CN117064968A

CN117064968A - Preparation method and application of betel nut total alkaloids

Info

Publication number: CN117064968A
Application number: CN202311233835.7A
Authority: CN
Inventors: 曾建国; 凌浩; 谢娟; 张峻恺; 刘英楷; 常冠宇; 邱集慧; 曾雨晴
Original assignee: Hunan Jiu'anhe Biotechnology Co ltd
Current assignee: Hunan Jiu'anhe Biotechnology Co ltd
Priority date: 2023-09-23
Filing date: 2023-09-23
Publication date: 2023-11-17

Abstract

The invention relates to the technical field of plant extracts, in particular to a preparation method and application of betel nut total alkaloids. The invention discloses a betel nut extract, which comprises betel nut base, nor betel nut base, betel nut hypobase and nor betel nut hypobase, wherein the total content of betel nut base, nor betel nut base and nor betel nut hypobase is 0.5-65.0% of the total mass fraction. The betel nut extract can obviously improve the production performance of livestock and aquatic animals, improve the oxidation resistance of organisms and promote intestinal health.

Description

Preparation method and application of betel nut total alkaloids

Technical Field

The invention relates to the field of cultivation input products, in particular to a preparation method and application of betel nut total alkaloids.

Background

The 194 th bulletin of agricultural rural area of the people's republic of China proposes that all growth-promoting medicine feed additive varieties except traditional Chinese medicines are withdrawn from 1 month and 1 day of 2020, and that feed manufacturers stop producing commodity feeds containing growth-promoting medicine feed additives (except traditional Chinese medicines) from 7 months and 1 day of the same year, and marks that China starts to enter the times of forbidden, limited and no resistance. Under the background of intensive and large-scale livestock and poultry cultivation, the demands of the market on feed-use tibetan products are increased, and at present, feed additives such as prebiotics, probiotics, organic acids, enzyme preparations, plant extracts and the like of the feed-use tibetan products with higher acceptance are mainly in a research or small-scale use stage, and the products which are popularized and applied on a large scale in actual production are fewer, and the main reasons are quite high price or unobtrusive use effect, so that the livestock and poultry cultivation cost is greatly increased, and the cost performance and the competitiveness of the products are low. Therefore, the development of low-cost, safe and efficient feed antibiotic alternatives is a hotspot in current research.

Areca catechu L is a evergreen arbor of Areca genus of Palmaceae, and is widely distributed in tropical and subtropical areas of south China and other countries such as south Asia and southeast Asia, and immature fruit peel (conventionally called Areca peel) is often processed into various color Areca catechu chewable products. Meanwhile, betel nuts are used as the first of four southern medicines in China, contain various active ingredients such as alkaloid, flavone, tannin, triterpene, steroid, polysaccharide, fatty acid, amino acid and the like, and have long medicinal history (Zhou Mingxi et al, 2022, jing Yongshuai et al, 2022); a number of in vitro cell tests and in vivo mouse model studies have shown that the functional components in betel nuts have certain activity in terms of antioxidation (Lee and Science,2010, korin, 2010), anti-inflammatory (Wu Jiangtao, 2016), bacteriostasis (topaz et al, 2009, zhao Ziwei, 2019), lipid metabolism regulation (He Juan, 2014, li Xing, 2013), digestion promotion (Baicang, 2003) and the like.

The invention CN116649471A discloses an application of betel nut products (betel nut extract or betel nut superfine powder) in improving the growth performance and meat quality of livestock and poultry animals, wherein betel nuts are used as water extract and superfine powder, the betel nut extract is focused on polyphenol content of 10-30%, and the detection content of betel nut total alkali is 1.57% +/-0.03%. The betel nut extract can be used as a breeding input product to be applied to target animals such as livestock, poultry, aquatic products and the like, and focuses on betel nut total alkali, and the content of betel nut extract can reach 65.0%.

The fresh betel nut described in the patent is an immature betel nut fruit, and the betel nut core is the seed of the immature betel nut fruit.

The technical problems of the feeding antibiotics and betel nut treatment technology at the present stage are as follows:

(1) Under the background of 'forbidden and limited' in China, the low-cost, safe and efficient feeding antibiotic substitute has large market demand and small product quantity.

(2) At present, seeds of immature betel nuts are not fully utilized in the market, and the betel nut green and sustainable development is promoted based on the comprehensive utilization of resources.

(3) At present, researches on the alkaloid functional components of betel nuts are mainly on in vitro cell and in vivo mouse models, and related researches on improving the production performance and the body health of target animals such as livestock, poultry, aquatic products and the like as breeding input products are rarely reported.

Reference to the literature

LEE K K,SCIENCE J C J I J O C 2010.The effects of areca catechu L extract on anti-aging.International Journal of Cosmetic Science[J],21:285-295.

Korean 2010 extraction and separation of antioxidant components from Arecae semen [ M ] university of Hainan.

He Juan 2014 Effect of arecoline on lipid metabolism of 3T3-L1 adipocytes [ M ] university of Nanhua.

Huang Yulin, yuan Lamei, and Equipped with Szechwan lewood, et al 2009, study of antibacterial activity of Areca catechu extract, food science [ J ]:3.

Jing Yongshuai, ma Yunfeng, pan Feibing, et al 2022, research progress on herbal examination, chemical composition and pharmacological action of betel nut, inferior traditional medicine [ J ],18:232-239.

Li Xing 2013 Effect of arecoline on 3T3-L1 preadipocyte differentiation [ M ] university of Nanhua.

Wu Jiangtao 2016 the anti-inflammatory activity of arecoline and its effect on NF- κB signaling pathway [ M ] agricultural university of North China.

Zhao Ziwei 2019 the influence of betel water extract on mouse immunity and intestinal flora [ M ] Hunan agricultural university.

Zhou Mingxi, guo Yichen, li Ke, et al 2022, arecae semen active ingredient and pharmaceutical and toxicological study progress Chinese patent medicine [ J ],44:878-883.

Baicang 2003 effects of Areca catechu on gastric motility and gastrointestinal hormone in normal rats and functional dyspepsia model rats [ M ]. University of Nanjing medical science.

Disclosure of Invention

In order to solve the technical problems, the invention provides the following technical scheme:

an extract of betel nut comprising total alkaloids of betel nut comprising arecoline, norarecoline, arecoline and norarecoline.

Preferably, the betel nut extract is betel nut core extract.

