CN109498665A - A kind of application method of saponin extract as fish immunity reinforcing agent - Google Patents
A kind of application method of saponin extract as fish immunity reinforcing agent Download PDFInfo
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- CN109498665A CN109498665A CN201811507397.8A CN201811507397A CN109498665A CN 109498665 A CN109498665 A CN 109498665A CN 201811507397 A CN201811507397 A CN 201811507397A CN 109498665 A CN109498665 A CN 109498665A
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Abstract
A kind of application method the invention discloses saponin extract as fish immunity reinforcing agent is specially that arasaponin extract is first configured to the water-soluble solution of suitable concentration, using misty spray pattern by it slowly, be equably sprayed into feed.So that effective component is uniformly distributed in feed, solve the problems, such as that powdered extract is difficult to Direct Uniform and admixes feed since additive capacity is humble.
Description
Technical field
The present invention relates to a kind of fish immunity reinforcing agent, especially a kind of saponin extract is as fish immunity reinforcing agent
Application method.
Background technique
Fish are frequently subjected to a variety of pathogen infections in the breeding process, improve the disease-resistant energy of fish by using immunopotentiator
Power is the important measures for reducing disease and occurring, safeguarding aquaculture healthy and sustainable development.
Arasaponin is the main active of Chinese medicinal plant Radix Notoginseng, has immunoregulation effect, can be used as Immune-enhancing effect
Agent uses.But there are problems that in concrete application, 1) assurance for amount not only raises cost, no if additive amount is too high
Conducive to promoting and applying in practice, and negative effect may cause to cultivated animals；If additive amount is too low, and expection is not achieved
Effect.
2), arasaponin extract is solid-state powder, and since additive capacity is humble, it is directly admixed to feed, and there are it
The problem of effective component is unevenly distributed in feed.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art places, and additive amount can be accurately held by providing one kind, and
It is able to achieve a kind of application method of the saponin extract being evenly distributed as fish immunity reinforcing agent.
A kind of application method of saponin extract as fish immunity reinforcing agent,
(1) arasaponin extract is provided；
(2) arasaponin extract is dissolved in phosphate buffer (PBS) or physiological saline or deionized water or pure water,
It is configured to the saponin aqueous solution that concentration is 0.05- 0.1mg/mL；
(3) per kilogram feed uniformly sprays 100n-200nmL saponin aqueous solution, by per kilogram feed addition arasaponin extract
10nmg is calculated, and wherein n is (1,2,3,4,5)；
(4) uniformly dry to be sprinkled upon particle basal feed surface first by suitable feed adhesive when feed is pellet, so
Appropriate saponin aqueous solution is packed into watering can afterwards, slowly, is equably sprayed on feed, is mixed, interior is dried or 60 DEG C of drying；Work as feeding
When material is powdery compound feed, appropriate saponin aqueous solution is packed into watering can, slowly, is equably sprayed in mash feed, is stirred
Uniformly, the particle of appropriate particle size, 60 DEG C of drying are made using minitype fodder-pelleting machine.
The preparation method of phosphate buffer are as follows: 0.01mol/L phosphate buffer (PBS) is prepared according to a conventional method, tool
Body is as follows: weighing 8g sodium chloride, 0.2g potassium chloride, 1.44g disodium hydrogen phosphate and 0.24 gram of potassium dihydrogen phosphate are dissolved in 800mL
In deionized water, the pH value of solution is adjusted to 7.40, adds deionized water to be settled to 1L, high pressure steam sterilization, room temperature is protected after cooling
It deposits.
Physiological saline is prepared according to a conventional method, specific as follows: 0.9 gram of sodium chloride is weighed, is dissolved in a small amount of deionized water,
100mL is diluted to get to 0.9% sodium-chloride water solution.
In summary, the present invention following advantage compared with prior art:
1, arasaponin powdered extract is configured to the water-soluble solution of debita spissitudo by the present invention, is used by tools such as watering cans
Misty spray pattern solves powdered extract since additive capacity is low so that effective component is uniformly distributed in feed
It is micro- and be difficult to the problem of Direct Uniform admixes feed.
2, application method of the present invention is easy to operate, Yi Shihang.
3, solvent of the present invention is common solvent, and preparation method is conventional, simple, is easy to get, and safe and non-toxic pair
Effect.
4, the dosage for the arasaponin extract that the present invention is recommended to use is humble, low in cost, is especially suitable for fish
This lesser cultivated animals of single individual use.
