Microorganism metabolites feed supplement cyclic decapeptide and application

Technical Field

The invention relates to a microorganism metabolic product from the new compound-cyclic decapeptide (C₃₅ H₅₀ N₁₀ O₁₀ · 5H₂ O), and the use of the compound.

Technical background

Since the 20 century since the medium term, with the rapid development of the aquaculture industry, antibiotic, grooves intersect, the quinolone drugs such as within the range of feed additive widely applied in the world. These additive the prevention of disease, the promotion of the growth of the animals is very obvious role of, and has in particular period the role of the filter should not be overlooked. As green, safety, health, environmental protection that have a theme of the human attention, people will be produced by this concern as to the safety of the food. Medicine feed additive of having a significant effect at the same time, also have residual, toxicity, "three is caused" (teratogenic, carcinogenic, mutagenic), and the like adverse effects, and is also very serious side effects, long-term use of the pathogenic bacteria produce drug resistance. Therefore, a residue pharmaceutical feed additive, pollution of the environment, such as threat to human health problems have now become the States culture workers and health workers hot issue of great concern.

Since for a long time, foreign and domestic livestock and poultry, aquaculture most feed supplement is mainly vitamin, such as trace elements, promoting growth feed additive forbidden [...] after such as ethanol, the feed of new research and growth-promoting additive in feed industry application become urgent need to solve the problem. As the microbial feed additive, it is taken from natural, natural used, has low toxicity, low residue, to the non-target biological poison is low, no pollution to the environment, and the like, the current can be reduced or avoided by the chemical feed additive "drug hazart", this is in line with green animal product, the direction of development of aquatic products, will also become a elevage, aquaculture research of new hot spots. Therefore, improving the animal's immunity, promotes culture animal growth, reduce the breeding animal disease, drug or medication not as far as possible, the production of the product, has great social and economic benefits.

According to the research, microbial active material mainly has: fungal polysaccharides, dextrans, lipopolysaccharide (LPS), peptidoglycan and peptidomimetic substances and the like. A large number of pharmacological research tests prove that, these microorganisms not only active substances able to activate the T cell, cell B, phagocytic, natural killer cell (NK), cytotoxic cell (CTL) and readygel factor activation of immune cells such as anti-cell (LAK), can also promote the cell factor to produce, role for complement, to the immune system play the mediation role in many aspects. Research found that certain bioactive peptide can promote the growth of beneficial bacteria in the intestinal tract, digestive absorption function is improved. Protein produced by hydrolysis of immune activity with certain small peptide. Small peptides (  peptides Small) is one kind of molecular mass is relatively small, loose conformate with multiple biological function of the peptide. A large number of the test indicates that: small peptides in animal husbandry can improve survival rate, improve the feed conversion ratio in the feed and to promote the utilization rate of the various mineral elements, with antibiotic, immune, peroxidaton and adjusting characteristics such as flavor meat products. At the same time in improving immunity of organism, but also has good anti-virus activity, and has low toxicity and low resistance, and the like. When in clinical use, any route of administration are no adverse reaction; in addition, some microbial active material can promote the growth of the animals for breeding, reduce the cost of feed, is a novel, environmental protection, environment-friendly feed additive, such as ethanol is expected to disable [...] promoting growth feed additive, microbial polypeptide is a microbial fermentation products, through the biological engineering technology can realize low-cost, large-scale production, the production will therefore be displayed when in use the prospect of increasingly wide.

Content of the invention

The purpose of this invention is to offer bdellophage (  bacteriovorus Bdellovibro) S0416 fermentation product cyclic decapeptide (C₃₅ H₅₀ N₁₀ O₁₀ · 5H₂ O). The fermentation product cyclic decapeptide can promote the growth of the animals for breeding, enhance immune function of breeding animals, the resistance of the animal to the disease, prevention of diseases, and can promote the speed of the growth of the animal breeding, overall improve the cultivation production performance, is the environment, green feed additive of food safety.

The second objective of this invention is to provide the above-mentioned cyclic decapeptide preparation method.

The invention also provides the above-mentioned cyclic decapeptide in the preparation of feed additive to enhance immune function, the application of the drug, and the promotion of the growth of the animals in the preparation of food, the application of the drug, containing cyclic decapeptide the phagemid bdellovibrio bacteriovorus (  bacteriovorus Bdellovibro) fermentation of the application of the crude as the feedstuff.

Has the following structural formula (1) the cyclic decapeptide:

The above-mentioned cyclic decapeptide can also contain 5 a crystal are.

The preparation method of the above-mentioned cyclic decapeptide through microbial phagemid bdellovibrio bacteriovorus (  bacteriovorus Bdellovibro) fermentation, separation is obtained.