Preferably, the betulin, nor betulin, betuline and nor betuline in the betulin extract have a total mass fraction of 0.5% -65.0%.

A method for preparing an extract of betel nut, the method comprising the steps of:

(1) Pretreatment: pouring fresh betel nuts into boiling water, steaming for 20-60 min, drying at 45-90 ℃ until the water content is 18-21%, respectively peeling off peel and taking out seeds to obtain betel nut peel and betel nut core;

(2) Extracting: putting the betel nut cores and pure water into a reflux device, and firstly, pressing 1: the feed liquid ratio of 6 to 14 is 1: 5-8, heating and refluxing for 2-3 times, extracting for 60-120 min at 100 ℃, and collecting betel nut water extract;

(3) Concentrating: concentrating the betel nut water extract under reduced pressure at 55-70 ℃ with vacuum degree less than 0.08MPa;

(4) Spray drying: adding 0.2-8.0 times of auxiliary materials into the betel nut water extract, mixing, and carrying out spray drying to obtain dry powder;

(5) Sieving: sieving with 50-80 mesh sieve to obtain betel nut core extract.

Preferably, the cooking time in step (1) is 20-30 mm; the drying temperature is 50-80 ℃; the feed liquid ratio of the first reflux in the step (2) is 1:8, the feed liquid ratio of the second reflux is 1:6, the extraction time is 120min each time. The feed liquid ratio of the third reflux is 1:6, extracting for 60min.

Preferably, the temperature of reduced pressure concentration in step (3) is 60 ℃; in the step (4), the auxiliary materials are common commercial auxiliary materials such as maltodextrin, soluble starch, corn starch, potato starch, dextrin and the like.

Preferably, in the step (4), the mass ratio of the betel nut extract to the maltodextrin is: 1:0.8; and (5) sieving the mixture through an 80-mesh sieve.

Through detection, the total alkali content of the betel nut core extract obtained by the method is 1.89% -3.51%.

(1) Pretreatment: pouring fresh betel nuts into boiling water, steaming for 20-60 min, drying at 45-90 ℃ until the water content is between 18-21%, respectively peeling off the peel and taking out the seeds to obtain betel nut peel and betel nut core.

(2) Drying and crushing: drying betel nut cores at 60-150deg.C to water content less than or equal to 10%, and pulverizing;

(3) Sieving: sieving with 50-80 mesh sieve to obtain betel nut core extract.

Preferably, the cooking time in step (1) is 20-30 mm; the drying temperature is 50-80 ℃.

Preferably, in step (2) the drying temperature is 100-120 ℃; and (3) sieving with a 80-mesh sieve.

Through detection, the total alkali content of the betel nut core extract obtained by the method is 0.5% -1%.

(3) Extracting: putting betel nut core powder into a reflux device, adding 8-14 times of pure water, heating and refluxing for 1-2 h, repeating for 2 times, and collecting betel nut water extract; concentrating the extract to 20% of the original solution, regulating the pH value to 7-10 with sodium hydroxide solution to obtain alkaline extract, adding Dichloromethane (DCM) for 2-4 times for extraction, combining the extract, concentrating under reduced pressure to 20% of the solvent volume, standing overnight, filtering out precipitate to obtain filtrate, and concentrating under reduced pressure.

(4) Purifying: loading the treated macroporous resin by a wet method, balancing by water, loading into a column, loading the sample with the sample loading amount of 1:20-30, the concentration of the loading solution of 3mg/ml, the pH of the loading solution of 7-8, the volume of the eluent of 60-70% ethanol of about 3 columns, collecting the eluent, concentrating under reduced pressure, and drying.

Preferably, in step (2) the drying temperature is 100-120 ℃; . In the step (3), the feed-liquid ratio is 1:14, the extraction time is 2 hours, the pH value is adjusted to 8-9 by sodium hydroxide, and DCM is extracted for 3 times.

Preferably, in the step (4), the macroporous resin is any one of H103, S-8, AB-8, ADS-8 and D3250; the eluent is 70% ethanol.

Preferably, in step (4), the macroporous resin is H103.

Through detection, the total alkali content of the betel nut core extract obtained by the method is more than 50 percent.

The application of the betel nut core extract in the cultivation input product has any one of the following functions:

(1) Reducing the feeding coefficient of fish or livestock and improving the production performance;

(2) Improving the total oxidation resistance of fish or livestock;

(3) Improving the digestion capability of fish or livestock;

(4) Improving the muscle quality of fish.

The invention has the beneficial effects that:

the areca seed extract containing biological active ingredients such as arecoline is prepared by hot water reflux extraction, concentration, drying, powdering, sieving and the like, and experimental research is carried out on grass carp and broiler chicken, so that the arecoline extract can obviously improve the production performance of livestock and aquatic animals, the oxidation resistance of organisms and intestinal health.

Drawings

FIG. 1 is a flow chart of a betel nut core extract production process 1;

FIG. 2 is a flow chart of process 2 for producing betel nut core extract;

FIG. 3 is a flow chart of the betel nut core extract production process 3;

FIG. 4 characteristic spectrum of Areca catechu extract

FIG. 5 effect of betel nut core extract on grass carp foregut tissue morphology; a: a CON group; ANE-1 group; ANE-2 group; ANE-3 group;

FIG. 6 effect of betel nut core extract on intestinal tissue morphology in grass carp; a: a CON group; ANE-1 group; ANE-2 group; ANE-3 group;

FIG. 7 effect of betel nut core extract on hindgut tissue morphology of grass carp; a: a CON group; ANE-1 group; ANE-2 group; ANE-3 group;

FIG. 8 effect of betel nut core extract on the morphology of the duodenum tissue of broiler chickens; a: a CON group; ANE-1 group; ANE-2 group; ANE-3 group;

FIG. 9 effect of betel nut core extract on jejunal tissue morphology of broiler chickens; a: a CON group; ANE-1 group; ANE-2 group; ANE-3 group;

FIG. 10 effect of betel nut core extract on ileal tissue morphology of broiler chickens; a: a CON group; ANE-1 group; ANE-2 group; ANE-3 group.

Detailed Description

In order to better understand the technical content of the present invention, the following provides specific examples to further illustrate the present invention.

Test one: preparation of betel nut core extract, product components and identification report

Example 1: process 1 for producing betel nut core extract

The production process flow is shown in the figure 1, and the specific contents are as follows:

1. pretreatment: pouring fresh Arecae semen into boiling water, steaming for 30min, oven drying at 70deg.C until water content is 18%, peeling off peel and taking out seeds to obtain Arecae semen peel and Arecae semen core.