5, the dosage of arasaponin extract of the present invention can effectively enhance fish immunity function, improve disease-resistant
Ability.By taking Tilapia mossambica as an example, Tilapia mossambica gamma interferon is can be improved in per kilogram feed addition 10mg and 30mg arasaponin extract
With Complement C_3 level, the death rate of Tilapia mossambica after artificial challenge Aeromonas hydrophila can be reduced by 40% or so；It is with Larimichthys crocea
Example, per kilogram feed addition 10mg arasaponin extract can reduce the death rate of Larimichthys crocea after artificial challenge vibrio alginolyticus
37% or so.
Specific embodiment
The present invention is described in more detail below with reference to embodiment.
Embodiment 1
A kind of application method of saponin extract as fish immunity reinforcing agent,
(1) arasaponin extract is provided；
(2) arasaponin extract is dissolved in phosphate buffer (PBS) or physiological saline or deionized water or pure water,
It is configured to the saponin aqueous solution that concentration is 0.05- 0.1mg/mL；
(3) per kilogram feed uniformly sprays 100n-200nmL saponin aqueous solution, by per kilogram feed addition arasaponin extract
10nmg is calculated), wherein n is (1,2,3,4,5)；
(4) uniformly dry to be sprinkled upon particle basal feed surface first by suitable feed adhesive when feed is pellet, so
Appropriate saponin aqueous solution is packed into watering can afterwards, slowly, is equably sprayed on feed, is mixed, interior is dried or 60 DEG C of drying；Work as feeding
When material is powdery compound feed, appropriate saponin aqueous solution is packed into watering can, slowly, is equably sprayed in mash feed, is stirred
Uniformly, the particle of appropriate particle size, 60 DEG C of drying are made using minitype fodder-pelleting machine.
(5) the not described part of the present embodiment is same as the prior art.
In order to verify effect of the invention, the present invention has carried out following test:
One: influence (experiment) of the arasaponin extract to Tilapia mossambica immunity function and disease-resistant former infection ability
Materials and methods
The preparation of 1.1 pharmaceutical chemistry:
Arasaponin extract is dissolved in PBS, compound concentration is the arasaponin aqueous solution of 0.1mg/mL, and appropriate be packed into is taken to spray
Pot.Fresh-water fishes particle basal feed surface first is sprinkled upon by suitable feed adhesive is uniformly dry, then with watering can by arasaponin
Aqueous solution is slow, is equably sprayed at pellet surface, mixes, interior is dried.
Test fish grouping and feeding management
Experiment is 100g or so with Tilapia mossambica original body mass.It is used before experiment in this research department's indoor full automatic circulating water culture system
Then fresh-water fishes basal feed handling 2 weeks is randomly divided into 3 groups (A0, A1, A2), every group of 60 tails.
A1 group per kilogram feed addition 100mL saponin aqueous solution (i.e. per kilogram feed arasaponin containing 10mg extract),
A2 group per kilogram feed addition 300mL saponin aqueous solution (i.e. per kilogram feed arasaponin containing 30mg extract), control group
(A0) arasaponin extract is free of in per kilogram feed addition 300mLPBS(, that is, feed).Culture experiment carries out 8 weeks, every the sky
Noon feeds 1 time, feeds 30g feed (weighing 0.5% by fish body to feed) every time.Daily observation is ingested and death condition, pulls out in time residual
Bait and dead fish.Water temperature control is in (19.5 ± 2.5) DEG C during raising.
The measurement of blood parameters
1.3.1 blood sampling
The 30th day after feeding experiment starts, 5 tails were fished at random from each group for trying Tilapia mossambica, tail bone venous blood sampling.4 DEG C of items
5000rpm is centrifuged 10min under part, collects upper serum, it is spare to be stored in -20 DEG C of refrigerators.
1.3.2 Biochemical Indexes
It is commented using superoxide dismutase (SOD), lysozyme (LZM), Complement C_3 (C3), interferon-γ (IFN-γ) as immune
Valence index.SOD, lysozyme build up the kit measurement of Bioengineering Research Institute using Nanjing, and Complement C_3 uses U.S. R&D
The measurement of Fish Complement Component 3 (C3) Elisa assay kit, interferon-γ use U.S. R&D Fish
The measurement of Interferon γ (IFN-γ) Elisa assay kit.Concrete operations are carried out in strict accordance with kit specification.
