CN101664107A - High-efficiency shrimp immunomodifier - Google Patents

Abstract

The invention discloses an active oligopeptide and polysaccharose composite immunomodifier using compound amino acid as a feed flavoring agent, which is prepared by making full use of waste from Paphia undulata processing plants, determining obvious influence factors through an orthogonal experiment, controlling a hydrolysis degree to be 34 to 40 percent and an average polypeptide length of about3, optimally extracting active sulfated polysaccharide gracilaria tenuistipitata by a ultrasonic method, and adding composite vitamin and other minor ingredient according to a formula design principle. The immunomodifier can induce the appetite of fish and shrimps to take food, improve appetite and growth, increase fish and shrimps yield, improve immunocompetence of fish and shrimps, reduce the fish and shrimp diseases, save cost and be directly mixed in feed with stirring and is convenient in batch charging.

Description

A kind of efficient prawn immunomodulator

One. technical field:

The present invention utilizes Ba Fei clam processing factory leftover bits and pieces hydrolytic degradation to obtain active oligopeptide, and the fragrant plant mentioned in ancient texts dish extracts and obtains activared carbon sulfur polysaccharide ester, the compound immunopotentiator of making.Belong to culture fishery nourishing additive agent industry.

Two. research background and purpose:

Remarkable social benefit and economic benefit have been created in the fast development of aquaculture cause, and meanwhile, the reality that water environment worsens has day by day also been brought a difficult problem for disease control in the aquaculture.The aquaculture waters is wide, and cultivated animals is ill under water to be difficult for being found, and is difficult in time execute control.Therefore, the expert proposes " anti-overweighting controlled " already.How improving aquatic livestock immune disease-resistance ability, is the culture fishery urgent problem.

China's feed addictive industrial development has a high potential, and the exploitation of feed addictive resource plays decisive role with application to feed product quality, feed feeding effect.At single additive professional not high enough to basestocks coupling, composite additive has begun to popularize and has used.Immunomodulator also belongs to the additive category.

Immunomodulator is the medicine that a class strengthens body's immunity non-specificly, and selectivity is not high, and often a kind of medicine has multiple function, also can belong to the feed addictive scope.

1, active peptide

Modern nutriology is discovered: animal ingestion protein is digested and assimilated with low peptide form after gastral enzyme effect mostly, and the ratio that absorbs with the free amino acid form is very little.Further test disclosed again the digestion of peptide specific ionization amino acid faster, absorb manyly, show that the biological value of peptide and nutritive value specific ionization amino acid are higher.This is the infinite glamour place of active peptide just also.Biologically active peptide is that 20 natural amino acids are formed with difference and the general name of the different peptide classes of the linearity from the dipeptides to the complexity of arrangement mode formation, loop configuration in the protein, is the multi-functional compounds that comes from protein.

Active peptide has multiple internal metabolism and physiological regulation function, easily digest and assimilate, effects such as promoting immunity, hormone are regulated, antibiotic, antiviral, hypotensive, reducing blood lipid is arranged, and edible safety is high, is the most popular research topic of current international food circle and the function factor that has development prospect.

The Ba Fei clam concentrates juice:

This concentrate is that Guangxi Ba Feiha processing factory leftover bits and pieces concentrates and to form, it contains abundant protein, nutriment such as compound amino acid and active peptides, can provide the abundant nutrition material for aquatic livestocks such as prawns, mainly comprise most important essential amino acid (lysine, glutamic acid), glutamic acid wherein, nucleotides and peptide matters can be as tasty agents, and glycine, the L-arginine, compound amino acids such as L-alanine are as the feed flavouring agent, lure the prawn feed, improve appetite and promote growth, improve prawn output, improve the prawn immunocompetence simultaneously, reduce that prawn disease is produced, save cost.

2, immunocompetence polysaccharide

Polysaccharide is the important natural active matter of a class, has the nospecific immunity facilitation of wide spectrum, can obviously strengthen the cellular immunity and the humoral immune function of body.