2. Extracting: (a) putting betel nut cores into a reflux device; (b) reflux extracting 2 times under heating, the first time according to 1:8, the feed liquid ratio is 1:6, adding pure water into a reflux device, extracting for 120min at 100 ℃, and collecting water extract.

3. Concentrating: concentrating under reduced pressure at 60deg.C under vacuum degree less than 0.08MPa to ensure that the concentrated solution does not adhere to wall.

4. Spray drying: (a) adding betel nut water extract into 0.8 times maltodextrin for mixing; (b) spray drying to obtain a dry powder.

5. Sieving: sieving with 50 mesh sieve.

The total alkali content of the betel nut core extract is 2.86 percent.

Example 2: process 1 for producing betel nut core extract

1. pretreatment: pouring fresh Arecae semen into boiling water, steaming for 25min, oven drying at 60deg.C until water content is 18%, peeling off peel and taking out seeds to obtain Arecae semen peel and Arecae semen core.

2. Extracting: (a) putting betel nut cores into a reflux device; (b) reflux extracting 2 times under heating, the first time according to 1:8, the feed liquid ratio is 1:6, adding pure water into a reflux device according to the feed-liquid ratio of 1:6 for the third time, extracting for 120, 120 and 60 minutes at the temperature of 100 ℃, and collecting water extract.

5. Sieving: sieving with 50 mesh sieve.

The total alkali content of the betel nut core extract is 3.51 percent.

Example 3: process 1 for producing betel nut core extract

1. pretreatment: pouring fresh Arecae semen into boiling water, steaming for 25min, oven drying at 90deg.C until water content is 18%, peeling off peel and taking out seeds to obtain Arecae semen peel and Arecae semen core.

2. Extracting: (a) putting betel nut cores into a reflux device; (b) reflux extracting 2 times under heating, the first time according to 1:14, the second time according to 1:10, adding pure water into a reflux device, extracting for 120 min and 60min at 100 ℃, and collecting water extract.

5. Sieving: sieving with 80 mesh sieve.

The total alkali content of the betel nut core extract is 1.89 percent.

Example 4:

the betel nut core extract and auxiliary materials are mixed according to different proportions and spray dried, and the results are shown in table 1.

TABLE 1 betel nut core extract and spray drying results of different adjuvants at different ratios

By comparing 5 common starch-bound betel nut core extracts for spray drying, the overall result shows that maltodextrin is optimal when the ratio of extract to maltodextrin is 1: the yield can reach 90.9% at 0.8.

Example 5: process for producing betel nut core extract 2

The production process flow is shown in the figure 2, and the specific contents are as follows:

1. pretreatment: pouring fresh Arecae semen into boiling water, steaming for 30min, oven drying at 80deg.C until the water content is 19%, respectively peeling off pericarp and taking out seeds to obtain Arecae semen pericarp and Arecae semen core.

2. Drying and crushing: (a) Drying betel nut cores at 120 ℃ to ensure that the moisture content is less than or equal to 10%; (b) pulverizing.

3. Sieving: sieving with 80 mesh sieve.

The total alkali content of the betel nut core extract is 0.5 percent.

Example 6: process for producing betel nut core extract 2

4. Pretreatment: pouring fresh Arecae semen into boiling water, steaming for 20min, oven drying at 60deg.C until water content is 19%, respectively peeling off pericarp and taking out seeds to obtain Arecae semen pericarp and Arecae semen core.

5. Drying and crushing: (a) Drying betel nut cores at 100deg.C to water content less than 10%; (b) pulverizing.

6. Sieving: sieving with 50 mesh sieve.

The total alkali content of the betel nut core extract product is 0.7 percent.

Example 7: process for producing betel nut core extract 2

7. pretreatment: pouring fresh Arecae semen into boiling water, steaming for 20min, oven drying at 50deg.C until the water content is 19%, respectively peeling off pericarp and taking out seeds to obtain Arecae semen pericarp and Arecae semen core.

8. Drying and crushing: (a) Drying the betel nut core at 60 ℃ to ensure that the moisture content is less than or equal to 10%; (b) pulverizing.

9. Sieving: sieving with 50 mesh sieve.

The total alkali content of the betel nut core extract is 1.0 percent.

Example 8: production process 3 of betel nut core extract

The production process flow is shown in (figure 3), and the specific contents are as follows:

(1) Pretreatment: pouring fresh betel nuts into boiling water, steaming for 20-30 min, drying at 50-80 ℃ until the water content is between 18-21%, respectively peeling off the peel and taking out the seeds to obtain betel nut peel and betel nut core.

(2) Drying and crushing: drying Arecae semen core at 100-120deg.C to water content of 10% or less, and pulverizing;

(3) Extracting: putting betel nut core powder into a reflux device, adding 14 times of pure water, heating and reflux extracting for 2h, repeating for 2 times, and collecting betel nut water extract; concentrating the extract to 20% of the original solution, adjusting pH to 8-9 with sodium hydroxide solution to obtain alkaline extract, adding Dichloromethane (DCM) for extraction for 3 times, mixing the extract, concentrating under reduced pressure to 20% of the solvent volume, standing overnight, filtering to remove precipitate to obtain filtrate, and concentrating under reduced pressure.

(4) Purifying: loading the treated macroporous resin H103 by a wet method, balancing by water, loading into a column, loading sample with a sample amount of 1:25g, loading column liquid with a concentration of 3mg/ml, loading column liquid with a pH of 7-8 and eluting with 70% ethanol with a volume of about 3, collecting the eluting solution, concentrating under reduced pressure, and drying.

The detection shows that the total alkali content of the betel nut core extract product is 65.0%.

Example 9: production process 3 of betel nut core extract

(3) Extracting: putting betel nut core powder into a reflux device, adding 10 times of pure water, heating and reflux extracting for 2h, repeating for 2 times, and collecting betel nut water extract; concentrating the extract to 20% of the original solution, adjusting pH to 8-9 with sodium hydroxide solution to obtain alkaline extract, adding Dichloromethane (DCM) for extraction for 3 times, mixing the extract, concentrating under reduced pressure to 20% of the solvent volume, standing overnight, filtering to remove precipitate to obtain filtrate, and concentrating under reduced pressure.