Attack malicious infection experiment
1.4.1 Bacteria Culture
Experiment with pathogen be this laboratory separation, conservation Aeromonas hydrophila (Aeromona hydrophila)ZN1.It will
ZN1 conservation liquid is recovered (28 DEG C, 200rpm is overnight) with TSB culture medium, and bacterium solution is centrifuged 10min with 3000rpm, collects precipitating bacterium
Body is resuspended with sterile PBS, is counted, and adjusts bacterial concentration according to pre-tapped poison experimental result.
Infection experiment
In the 8th week of feeding experiment, the Tilapia mossambica point for fishing for body colour health at random from each group was supported in 3 water vats, every 15 tail of cylinder
Fish carries out challenge test.Aeromonas hydrophila bacterium solution (3.0 × 10 is injected intraperitoneally in every tail8Cfu/mL) 0.5mL.Every group is continued to throw
Original feed is fed, is observed 2 weeks, and count the death rate.
Serum antibody titer measurement
2 weeks after attacking poison, the Tilapia mossambica for taking 5 tails to survive at random from each group, blood sampling prepares serum, the total antibody of detection serum and
Aeromonas hydrophila specific antibody titres, while measuring the Total albumen content.
The total antibody of serum is surveyed using enzyme-linked immunosorbent assay (ELISA) method.Serum is acted as 2 times from 1:500 times with PBS
Than dilution, every 50 μ L of hole is coated with 96 hole elisa Plates, while making blank control with PBS, and 4 DEG C overnight.Next day abandons coating buffer, every hole drop
Add PBS-Tween20 of the 100 μ L containing 2%BSA, 37 DEG C of closing 90min.PBS-Tween20 is washed 3 times, and 50 μ L1 are added in every hole:
1000 diluted anti-this laboratory the Larimichthys crocea wide spectrum monoclonal antibody 2F5H4(self-controls of mouse), 37 DEG C of reaction 60min.After ibid washing, every hole
The diluted sheep anti-mouse igg Horseradish Peroxidase Conjugates (sigma) of 50 μ L1:10000,37 DEG C of reaction 60min are added.
PBS-Tween20 is washed 3 times, H2After O washes 1 time, the reaction substrate OPD(sigma of 50 μ L Fresh is added in every hole), room temperature is kept away
15 min of light colour developing.Finally use 2mol ﹒ L-1H2SO4Reaction is terminated, reads the OD value at 495nm with microplate reader (biorad).
Serum specific antibody potency is using conventional ELISA method operation.Envelope antigen is the thermophilic aqueous vapor list of ultrasonication
Born of the same parents bacterium ZN1, blank control are coated with PBS, and every 50 μ L of hole, 4 DEG C overnight.Primary antibody is that (1:50 times is played multiple proportions to tilapia serum to be measured
Dilution), every 50 μ L of hole.Secondary antibody is the anti-Larimichthys crocea wide spectrum monoclonal antibody 2F5H4(1:1000 dilution of mouse), every 50 μ L of hole.Three resist for goat-anti
Mouse IgG Horseradish Peroxidase Conjugates (1:10000 dilution), every 50 μ L of hole.Reaction substrate OPD(sigma is added) after, room
Temperature is protected from light 15 min of colour developing, with 2mol ﹒ L-1H2SO4Reaction is terminated, reads the OD value at 495nm with microplate reader (biorad).
The antibody for being greater than maximum serum diluting multiple as the serum of 2.1 times of blank control OD value using serum OD value is imitated
Valence.To all antibody titer Qu ㏒2Data processing is carried out after value.
Total serum protein uses Coomassie brilliant blue decoration method (Bradford method).The sterile PBS of test serum is made into series
Dilution.2 μ L serum dilutions are added in every hole on ELISA Plate, and then every hole adds 200 μ L Coomassie brilliant blue solution, uses microplate reader
(Biorad) the OD value at 595nm is read.Protein concentration is calculated according to standard curve.
Data statistics and processing
Experimental data is indicated with sample mean ± standard deviation (mean ± SD), using one-in 13.0 statistical software of SPSS
Way ANOVA variance analysis and DuncanShi mean value Multiple range test are analyzed and processed experimental data, and P < 0.05 thinks have
Significant difference.
As a result with analysis
Influence of 2.1 arasaponins to tilapia serum biochemical indicator
After feeding arasaponin 30, part relevant to nonspecific mental retardation biochemistry refers in each experimental group tilapia serum
Target testing result is shown in Table 1.As can be seen from Table 1, A1, A2 group SOD in serum enzyme activity are slightly above control group, but the not significant (P of difference
> 0.05).A1, A2 group serum interferon γ, Complement C_3 and total protein content are all remarkably higher than control group (P < 0.05).
