] 2.1 Determination of production performance

[0170] On the first and 60th day of the experiment, the fattening pigs were weighed before morning feeding and recorded as the initial weight and the final weight of the experiment. The feed consumption of each replicate fattening pig was accurately recorded every day. The average daily feed intake, average daily weight gain and feed-to-weight ratio were statistically analyzed.

[0171] Average daily feed intake = total feed intake per replicate / (number of experimental days × number of fattening pigs per replicate);

[0172] Average daily weight gain = (weight at the end of the test - weight at the beginning of the test) / number of days of the test;

[0173] Feed-to-weight ratio = average daily feed intake / average daily weight gain.

[0174] 3 Results and discussion

[0175] The effects of different amounts of sweet orange extract on the growth performance of finishing pigs are shown in Table 10. The results showed that sweet orange extract can significantly increase the final weight, average daily gain and average daily feed intake of finishing pigs (p<0.01). With the increase of sweet orange extract dosage, the feed-to-weight ratio of growing and finishing pigs decreased linearly.

[0176] In summary, adding 1g / kg to 3g / kg of sweet orange extract to feed can improve the growth performance of fattening pigs, among which 3g / kg is the best.

[0177] Table 10 Effects of different amounts of sweet orange extract on the growth performance of fattening pigs

[0178]

[0179] Note: Data in the same row without letters or with the same letters indicate no significant difference (P>0.05), while data with different letters indicate significant difference (P<0.05).

Claims

1. A sweet orange extract, characterized in that The preparation method of the sweet orange extract comprises the following steps: (1) Extraction: crush the sweet orange raw material, add alkaline solution for extraction, extract 2-3 times, 1h / time-1.5h / time, solid-liquid ratio 1:6-10, extraction temperature 70°C-90°C, filter, collect the extract, and mix; (2) concentrating the extract at a concentration temperature of 55° C. to 75° C., concentrating, filtering, and collecting the clear liquid; (3) passing the clear solution through an anion exchange resin and eluting with water, alkaline water, and alkaline ethanol in sequence; (4) collecting the alkaline ethanol portion, concentrating it until there is no alcohol taste, adding acid to adjust the pH, filtering, and collecting the filter residue; (5) drying the filter residue at a temperature of 60° C.-70° C. to obtain a sweet orange extract.

2. The sweet orange extract according to claim 1, characterized in that The alkaline solution is a calcium hydroxide solution.

3. The sweet orange extract according to claim 2, characterized in that The pH of the calcium hydroxide solution is 10-11.

4. The sweet orange extract according to claim 1, characterized in that The alkaline water is sodium hydroxide solution.

5. The sweet orange extract according to claim 1, characterized in that The anion exchange resin is D941 resin.

6. The sweet orange extract according to claims 1-5, characterized in that The preparation method of the sweet orange extract is: The dried sweet orange fruit is crushed, and 10 times of calcium hydroxide solution with pH=10 is added, and the extraction is performed twice, 1 hour / time, and the extraction temperature is 70°C. After extraction, it is filtered, and all the extracts are mixed and collected, and concentrated at a concentration temperature of 65°C, and concentrated to a solid concentration of 10%, filtered, and the clear liquid is collected, and the clear liquid is passed through D941 resin, and eluted with 2BV of water, 2BV of sodium hydroxide solution with pH=8, and 2BV of 60% ethanol with pH=11 in sequence, and the 60% ethanol segment with pH=11 is collected, concentrated until there is no alcohol taste, hydrochloric acid is added to adjust the pH to 5, filtered, and the filter residue is collected, and the filter residue is dried at 60°C to obtain a sweet orange extract.

7. Use of the sweet orange extract according to any one of claims 1 to 6 in aquaculture inputs, characterized in that: The present invention has any one of the following functions: (1) Promote the growth of livestock, poultry and aquatic animals and improve production performance; (2) Prepare products that promote the growth of livestock, poultry and aquatic animals and improve production performance.

8. The use according to claim 7, characterized in that: The product is a feed additive.

9. The use according to claim 8, characterized in that: The added amount of the sweet orange extract in the feed additive is 0.1-4.0 g / kg.

10. The use according to claim 7, characterized in that: The livestock, poultry and aquatic animals are bullfrog, chicken, pig, grass carp and Chinese soft-shelled turtle.

11. The use according to claim 10, characterized in that: When the animal is bullfrog, the amount of sweet orange extract added to the basic feed is 0.5-4.0 g / kg; When the animal is a chicken, the amount of sweet orange extract added to the basic feed is 1.0-3.0 g / kg; When the animal is a pig, the amount of sweet orange extract added to the basic feed is 1.0-3.0 g / kg; When the animal is grass carp, the amount of sweet orange extract added to the basic feed is 0.1-2.0 g / kg; when the animal is Chinese soft-shelled turtle, the amount of sweet orange extract added to the basic feed is 1.5-2.5 g / kg.

12. The use according to claim 10, characterized in that: When the animal is bullfrog, the amount of sweet orange extract added to the basic feed is 1.0 g / kg; When the animal is a chicken, the amount of sweet orange extract added to the basic feed is 2.0 g / kg; When the animal is a pig, the amount of sweet orange extract added to the basic feed is 3.0 g / kg; When the animal is grass carp, the amount of sweet orange extract added to the basic feed is 1.0 g / kg; When the animal is Chinese soft-shell turtle, the amount of sweet orange extract added to the basic feed is 1.5 g / kg.

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