(4) Purifying: loading the treated macroporous resin S-8 into a column by adopting a wet method, and loading the column after water washing and balancing, wherein the loading amount is 01:30, the concentration of the upper column liquid is 3mg/ml, the pH of the upper column liquid is 7-8, the volume of the eluent is about 3 columns of 60% ethanol, the eluent is collected, and the eluent is dried after decompression concentration.

The detection shows that the total alkali content of the betel nut core extract product is 56.3 percent.

Example 10: production process 3 of betel nut core extract

(3) Extracting: putting betel nut core powder into a reflux device, adding 8 times of pure water, heating and reflux extracting for 2h, repeating for 2 times, and collecting betel nut water extract; concentrating the extract to 20% of the original solution, adjusting pH to 8-9 with sodium hydroxide solution to obtain alkaline extract, adding Dichloromethane (DCM) for extraction for 3 times, mixing the extract, concentrating under reduced pressure to 20% of the solvent volume, standing overnight, filtering to remove precipitate to obtain filtrate, and concentrating under reduced pressure.

(4) Purifying: loading the treated macroporous resin AB-8 into a column by adopting a wet method, and loading the column after water washing and balancing, wherein the loading amount is 1:20, the concentration of the upper column liquid is 3mg/ml, the pH of the upper column liquid is 7-8, the volume of the eluent is about 3 columns of 70% ethanol, the eluent is collected, and the eluent is dried after decompression concentration.

The detection shows that the total alkaloid content of the betel nut core extract product is 51.7%.

Example 11: identification test of betel nut core extract

Liquid chromatography identification

1 reagents or materials

Acetonitrile: chromatographic purity; triethylamine: analytically pure; phosphoric acid: analytically pure; 0.24% phosphoric acid solution (ph=2.18): taking 0.5mL of phosphoric acid, fixing the volume to a 1L volumetric flask with water, and then adjusting the pH value of the 0.24% phosphoric acid solution to 2.18 with triethylamine; arecoline hydrobromide, arecoline, norarecoline standard: the content is more than or equal to 98.0 percent.

2 instrument apparatus

A high performance liquid chromatograph and an ultraviolet detector; analytical balance: the sensing amount is 0.1mg and 0.01mg; an ultrasonic cleaner.

3 test procedure

3.1 standard stock solution: 553.06ug/ml arecoline hydrobromide (actual concentration of arecoline), 205ug/ml arecoline, 202ug/ml desmethyl arecoline and 243ug/ml arecoline total alkali mixed reference solution are prepared. And storing in a sealed manner at 2-8 ℃ in a dark place for one month.

3.2 standard series working solutions: accurately measuring 0.5mL of each stock solution of the mixed reference substance, gradually diluting the stock solution into 0.10mg/mL, 0.075mg/mL, 0.050mg/mL, 0.025mg/mL, 0.0125mg/mL, 0.010mg/mL, 0.005mg/mL and 0.0025mg/mL, and preparing the stock solution for clinical use.

3.3 preparation of sample solution: two experiments were performed in parallel. About 10mg of the sample is weighed, placed in a 10mL brown volumetric flask, 10mL of methanol is added for dissolution and volume fixing to a scale, and the mixture is shaken uniformly for later use.

3.3.1 liquid chromatography reference conditions

The column was PCP SCX 5um 4.6mm x 250mm (zhejiang hua spectrum new technology limited), mobile phase a acetonitrile: b0.24% phosphoric acid water (triethylamine adjusts ph=2.18) 1:9, the flow volume is 1ml/min; the sample injection amount is 15ul; the column temperature is 40 ℃, and the detection wavelength is 215nm.

3.3.2 determination of Standard solution and sample solution

Under the optimal condition of the instrument, respectively taking a sample solution and a standard solution, filtering with a 0.22 mu m organic microporous filter membrane, and measuring by a machine.

4 test results

The sample solution pattern should show 4 characteristic peaks corresponding to the characteristic pattern (figure 4), and peak 1, peak 2, peak 3, peak 4 should be consistent with the standard solution retention time of demethylated arecoline, peak 4 (arecoline) is set as S peak, and the relative retention time of peaks 1,2, 3, 4 and S peak should be within + -5% of the specified value.

(3) Product component of betel nut core extract

1. Betel nut core extract effective component and its content

The content of total alkaloids (arecoline, nor arecoline) in the extract is 0.5-65.0%.

(1) Betulin

Chemical name: n-methyl-1, 2,5, 6-tetrahydropyridine-3-carboxylic acid methyl ester

CAS number: 63-75-2

The molecular formula: c (C) ₈ H ₁₃ NO ₂ Molecular weight: 155.1970

Structural formula:

(2) alkaline name of norareca catechu: methyl 1,2,5, 6-tetrahydropyridine-3-carboxylate hydrochloride CAS no: 6197-39-3

The molecular formula: c (C) ₇ H ₁₂ CINO ₂ Molecular weight: 177.62868

Structural formula:

(3) betel secondary alkali chemical name: 1-methyl-1, 2,5, 6-tetrahydro-3-picolinic acid CAS No.: 499-04-7

The molecular formula: c (C) ₇ H ₁₁ NO ₂ Molecular weight: 141.16774

Structural formula:

(4) nor betel secondary basicity name: 1,2,5, 6-tetrahydropyridine-3-carboxylate CAS number: 498-96-4

The molecular formula: c (C) ₆ H ₁₀ CINO ₂ Molecular weight: 127.1412

Structural formula:

2. other components and the content thereof

The betel nut core extract contains not only betulin, nor betulin, but also flavone, tannin, polysaccharide, microelements, protein, fat, fiber, fatty acid, amino acid, etc.

Example 12: application of betel nut core extract in grass carp

(1) Test materials

Materials: betel nut core extract (betel nut total alkali content 2.86%) is provided by the major laboratory of Hunan province, a veterinary drug of Hunan agricultural university; 480 tails of healthy grass carp with similar body size and weight; the test feed is prepared by self according to the proportioning requirement of GB/T36205-2018 grass carp compound feed, and does not contain any drug additive. The ration and nutrition levels of the feed are shown in Table 2.

Table 2 tests of ration and nutrient level (dry matter basis) of grass carp feed

Nutritional ingredients

Note that: the premix is provided for each kilogram of daily ration: VA 120000IU, VD ₃ 40000IU、VE 480mg、VK ₃ 200mg、VB ₁ 200mg、VB ₂ 280mg、VB ₈ 240mg、VB ₁₂ 0.6Mg, 720Mg of calcium pantothenate, 1000Mg of nicotinic acid, 60Mg of folic acid, 1.2Mg of biotin, 6850Mg of VC phosphate, 3200Mg of inositol, 4000Mg of Mg, 4800Mg of Fe, 2000Mg of Zn, 800Mg of Mn, 160Mg of Cu, 12Mg of Co, 4Mg of Se and 40Mg of I. The nutritional ingredients are all measured values.