Influence of 1 arasaponin of table to tilapia serum biochemical indicator
| Group | SOD (U/mL) | IFN-γ (ng/L) | C3(ng/L) | Total protein (mg/mL) |
| A0 group | 27.29±13.93a | 41.83±2.84a | 78.83±38.35a | 2.52±0.32a |
| A1 group | 37.77±12.23ab | 58.89±6.47b | 163.53±44.12b | 3.00±0.05b |
| A2 group | 37.27±18.24ab | 72.87±6.85c | 181.58±8.58b | 3.17±0.15b |
Note: the female difference person of same column shoulder marking-up indicates significant difference (P < 0.05).Following table is same.
Influence of the arasaponin to tilapia serum antibody titer
Table 2 shows experiment each group tilapia serum antibody titer ㏒2Value.The total antibody titer of A1, A2 group serum and specific antibody effect
Valence is all remarkably higher than control group (P < 0.05).
2 arasaponin of table is to tilapia serum antibody titer (㏒2Value) influence
| Group | Total antibody titer | Specific antibody potency |
| A0 group | 8.17±0.45a | 5.84±0.45a |
| A1 group | 13.77±0.84b | 9.84±0.45c |
| A2 group | 14.57±0.55b | 10.24±0.45c |
The influence of 2.3 arasaponins former infection ability disease-resistant to Tilapia mossambica
Death condition after experiment fish artificial challenge Aeromonas hydrophila is shown in Table 3.Control group is average in 2 weeks after infection pathogen
Cumulative mortality is 57.78%, the experimental group (A1 and A2 group) of per kilogram feed addition 10mg and 30mg arasaponin extract
The average cumulative death rate is respectively 11.11% and 17.78%, reduces by 46.67% and 40% respectively than control group.A1 and A2 group is average dead
The rate difference of dying is not significant (P > 0.05), but substantially lower than control group (P < 0.05).Therefore, suitable dose is added in feed
Arasaponin extract can effectively improve the ability that Tilapia mossambica resists pathogen.
3 arasaponin of table infects Tilapia mossambica the protective effect after Aeromonas hydrophila
| Group | Attack malicious quantity (tail) | Attack toxic dose (cfu/ tail) | Average mortality (%) |
| A0 group | 15×3 | 1.5×108 | 57.78±3.85b |
| A1 group | 15×3 | 1.5×108 | 11.11±3.85a |
| A2 group | 15×3 | 1.5×108 | 17.78±3.85a |
3, brief summary
Per kilogram feed addition 10mg and 30mg arasaponin extract (i.e. per kilogram feed addition 100mL and 300mL Radix Notoginseng soap
Glucoside aqueous solution) salivary lysozymes such as tilapia serum interferon gamma, Complement C_3 can be significantly improved and test fish after attacking poison
The death rate after Tilapia mossambica artificial challenge Aeromonas hydrophila can be reduced by 40% or more by internal serum antibody titer.
Test two: application (cultivation practical application) of the arasaponin extract in rheum officinale fish culture
The preparation of test group bait: it is water-soluble that arasaponin extract with PBS is configured to the arasaponin that concentration is 0.1mg/mL
Liquid.It is calculated by per kilogram feed addition 10mg arasaponin extract, i.e. per kilogram feed addition 100mL saponin aqueous solution.With
Arasaponin aqueous solution slowly, is equably sprayed in compound fish bait used specially for pseudosciaena crocea by watering can, is stirred evenly, is raised using small particle
The particle of diameter 2mm is made in material machine, and 60 DEG C dry and are stored in -20 DEG C.
Compareing bait is that per kilogram mixed feed sprays 100mLPBS, remaining is the same as test group bait preparation method.
Medicine-feeding test: Fujian Province Ningde richness flood produce Co., Ltd Larimichthys crocea mariculture area set 2 aquaculture net cages (3m ×
3m × 3m), each net cage supports 4000 tails or so, and every urosome weighs 100 grams or so.One of net cage is set as control group, feeds base
Plinth mixed feed；Another net cage is set as test group, feeds the mixed feed of addition saponin aqueous solution, additive capacity is per kilogram
The feed extract of arasaponin containing 10mg.Two groups feed 2 times daily, and each feedstuff feeding amount weighs 2% calculating by fish body.It feeds
Therapy lasted 3 weeks, first continuously feed 7 days test bait within test group first week, second week continuously feeds 7 days control bait, third
Week continuously feeds 7 days test bait again.Period control group continuously feeds control bait.