River hedge (Gracilaria) is the marine alga that dwells at the large-scale end, belong to Rhodophyta (Rhodophyta). be used as medicine with titles such as " asparagus ", " pelvetia silquosa ", " phoenix tail dishes " from ancient times, this medicine of medicine work description in ancient times is born on the stone of seashore, the southeast, and it is sweet cold in nature to distinguish the flavor of, and the effect of softening and eliminating sputum, clearing away heat and promoting diuresis is arranged.The fragrant plant mentioned in ancient texts dish is not only a kind of natural iodine feed addictive, and contains several amino acids, vitamin, mineral matter, also contains the organic compound (algal polysaccharides etc.) of a large amount of non-nitrogenous biologically actives and the active material that stimulates growth of animal.It simultaneously is again the raw material that industry generates agar.

The fragrant plant mentioned in ancient texts dish is the huge economic algae of a class in the red algae, and output is huge, all is basically at present to use as feed or food, does not have deep processed product, and added value is low.The marine algae raw material sources that the present invention utilizes are extensive, hold concurrently again to be the medicine-food two-purpose kind, and security is good, the production technology simple and effective.

Wherein sulfur agar is counted as the discarded object that extracts the agarose for a long time.Along with further investigation, find that the Gracilaria polysaccharide sulfate all has blood coagulation resisting function, physiologically active such as antiviral, antitumor in vivo and in vitro to the seaweed polysaccharide sulfate biological function.Polysaccharide has many-sided biologically active, such as antiviral, antitumor and immunological regulation etc.; And organism normal cell do not had toxic and side effect, may become desirable medicament sources.Utilize the immunoloregulation function of fragrant plant mentioned in ancient texts dish polysaccharide sulfate, can be applicable in the aquaculture, improve the prawn immunocompetence, reduce that prawn disease is produced, save cost.

Main purpose of the present invention is to make full use of the Ba Fei clam to concentrate compound being prepared into of the active component of juice and fragrant plant mentioned in ancient texts dish polysaccharide sulfate and a kind ofly have the prawn of promotion and grow, and reduces the immunopotentiator that prawn disease is produced.

Summary of the invention:

The technology of the present invention main points: make full use of Ba Fei clam processing factory leftover bits and pieces, by acid hydrolysis, strict control hydrolysis degree, the maintenance hydrolysis degree is 30-40%, is degraded into the average peptide chain length and is about 3 the little peptide with immunoregulation effect; Prepare fragrant plant mentioned in ancient texts dish polysaccharide sulfate by process optimization.

Efficient prawn immunomodulator, composed of the following components by weight percentage:

Ba Fei clam hydrolysis amino acid and active oligopeptide 70-80%

Fragrant plant mentioned in ancient texts dish polysaccharide sulfate 4-5%

B B-complex 3-4%

Oxygenation agent 3-4%

Sodium alginate is an amount of

Anticorrisive agent is an amount of

Concrete steps:

1, Ba Fei clam hydrolysis amino acid and active oligopeptide preparation:

(1) technological process: Ba Fei clam condensed cream → → hydrochloric acid hydrolysis → → catch up with acid → → alkali neutralization → → the dialysis desalination → → debitterize → → be condensed into cream

(2) protein degree is measured: ninhydrin colorimetry, and the computing formula of degree of hydrolysis is:

$\mathrm{DH}=\frac{\mathrm{AN}}{\mathrm{TN}}\×100\%$

In the formula: DH is a degree of hydrolysis; AN is the amino nitrogen content in the zymolyte; TN is the nitrogen pool of sample.

(3) the amino acid kind is an assay: Amino Acids Analysis System (DIONEX ICS-2500 ion chromatograph)

Amino acid kind and content in the table 1. Ba Fei clam leftover bits and pieces inspissated juice dry powder

(4) average peptide chain length=1/DH

By research acidity, hydrolysis time, material ratio, four factors of hydrolysis temperature are determined factor factor level table to the influence of acid hydrolysis mixed protein degree, pass through L ₉3 ⁴Orthogonal experiment is determined optimum hydrolysis technology, and the control hydrolysis degree is 34%-40%, and average polypeptide length is about 3.

2, extract separation by the sour water ultrasonic wave and obtain fragrant plant mentioned in ancient texts dish polysaccharide sulfate.