(2) Test method

1. Grouping

480 healthy grass carp bodies with similar body weight are randomly divided into 4 groups, 3 replicates of each group, and 40 replicates of each group. Wherein 3 test groups (ANE-1, ANE-2, ANE-3) respectively added with 0.25g/kg, 0.5g/kg, 1g/kg of betel nut core extract into basic diet; 1 blank group (CON group), feeding basic diet, and adding 0.25g/kg, 0.5g/kg, and 1.0g/kg of ANE-1, 2, and 3 groups respectively without adding Areca catechu extract.

2. The feeding management feeding test is carried out in a practice base of aquatic product scientific research and teaching in Hunan agricultural university of the West Dongting management area of the Ding urban area of the Changde city of 7-9 months in 2022, and cage culture is adopted. After the test fish is purchased and domesticated for 10 days by using basic diet, the test fish is randomly divided into 12 net boxes (1.5 m multiplied by 2.0 m) according to 40 tails of each net box, and 3 net boxes of each treatment group are subjected to formal test for 8 weeks and 66 days. Feeding is carried out for 1 time in a test period of 08:30 and 16:30 each day, feeding amount is adjusted according to weather, water temperature and feeding conditions by taking 3% -5% of the weight of grass carp as a standard in a satiation mode, a small amount of residual materials are ensured after 15 minutes of feeding each time, and daily feeding and death conditions are accurately recorded.

3. Measurement index

3.1, during the test period of growth performance, accurately recording initial weight, final weight and death grass carp mantissa of grass carp, calculating indexes such as weight gain rate, feeding rate, feed conversion efficiency, survival rate and the like, and calculating the following formula:

survival rate (survivin rate, SR%) =100×test end grass carp mantissa (tail)/test initial grass carp mantissa (tail) Feeding rate (Feeding rate, FR%) =100×total dry matter feed intake (g)/{ [ initial mass (g) +final mass (g) ]×number of days of cultivation/2 }

Feed conversion efficiency (FE%) =100×dry matter Feed intake (g)/[ final body weight (g) -initial body weight (g) ]

Weight gain rate (WGR%) =100× (Wt-Wo)/Wo

Note that: wo is the initial weight of the fish body; wt is the final weight of the fish body; t is the number of days of feeding.

3.2 body index

When the test is finished, measuring the body length of the grass carp to calculate the fullness degree of the grass carp; and weighing viscera disc, liver and mesenteric fat of grass carp to calculate the indexes such as viscera body ratio, liver body ratio and intestinal fat ratio, wherein the calculation formula is as follows:

fullness (g/cm 3) =100×body weight (g)/body length (cm) 3;

visceral volume ratio (%) = 100 x visceral weight (g)/body weight (g);

liver body ratio (%) =100×liver weight (g)/body weight (g);

Intestinal lipid ratio (%) =100×intestinal lipid weight (g)/body weight (g).

3.3 muscle texture Properties

3 grass carps are randomly taken from each net cage, the back muscles are collected and stored at a low temperature of 4 ℃, and the texture characteristics of the muscles are measured within 24 hours.

3.4 liver antioxidant

At the end of the test, grass carp liver tissue is collected, and the total antioxidant capacity (T-AOC), catalase Activity (CAT), superoxide dismutase activity (SOD) and the like are measured.

3.5 serum antioxidant

And after the test is finished, blood is collected from the tail vein of the test grass carp by using a disposable vacuum blood collection tube, after centrifugation (3500 rpm for 15 min), a pipetting gun is used for sucking the supernatant of the blood sample, the supernatant is subpackaged into a centrifuge tube, and after quick freezing by liquid nitrogen, the blood sample is transferred into a refrigerator at-80 ℃ for preservation, and is used for measuring the antioxidant index of serum.

3.6 intestinal morphology

At the end of the experiment, the content-free foregut, midgut, hindgut were dissected out, fixed with 4% formaldehyde solution, stained with conventional hematoxylin-eosin (HE), observed under an optical microscope, measured for Villus Height (VH), crypt Depth (CD), and the intestinal villus/crypt ratio was calculated.

4. Analysis of test data

Data were statistically analyzed using the ANOVA one-way variance program of SPSS25 software, test results were expressed using "mean and standard error" with P >0.05 being insignificant and P <0.05 being significant.

(3) Test results

1. Effect of betel nut core extract on grass carp growth performance

The effect of betel nut core extract on grass carp productivity is shown in table 3. Compared with CON group, the feed coefficient of ANE-1 group grass carp is obviously reduced (P < 0.05) and other indexes have no obvious influence (P > 0.05).

The betel nut core extract is added into the feed to improve the production performance of grass carp.

TABLE 3 Effect of betel nut core extract on grass carp growth Properties

Note that: the same row of data shoulder marks have no letters or the same letters indicate that the difference is not significant (P > 0.05), and the letters indicate that the difference is significant (P < 0.05). The table below is the same.

2. Effect of betel nut core extract on grass carp body index

The effect of betel nut core extract on grass carp body index is shown in table 4. Compared with CON group, the fullness of ANE-1 group is obviously improved (P < 0.05), the dirty body ratio of ANE-2 group is obviously reduced (P < 0.05), the liver body ratio of ANE-2 group is obviously improved (P < 0.05), and other indexes have no obvious influence (P > 0.05).

The betel nut core extract is added into the feed to improve the body index of grass carp to a certain extent.

TABLE 4 Effect of betel nut core extract on grass carp body index

3. Influence of betel nut core extract on muscle texture characteristics of grass carp

The effect of betel nut core extract on muscle texture characteristics of grass carp is shown in table 5. The muscle hardness, viscosity and masticability of the grass carp muscle of ANE-2 and ANE-3 groups were significantly improved (P < 0.05) compared to CON group, and the concentration-dependent tolerance was linearly changed (linear, P < 0.01), and other indexes had no significant effect (P > 0.05).

The betel nut core extract is added into the feed to improve the texture characteristics of grass carp muscles.