Challenge viral dosage: after 3 weeks feed, fishing for 150 tails from each net cage at random respectively, divides to support and support in 3 recirculated waters
Pond is grown, each 50 tail of pond carries out challenge viral dosage after a week.It is vibrio alginolyticus that poison, which is attacked, with pathogen, bacterial concentration is 1 ×
1080.2mL is injected intraperitoneally in CFU/ mL, every tail.It is observed continuously 10 days, counts the death rate.
Challenge viral dosage result: the control group average cumulative death rate is 62.67%, and the test group average cumulative death rate is
25.33%, 37.34% is reduced than control group.This illustrates per kilogram feed addition 10mg arasaponin extract (i.e. per kilogram feed
Addition 100mL arasaponin aqueous solution) death rate of Larimichthys crocea after vibrio alginolyticus attack can be effectively reduced.
Claims (3)
1. a kind of application method of saponin extract as fish immunity reinforcing agent, which is characterized in that the specific steps are that:
(1) arasaponin extract is provided；
(2) arasaponin extract is dissolved in phosphate buffer (PBS) or physiological saline or deionized water or pure water,
It is configured to the saponin aqueous solution that concentration is 0.05- 0.1mg/mL；
(3) per kilogram feed uniformly sprays 100n-200nmL saponin aqueous solution, by per kilogram feed addition arasaponin extract
10nmg is calculated), wherein n is (1,2,3,4,5),
(4) uniformly dry to be sprinkled upon particle basal feed surface first by suitable feed adhesive when feed is pellet, so
Appropriate saponin aqueous solution is packed into watering can afterwards, slowly, is equably sprayed on feed, is mixed, interior is dried or 60 DEG C of drying；Work as feeding
When material is powdery compound feed, appropriate saponin aqueous solution is packed into watering can, slowly, is equably sprayed in mash feed, is stirred
Uniformly, the particle of appropriate particle size, 60 DEG C of drying are made using minitype fodder-pelleting machine.
2. a kind of application method of the saponin extract according to claim 1 as fish immunity reinforcing agent, feature exist
In the preparation method of phosphate buffer are as follows: 0.01mol/L phosphate buffer (PBS) is prepared according to a conventional method, specifically such as
Under: weigh 8g sodium chloride, 0.2g potassium chloride, 1.44g disodium hydrogen phosphate and 0.24 gram of potassium dihydrogen phosphate be dissolved in 800mL go from
In sub- water, the pH value of solution is adjusted to 7.40, deionized water is added to be settled to 1L, high pressure steam sterilization, room temperature preservation after cooling.
3. a kind of application method of the saponin extract according to claim 1 as fish immunity reinforcing agent, feature exist
In physiological saline is prepared according to a conventional method, specific as follows: to weigh 0.9 gram of sodium chloride, be dissolved in a small amount of deionized water, dilute
To 100mL to get to 0.9% sodium-chloride water solution.
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| CN201811507397.8A CN109498665B (en) | 2018-12-11 | 2018-12-11 | A fish feed containing saponin extract |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106729050A (en) * | 2016-12-19 | 2017-05-31 | 福建万亿店中店电子商务有限责任公司 | A kind of compound immune enhancing oral liquid and preparation method thereof |
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2018
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106729050A (en) * | 2016-12-19 | 2017-05-31 | 福建万亿店中店电子商务有限责任公司 | A kind of compound immune enhancing oral liquid and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| 刘耕陶主编: "《当代药理学 第2版》", 1 May 2008 * |
| 国晶晶 等: "三七主要成分及其免疫调节作用的研究进展", 《中药学》 * |
| 陈静 摘: "三七中的新皂苷及其免疫增强活性", 《国外医药.植物药分册》 * |
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Address after: 350000 No. 54, 247 Gulou District, Fujian, Fuzhou Patentee after: BIOLOGICAL TECHNOLOGY INSTITUTE OF FUJIAN ACADEMY OF AGRICULTURAL SCIENCES Patentee after: NINGBO FUFA AQUATIC PRODUCTS Co.,Ltd. Address before: 352300 fish pond in qiuzhu village, Sandu Town, Jiaocheng District, Ningde City, Fujian Province Patentee before: NINGBO FUFA AQUATIC PRODUCTS Co.,Ltd. Patentee before: BIOLOGICAL TECHNOLOGY INSTITUTE OF FUJIAN ACADEMY OF AGRICULTURAL SCIENCES |
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