(1) technological process: the fragrant plant mentioned in ancient texts dish → → ether/absolute ethyl alcohol decolouring degreasing → → sour water heat extracts → → centrifugation → → decompression concentrates → → precipitation with alcohol → → acetone, ether, absolute ethanol washing → → vacuum drying → → be ground into dry powder

(2) measurement of the polysaccharide content (phenolsulfuric acid method)

(3) the Dodgson standard measure of sulfate is measured

Take by weighing the fragrant plant mentioned in ancient texts dish, HCl, extraction time, material ratio and the extraction temperature with variable concentrations carried out single factor experiment respectively under the effect of power 500W ultrasonic wave.For the optimum process condition of determining that ultrasonic acid is carried, according to the experiment of single factor result, design L ₉3 ⁴Orthogonal experiment.

3, according to Chinese prawn the demand of vitamin is added each vitamin: in the 1kg compound, add LAPP (vitamin C condensed phosphate) 4g, inositol 4g, choline 4g, V _A10,000 IU, V _D5000IU, V _E350mg, V _B540mg, V _B3100mg, V _B1110mg

4, add other auxiliary materials, directly grind interpolation and fully be mixed and made into bottled product, low temperature is preserved.

Case study on implementation one

1, Ba Fei clam hydrolysis amino acid and active oligopeptide preparation:

By research acidity 2-6mol/L, hydrolysis time 1-5 hour, material ratio 1: 5-8, hydrolysis temperature 50-90 ℃ of four factor are determined factor factor level table to the influence of acid hydrolysis mixed protein degree, pass through L ₉3 ⁴Orthogonal experiment is reached a conclusion: influence is in proper order to degree of hydrolysis: time＞temperature＞acidity＞material ratio.Its optimised process preparation: hydrolytic acidity (hydrochloric acid) concentration is 4-5mol/L, and the time is 4-5 hour,, temperature is 70-80 ℃, finally degree of hydrolysis is controlled at 30-40%, promptly the average peptide chain length approximates 3.Prepare Ba Fei clam amino acid active oligopeptides condensed cream 1000-1200 gram according to optimum process condition, hydrolysis, catch up with acid, alkali neutralization, dialysis desalination, active carbon debitterize, decompression at last is condensed into the lark paste with the suitable total amount of raw material.

2, extract polysaccharide sulfate by ultrasonic wave acid system Congjiang Li dish, by single factor experiment, investigated acid concentration, time, material when temperature to the influence of polysaccharide sulfate recovery rate, determine the orthogonal test scheme, drawing optimum process condition is: acidity (hydrochloric acid) is 0.01-0.02mol/L, and ultrasonic wave extraction time is 20-25min, and temperature is 60-70 ℃, it is 20-21.70% that ethanol precipitation is worth polyoses content, and sulfate radical content is the fragrant plant mentioned in ancient texts dish polysaccharide sulfate of 10-10.23%.According to optimum process condition, get 500g degreasing river hedge dish powder, sour water heat is extracted in the ultrasonic wave extractor of power 500W, centrifugation, decompression concentrates the adding concentration of alcohol and reaches the 60-70% precipitation, acetone, ether, absolute ethanol washing, vacuum drying, pulverizing obtains dun fragrant plant mentioned in ancient texts polysaccharide sulfate and is about 45g.The fragrant plant mentioned in ancient texts polysaccharide sulfate is the nonreducing sugar that a class contains sulfate group, the heteroglycan of no starch and non-polyphenols.

3, according to Chinese prawn the demand of vitamin is added each vitamin: get LAPP (vitamin C condensed phosphate) 4g, inositol 4g, choline 4g, V _A10,000 IU, V _D5000IU, V _E350mg, V _B540mg, V _B3100mg, V _B11Mixing such as 10mg, a spot of again sodium alginate and anticorrisive agent mixed grinding.

4, the compounds of case study on implementation 2 and 3 preparations are joined paste after the hydrolysis of Ba Fei clam respectively, stir, in the plastic cement bottle of dress as 500ml, finished product.

Case study on implementation two

Select healthy, Chinese prawn 3.5-4.5g of uniform size, if 5 test group and 2 control groups are put shrimp 20 tails for every group, divide and support in the aquarium of 100cm * 50cm * 60cm, test group is thrown something and fed respectively and is contained 1% immunopotentiator of the present invention, the control group basal feed of throwing something and feeding.Experimental period control water temperature is at 25-28 ℃, and 24h inflates, and every day, soil pick-up changed water once.Raise the mensuration of carrying out relevant immune indexes after 15 days.