TABLE 5 influence of betel nut core extract on muscle texture characteristics of grass carp

4. Effect of betel nut core extract on antioxidant capacity of grass carp liver

The effect of betel nut core extract on the oxidation resistance index of grass carp liver is shown in table 6. Compared with CON group, the liver total antioxidant capacity of ANE-2 group and ANE-3 group grass carp is obviously improved (P < 0.05), and has certain concentration dependence and linear change (P < 0.01); liver catalase activities (P < 0.05) of the grass carp in the ANE-1 group and the ANE-2 group are obviously improved, and the grass carp has certain concentration dependence and shows secondary change (P < 0.01); the liver superoxide dismutase activity of the grass carp in the ANE-1 group, the ANE-2 group and the ANE-3 group is obviously improved (P < 0.05), and the grass carp has certain concentration dependence and is in linear and secondary change (linear, P <0.01; numeric, P < 0.05); liver glutathione peroxidase activity of ANE-3 group grass carp is obviously improved (P < 0.05), and has certain concentration dependence and linear change (P < 0.01); liver malondialdehyde content of ANE-2 group grass carp was significantly increased (P < 0.05); other indices had no significant effect (P > 0.05).

The betel nut core extract is added into the feed to improve the antioxidant capacity of the grass carp liver.

TABLE 6 Effect of betel nut core extract on antioxidant capacity of grass carp liver

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5. Effect of betel nut core extract on antioxidant capacity of grass carp serum

The effect of betel nut core extract on the index of antioxidant capacity of grass carp serum is shown in table 7. The total antioxidant capacity and catalase activity of serum of the grass carp of ANE-2 group are significantly improved (P < 0.05) compared with the CON group, and the serum is in linear and secondary changes (linear, P <0.05; squaric, P < 0.01); serum catalase activity of ANE-3 group grass carp is obviously reduced (P < 0.05); serum superoxide dismutase activity of the grass carp in the ANE-2 group and the ANE-3 group is obviously improved (P < 0.05), and the grass carp has concentration dependence and linear change (P < 0.01); serum malondialdehyde levels were significantly reduced (P < 0.05) in the ANE-1 and ANE-3 group grass carp and had a concentration dependence with a linear change (P < 0.01); other indices had no significant effect (P > 0.05).

The betel nut core extract is added into the feed to improve the antioxidant capacity of grass carp serum.

TABLE 7 Effect of betel nut core extract on antioxidant capacity of grass carp serum

6. Effect of betel nut core extract on grass carp's front intestinal tissue morphology

The effect of betel nut core extract on grass carp foregut tissue morphology is shown in table 8 and fig. 5. Microscopic observation and data statistical analysis show that the villus height of the foregut of the ANE-2 group grass carp is obviously higher than that of the CON group and is changed secondarily (P < 0.01); the foregut villus/crypt ratio was significantly higher for the ANE-1 and ANE-2 grass carp than for the CON group (P < 0.05), with linear and quadratic changes (linear, P <0.01; squaric, P < 0.05). Other indices were not significantly different (P > 0.05).

The areca catechu extract is added into the feed to improve the tissue morphological development of the grass carp foreintestines.

TABLE 8 Effect of betel nut core extract on grass carp foregut tissue morphology

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7. Effect of betel nut core extract on intestinal tissue morphology in grass carp

The effect of betel nut core extract on the intestinal tissue morphology of grass carp is shown in Table 9 and FIG. 6. Microscopic observation and data statistical analysis show that the crypt depth of the midgut of the grass carp in the ANE-1 group and the ANE-3 group is obviously lower than that of the CON group and is in linear change (P is less than 0.01); the villus/crypt ratio of the middle intestine of the grass carp in ANE-1 group and ANE-3 group was significantly higher than that in CON group (P < 0.05), and varied linearly (linear, P < 0.05). There was no significant difference in fluff height between groups (P > 0.05).

The areca catechu extract is added into the feed to improve the tissue morphological development of the midgut of grass carp.

TABLE 9 Effect of betel nut core extract on the morphology of grass carp midgut tissue

8. Effect of betel nut core extract on intestinal tissue morphology after grass carp

The effect of betel nut core extract on the hindgut tissue morphology of grass carp is shown in Table 10 and FIG. 7. Microscopic observation and data statistical analysis show that compared with the CON group, the heights of villi of the hindintestines of the grass carp in the ANE-1 group, the ANE-2 group and the ANE-3 group are obviously increased (P < 0.05) and are linearly changed (linear, P < 0.01); the villus/crypt ratios of the hindgut of the grass carp in ANE-1, ANE-2 and ANE-3 groups were significantly increased (P < 0.05) with linear and quadratic changes (linear, P <0.05; squaric, P < 0.01). Other indices were not significantly different (P > 0.05).

The areca catechu extract is added into the feed to improve the tissue morphological development of the grass carp hindgut.

TABLE 10 Effect of betel nut core extract on the morphology of grass carp hindgut tissue

(4) Conclusion(s)

1) In the test, the betel nut core extract is added into the feed to improve the weight gain rate and the final weight of the grass carp, reduce the feed coefficient and improve the production performance.

2) In the test, the betel nut core extract is added into the feed, so that the fullness of grass carp can be obviously improved, the dirty body ratio can be reduced, and the body index can be improved.

3) In the test, the betel nut core extract is added into the feed, so that the hardness, the tackiness and the chewiness of the grass carp muscles can be obviously improved, and the muscle quality can be improved.

4) In the test, the betel nut core extract can inhibit the oxidation injury of the liver to a certain extent by improving the total antioxidant capacity, the catalase activity and the superoxide dismutase activity of the liver of the grass carp, so that the liver health is protected.

5) In the test, the betel nut core extract can improve the total antioxidant capacity, the catalase activity and the superoxide dismutase activity of the grass carp serum, reduce the content of malondialdehyde in the serum and improve the body health of the grass carp.

6) In the test, the betel nut core extract is added into the feed to obviously improve the intestinal villus height of the grass carp, and the crypt depth also shows a descending trend, which shows that the betel nut core extract can promote the morphological development of intestinal tissues of the grass carp to a certain extent and improve the intestinal health.

Example 13: application of betel nut core extract in broiler chickens

(1) Test materials

Betel nut core extract (betel nut total alkali content 2.86%) is provided by the major laboratory of Hunan province, a veterinary drug of Hunan agricultural university; 240 healthy white feather broilers (Aijigao, AA broilers) with similar weight and 1 day old are half of the male and female broilers; the test feed is prepared by self according to the proportioning requirement of the feeding standard NYT33-2014 of the chickens in the agricultural department. The ration and nutrition levels are shown in Table 11.

Table 11 test broiler basal ration and nutrient level (dry matter basis)

Note that: ¹⁾ the premix is provided for each kilogram of daily ration: VA12000IU, VD35000IU, VE80 IU, VK33.2mg, VB1 3.2mg,VB2 65mg,VB6 4.3mg,VB12 0.017mg, pantothenic acid 20mg, biotin 0.22mg, folic acid 2.2mg,I 1.25mg,Fe 20mg,Mn 120mg,Se 0.3mg,Zn 110mg. ²⁾ The nutrition index is calculated value.

(2) Test method

1. Test design

240 healthy AA young chickens of 1 day old are selected and randomly divided into 4 treatments according to the principle of no difference of initial weight, 6 replicates are set for each treatment, and 10 chickens are repeated. The feeding period was 42d. The method is divided into a front stage (1-21 d) and a rear stage (22-42 d). The test groups are shown in Table 12.

Table 12 test group

2. Feeding management

Raising experiments are carried out in the technical institute of Shang county in 8 months to 10 months in 2022, the experimental animals are subjected to cage raising, the chicken houses and cages are cleaned before the experiments, the chicken houses and cages are sterilized by using 1% sodium hydroxide solution, the chicken houses and cages are cleaned by clear water after 24 hours, then the chicken houses, the charging trays, the charging barrels and the drinking water equipment are subjected to 48-hour fumigation sterilization by using formalin and potassium permanganate (30 ml of formalin and 15g of potassium permanganate are added per cubic meter), and the chicken houses are ventilated for five days. The test adopts the combination of natural illumination and artificial illumination, free ingestion and drinking water, and the health condition, ingestion, drinking water, excrement and the like of the test chicken flock are observed in the morning and evening every day, and disinfection and immunization are carried out according to the conventional procedure.

3. Measurement index

3.1 production performance is respectively 1 day old, 21 days old and 42 days old, single chickens are weighed on an empty stomach, the residual material amount is weighed, and the death and the diarrhea number are recorded. The Average Daily Feed Intake (ADFI), average Daily Gain (ADG) and feed-to-meat ratio (F/G) of each group in the early stage of growth (1-21 days old), the late stage of growth (22-52 days old) and the full stage of growth (1-42 days old) were calculated.

3.2 collecting liver tissues of broiler chickens when the liver antioxidant test is finished, and measuring total antioxidant capacity (T-AOC), catalase Activity (CAT), superoxide dismutase activity (SOD) and the like.

3.3 serum antioxidant

And after the test is finished, blood is collected from the neck veins of the broiler chickens by using a disposable vacuum blood collection tube, after centrifugation (3500 revolutions per minute for 15 min), a pipetting gun is used for sucking the supernatant of the blood sample, the supernatant is subpackaged into a centrifuge tube, and after quick freezing by liquid nitrogen, the chickens are transferred into a refrigerator at the temperature of minus 80 ℃ for preservation, and the serum antioxidant index is measured.

3.4 intestinal morphology

At the end of the experiment, the duodenum, jejunum and ileum without contents were dissected out, fixed with 4% formaldehyde solution, stained with conventional hematoxylin-eosin (HE), observed under an optical microscope, measured for Villus Height (VH), crypt Depth (CD), and the intestinal villus/crypt ratio was calculated.

(3) Test results

1. Effect of betel nut core extract on growth performance of broiler chickens

The performance of betel nut core extract on white feather broiler production is shown in table 13. The 42 day old and 1-42d average day gain of ANE2 and ANE3 groups increased significantly (P < 0.05) compared to CON group; the 21-42d feed weight ratio of ANE1 group and ANE3 group was significantly reduced (P < 0.05); the 1-42d feed weight ratio of ANE3 group is obviously reduced (P is less than 0.05), and other indexes have no obvious difference (P is more than 0.05).

In the test, the betel nut core extract is added into the diet, so that the average daily gain of white feather broilers can be increased to different degrees, the feed conversion ratio is reduced, and the production performance is improved.

Table 13 influence of betel nut core extract on broiler chicken productivity

Note that: the same row of numerical shoulder marks have no letters or the same letters indicate that the difference is not significant (P > 0.05), and different lower case letters indicate that the difference is significant (P < 0.05), and the following table is the same.

2. Influence of betel nut core extract on anti-oxidation index of broiler serum

The effect of betel nut core extract on the antioxidant function of white feather broiler serum is shown in table 14. Compared with CON group, the serum superoxide dismutase activity of the ANE3 group broiler is obviously reduced (P is less than 0.05), the total antioxidant capacity is obviously improved, the serum catalase activity of the ANE1, ANE2 and ANE3 group broiler is obviously increased (P is less than 0.05), and the serum superoxide dismutase activity is changed secondarily (P is less than 0.01). The differences among the other index groups are not significant (P > 0.05).

In the test, the betel nut core extract is added into the diet, so that the activity of the serum catalase of the broiler chickens can be increased, and the oxidation resistance of organisms can be improved.

Table 14 influence of betel nut core extract on the antioxidant index of broiler serum

3. Influence of betel nut core extract on oxidation resistance index of liver of broiler chickens

The effect of betel nut core extract on liver antioxidant function of white feather broiler is shown in table 15. Compared with CON group, the liver glutathione peroxidase and superoxide dismutase activity of the ANE1, ANE2 and ANE3 broiler chickens are obviously improved (P is less than 0.05), the concentration dependence is linear (linear, P is less than 0.01), and the liver catalase activity of the ANE1 broiler chickens is obviously improved (P is less than 0.05). The differences among the other index groups are not significant (P > 0.05).

In the experiment, betel nut core extract is added into diet, so that the activity of glutathione peroxidase, superoxide dismutase and catalase in the liver of broiler chickens can be increased, and the oxidation resistance of the liver can be improved.

Table 15 influence of betel nut core extract on the antioxidant index of the liver of broiler chickens

4. Effect of betel nut core extract on the morphology of the duodenum of broiler chickens

The effect of betel nut core extract on the morphology of the duodenum of broiler chickens is shown in table 16 and fig. 8. The duodenal villi length was significantly increased (P < 0.05) in the ANE2 and ANE3 group broilers compared to the CON group. The differences among the other index groups are not significant (P > 0.05).

In the test, the betel nut core extract is added into the diet, so that the length of the dorking duodenal villus can be increased, and the morphological development of the duodenal tissue can be promoted.