1, immunomodulator is to the influence of immunity function of shrimp

(1) phagocytosis of haemocyte

Get shrimp 20 tails for every group, draw anti-coagulants 0.2ml, insert prawn and enclose heart extracting blood 0.2ml, add sodium chloride solution and make anticoagulation, detect with reference to Chen Changfu method improvements such as (1995) with the 1ml asepsis injector.The results are shown in Table 2.

Table 2. immunopotentiator is engulfed the influence of vigor to the Chinese prawn haemocyte

(2) serum phenol oxidase vitality test

Get shrimp 20 tails for every group, enclose heart extracting blood 0.2ml from prawn, place the 1ml vial with the 1ml asepsis injector, centrifugal, get upper serum, detect result such as table 3 with reference to the method for Ashida.

Table 3. immunopotentiator is to the influence of Chinese prawn antalzyme activity and phenol oxidase vigor

(3) challenge test

Behind the bait 15d that throws something and feeds, carry out.The malicious concentration of attacking of Aeromonas hydrophila is 6 * 105/mL, carries out intramuscular injection (0.02mL/ tail) at the 2-3 uromere of pond crayfish, attack poison after, shrimp inflated respectively supports in bucket, observe and write down the result at any time.Protective rate computing formula: (the control group death rate-test group the death rate)/control group death rate * 100%.The results are shown in Table 4.

Table 4. immunopotentiator is to the influence of Chinese prawn immanoprotection action

The immunopotentiator that the present invention makes can activate the nonspecific immunity system of Chinese prawn body, strengthens immune resistivity effectively, reaches the purpose of defence disease invasion.

2, immunomodulator influence that Chinese prawn is grown

Select for use body length to be the shrimp seedling of 12-14mm, fed 1% immunopotentiator of the present invention and normal diet since first day respectively, it is long to measure shrimp seedling body after 15 days, the results are shown in Table 5.

Table 5. immunopotentiator is to the influence of Chinese prawn seedling development

The immunopotentiator that the present invention makes can promote growing of juvenile prawn.

3, immunomodulator is to the influence of prawn water quality

Table 6. immunopotentiator is to the influence of Chinese prawn shrimp pond water quality

The immunopotentiator that the present invention makes can also improve the water quality on the shrimp pool.

Claims (5)

1, efficient prawn immunomodulator, composed of the following components by weight percentage:

Ba Fei clam hydrolysis amino acid and active oligopeptide 70-80%

Fragrant plant mentioned in ancient texts dish polysaccharide sulfate 4-6%

B B-complex 4-6%

Oxygenation agent 3-5%

Sodium alginate is an amount of

Anticorrisive agent is an amount of

2, according to patent claim 1 described prescription design, it is characterized in that: Ba Fei clam hydrolysis amino acid and active oligopeptide prepare according to following optimised process: hydrolytic acidity (hydrochloric acid) concentration is 4-5mol/L, time is 4-5 hour, temperature is 70-80 ℃, finally degree of hydrolysis is controlled at 30-40%, promptly the average peptide chain length approximates 3.

3, according to patent claim 1 described prescription design, it is characterized in that: fragrant plant mentioned in ancient texts dish polysaccharide sulfate prepares according to following optimised process: acidity (hydrochloric acid) is 0.01-0.02mol/L, ultrasonic wave extraction time is 20-25min, temperature is 60-70 ℃, it is 20-21.70% that ethanol precipitation is worth polyoses content, and sulfate radical content is the fragrant plant mentioned in ancient texts dish polysaccharide sulfate of 10-10.23%.

4, according to patent claim 1 described prescription design, it is characterized in that: utilize Ba Fei clam compound amino acid and oligopeptides, have the active formulated thick paste of fragrant plant mentioned in ancient texts dish polysaccharide sulfate with nutritional labeling and immunoregulation effect.

5, according to patent claim 1 described prescription design, it is characterized in that: what the present invention made is a kind of feed flavouring agent, can lure the prawn feed, improve appetite and promote growth, improve prawn output, improve the prawn immunocompetence simultaneously, minimizing produces prawn disease, saves cost.