Table 16 influence of betel nut core extract on the morphology of the duodenal tissue of broilers

5. Effect of betel nut core extract on jejunal morphology of broiler chickens

The effect of betel nut core extract on jejunal morphology of broiler chickens is shown in table 17 and fig. 9. The betel nut core extract is added into the diet, and has no obvious influence (P is more than 0.05) on the morphological index of jejunum group of the broiler chickens.

Table 17 influence of betel nut core extract on jejunal tissue morphology of broiler chickens

6. Effect of betel nut core extract on ileal histology of broiler chickens

The effect of betel nut core extract on the ileal histology of broiler chickens is shown in table 18 and fig. 10. The betel nut core extract is added into the diet, and has no obvious effect (P is more than 0.05) on the ileum morphology and morphology indexes of the broiler chickens.

Table 18 effect of betel nut core extract on ileum tissue morphology of broiler chickens

(4) Conclusion(s)

1) In the test, the betel nut core extract is added into the diet, so that the average daily gain of white feather broilers can be increased to different degrees, the feed conversion ratio is reduced, and the production performance is improved.

2) In the test, the betel nut core extract is added into the diet, so that the activity of the serum catalase of the broiler chickens can be increased, and the oxidation resistance of organisms can be improved.

3) In the experiment, betel nut core extract is added into diet, so that the activity of glutathione peroxidase, superoxide dismutase and catalase in the liver of broiler chickens can be increased, and the oxidation resistance of the liver can be improved.

4) In the experiment, the betel nut core extract is added into the diet, so that the height of the duodenal villus of the broiler chickens can be increased, the intestinal tissue development of the broiler chickens is promoted, and the intestinal health of the broiler chickens is improved to a certain extent.

The application aims at comprehensively utilizing and developing betel nuts with multi-dimensional resources of different parts and different functions, focuses on the development and utilization of the alkaloid bioactive components of betel nuts, fully digs the active functions and feeding values of betel nuts, creates low-cost cultivation input products capable of improving the production performance, meat quality and organism oxidation resistance of livestock and poultry and aquatic animals, and comprehensively utilizes the boosting betel nuts resources and sustainably develops the livestock industry.

Note that: the foregoing is merely a preferred embodiment of the present application and it should be noted that numerous changes, modifications, substitutions and variations could be made herein by those skilled in the art without departing from the spirit and scope of the application as defined by the appended claims.

Claims

1. An extract of betel nut, characterized in that the extract of betel nut comprises betel nut total alkaloids, which comprise arecoline, norarecoline, arecoline and norarecoline.

2. The betel nut extract according to claim 1, characterized in that the betel nut extract is betel nut core extract.

3. The betel nut core extract according to claim 2, characterized in that the total mass fraction of betulin, norbetulin, betulin and norbetulin is 0.5-65.0%.

4. A process for preparing the betel nut extract according to any one of claims 1 to 3, characterized in that it comprises the following steps:

(5) Sieving: sieving with 50-80 mesh sieve to obtain the betel nut extract.

5. The method according to claim 4, wherein the cooking time in step (1) is 20 to 30 mm; the drying temperature is 50-80 ℃; the feed liquid ratio of the first reflux in the step (2) is 1:8, the feed liquid ratio of the second reflux is 1:6, extracting for 120min each time; the auxiliary material is at least one of maltodextrin, soluble starch, corn starch, potato starch and dextrin.

6. The method according to claim 4, wherein the temperature of the reduced pressure concentration in step (3) is 60 ℃; in the step (4), the mass ratio of the betel nut core extract to the maltodextrin is as follows: 1:0.8; and (5) sieving the mixture through an 80-mesh sieve.

7. A process for preparing the betel nut extract according to any one of claims 1 to 3, characterized in that it comprises the following steps:

(2) Drying and crushing: drying betel nut cores at 60-120 deg.c to water content less than 10%, and crushing;

(3) Sieving: sieving with 50-80 mesh sieve to obtain the betel nut extract.

8. The method according to claim 7, wherein the cooking time in step (1) is 20-30 mm; the drying temperature is 50-80 ℃; in the step (2), the drying temperature is 100-120 ℃, and the mixture is sieved by a 80-mesh sieve.

9. A process for preparing the betel nut extract according to any one of claims 1 to 3, characterized in that it comprises the following steps:

(1) Pretreatment: pouring fresh betel nuts into boiling water, steaming for 20-60 min, drying at 45-90 ℃ until the water content is between 18-21%, respectively peeling off peel and taking out seeds to obtain betel nut peel and betel nut core;

(3) Extracting: putting betel nut core powder into a reflux device, adding 8-14 times of pure water, heating and refluxing for 1-2 h, repeating for 2 times, and collecting betel nut water extract; concentrating the extract to 20% of the original solution, regulating the pH value to 7-10 with sodium hydroxide solution to obtain alkaline extract, adding Dichloromethane (DCM) for 2-4 times for extraction, combining the extract, concentrating under reduced pressure to 20% of the volume of the solvent, standing overnight, filtering out precipitate to obtain filtrate, concentrating under reduced pressure;

(4) Purifying: loading the treated macroporous resin by a wet method, washing and balancing, loading into a column, loading sample with a sample loading amount of 1:20-30, loading column liquid with a concentration of 3mg/ml, loading column liquid with a pH of 7-8 and eluting agent with a volume of 60-70% ethanol of 3, collecting the eluting solution, concentrating under reduced pressure, and drying to obtain the betel nut extract.

10. The method according to claim 9, wherein the cooking time in step (1) is 20-30 mm; the drying temperature is 50-80 ℃; in the step (2), the drying temperature is 100-120 ℃.

11. The method according to claim 9, wherein in step (3), the feed-to-liquid ratio is 1:14, the extraction time is 2 hours, the pH is adjusted to 9 with sodium hydroxide, and the dcm is extracted 3 times; in the step (4), the macroporous resin is any one of H103, S-8, AB-8, ADS-8 and D3250; the eluent is 70% ethanol; in step (4), the macroporous resin is H103.

12. Use of the betel nut extract according to any one of claims 1-3 or the betel nut extract prepared by the method according to any one of claims 4-11 in a cultivation input, characterized in that the cultivation input has any one of the following functions:

(2) Improving the total oxidation resistance of fish or livestock;

(3) Improving the digestion capability of fish or livestock;

(4) Improving the muscle quality of fish.