Compared with the prior art, this invention has the following advantages:

1) phagemid bdellovibrio bacteriovorus S0416 (  bacteriovorus Bdellovibro) generate cyclic decapeptide, can promote culture animal (Pig, chicken, fish, shrimp and other) growth, improve the production performance, reduces the cultivation cost;

2) cyclic decapeptide added to the culture in the feed of the animal, which can enhance the immunological function of the feeding animals, the disease-resistant ability, the cultivation of animal taking medicine, the role of the substitute some antibiotics, reduce the breeding animal disease resulting economic loss, and improve the quality and food safety;

3) as a microorganism metabolic product of the cyclic decapeptide, derived from nature, is apt to degrade in a natural, pollution-free to the environment. At the same time, of the product to the animal food is safe.

Description of drawings

Figure 1 strain S0416   16SrDNAPCR electrophoretic Image of the amplification product;

Figure 2 based on 16SrDNA sequence constructed S0416 system cladogram ;

Figure 3 this invention cyclic decapeptide time-of-flight mass spectrometry;

Figure 4 this invention cyclic decapeptide molecular formula.

Mode of execution

The following combination of the Figure the inventor to provide further explanations and specific embodiments of the present invention and the beneficial effects of cyclic decapeptide preparation method.

Embodiment 1 : (  bacteriovorus Bdellovibro) bacteriophagic Bdellovibrio S0416 separation and identification

Strain sources Shaanxi XI ' an Dongdaemun onsen water from sludge (water temperature of the 76 [...]). Separation, purification is obtained strain number S0416, after physiological biochemical, 16s   rDNA bacteriophagic Bdellovibrio identified to be (  bacteriovorus Bdellovibro). The strain in Petzhold Stolp and initially by 1962 years obtained by the separation of a from the soil, is a specialized to ravin bacterial of parasitic bacteria, phage function similar, is also my the Ministry of agriculture announced the animals can be use in the cultivation of a microorganism. Inventor separating strain S0416 can at the ordinary broth culture free growth, 16S the size of the amplification product   rDNA 400bp left and right, 1% agarose gel electrophoresis results see Figure 1. Sequence determination using two-way measurement, to obtain a total of 1029 base. Sequence determination the results are as follows:

   GCATAAGACC GGATAACTAG     AGCTAATACC     TCGAAAGATT

 CGGCTCTAGG ACAGGAGCTG

   CGCGTAAGAT GGTCAAAGGT     AAGATGAGTC     TTTTCGCTCT

 GTGAGGTAAC TAGCTAGTTG

   CTGAGAGGAT GGCTCACCAA     TTTAACTGGT     GGCGACGATC

 ACTGGAACTG GATCAGTCAC

   CAGTAGGGAA AGACACGGTC     CGGGAGGCAG     CAGACTCCTA

 TGGAGGAAAC TATTGCACAA

   AGGCCTTCGG TCTGATGCAG     TGAGTGATGA     CGACGCCGCG

 TCTGTCGCAG GTCGTAAAGC

   AAAGGATCGG GGGAATAACA     ACCCTGTAAG     CAATGAATGT

 GCCAGCAGCC CTAACTTCGT

   GGAATTATTG GCGGTAAGAC     AGCGTTGTTC     GAGGGATCCT

 GGATGTAGGT GGCGTAAAGC

   GCTCAACCCT GGCTTTGTAA     AAAGCCCAGG     GTCAGATGTG

 TTGATACTGC GGAAGTGCAT

   TTGTTGGTGT GAAGCTTGAG     GTTACTAGAA     TGTCGGAGAG

 TACGTAGATA AGTGGTGAAA

   AACTGGCCGA TCAACAGGAA     GAAGGCGTGT     TACCGGGAGC

 TGAGATCCGA ACACTGACAC

   CTGGTAGTCC AAGCGTGGGG     ATTAGATACC     ATCAAACAGG

 CGATGGATAC ACGCCGTAAA

   ACGAAGCTAA TTGTTGTTGG     CCCTTCAGTG     AGGTATTGAC

 ATCCCGCCTG CGCGTTAAGT

   GAAATTGACG GGGAGTACGG     AAAACTCACA     TCGCAAGATT

 CAAGCGGTGG GGGGCCCGCA

   GAACCTTACC AGCATGTGGT     GCAACGCGAA     TTAATTCGAT

 ATGTACTGGA TAGGCTTGAC

   GTCGGTACAC AGACTGGCAG     GCCCGCAAGG     AAATGTCGTC

 TGGCTGTCGT AGTTGCTGCA

   CCCGCAACGA CAGCTCGTGT     TGGGTTAAGT     CGTGAGATGT

 TGCATTTAGT GCGCAACCCC

   ACTGCCGGTG TGCCAGCATT     CTCTAGATGG     CAGTTGGGCA

 GGAAGGTGGG TTAAACCGGA

GATGACGTC

Transmitting the measured separation plant 16S the   rDNA partial sequence in the database GenBank 16S rDNA sequence comparison, application MEGA   3.1 software analysis, based on the constructed 16S   rDNA cladogram system development (see Figure 2). Analysis shows that the phages bdellovibrio bacteriovorus (Bdellovibrio bacteriovorus) between two relatively close distance is separated by the evolution of, similar rate up to 100%.

Embodiment 2: bacteriophagic leech vibrionales S0416 fermentation, active ingredient tracking separation

(A) materials and methods

1. Instrument

Glass layer chromatography column: 8 cm × 100 cm; X-6 microscopic melting point meter : Beijing tyack instrument co Ltd; Perkin   Elmer   240C element analyzer; time-of-flight mass spectrometer, the United States Company HP; Nicolet AVATAR   360   ft-IR-type infrared spectrometer: the United States Company Nicolet;   Mercury Varian   300   BB-type nuclear magnetic resonance apparatus, the United States Company VARIAN; Brucker   AM   400 superconducting NMR, Brucker Corporation of the United States;

2. Reagent

Lymphocyte separating liquid (Ficoll-Hypaque): Shanghai hua Jing biological high tech Company limited, lot number: 060321. Heparin sodium injection: tianjin biological chemical pharmaceutical, lot number: 20061009.

MEM culture medium: Gibco. Weighing 480 mg   MEM, using 50 ml three-steaming water lucifugous dissolved, for the 0.22   m microporous filter sterilization, packaging, 4 the preservation [...].

[...] (MTT): Sigma. Weighing 250 mg   MTT, is placed in a small beaker, add 50 ml PBS (0.01mol/L, pH7 . 4) of the magnetic stirrer to stir for 30 min, for the 0.22   m microporous filter sterilization, packaging, 4 the preservation [...].

Heparin sodium injection: tianjin biological chemical pharmaceutical, lot number: 20041009.

Hank ' s liquid: NaCl   8.0g, KCl   0.4g, Na₂ HPO₄. 12H₂ O   0.12g, KH₂ PO4 0.06g, glucose 1.0g, double-distilled water 1000 ml, 1% phenol red fluid 1D, after mixing the above-mentioned ingredient dissolved, the bottle, 8 pounds 15 min sterilization, 4 the refrigerator for preserving [...] , a near using to pH 7.3-7.6.

0.4% blue solution the liver moss hoped : Sino-American   Biotec. Weighing 4g the liver moss hopes blue , a small amount of distilled water and grinding, and double distilled water to 1000 ml, filter paper filtering, 4 the preservation [...] , when in use, the PBS diluted to 0.4%.

2% Tween -20 solution: Shanghai yamaura chemical ltd., lot number: 20040218. Diluted with distilled water to the 2%.

PBS (0.01mol/L, pH7 . 4), 0.9% physiological saline, N/10 such as hydrochloric acid.

3. Experimental animal and material

Kunming is 4th military medical University in MICE experimental animal center, average weight (18.7 ± 0.6) g; Escherichia coli (Escherichia   coli) the north-west of agriculture and forestry preservation aquatic science laboratory of the University of science and technology.

4. Fermentation culture and separation

With the ordinary culture nutrient broth (peptone 10g, beef extract 3g, NaCl5g, distilled water 1000 ml, pH7 . 2,121 the sterilizing [...] 30 min) as a culture medium inoculated bacteriophagic leech vibrionales, fermentation for fully-automatic in fermentation tank 7d. The fermented solution CR22G high-speed centrifuge the 4 [...] , 6000r/min, centrifugal 10 min 2 times, supernatant fluid is collected, through the 0.22   m Millipore filter (D=15cm) filtering to obtain the supernatant.

(1) active tracking preliminary separation

Soaked in the above-mentioned filtrate LSA-10 macroporous resin adsorption 24h, will be absorbed in the fill of the supernatant fluid of the chromatographic column, the column washing several times repeatedly of methanol, collecting methanol solution, vacuum distillation to recycle the methanol, concentrating, drying, obtain extract. After thin-layer chromatography (TLC) test, to determine with ethyl acetate-methanol (1:0, 4:1, 3:2, 1:2 and methanol, finaliy laundering column) as the elution system, for gradient elution.

In the chromatography column elution process, every 300 ml collect a fraction, collected a total of 29 a fraction (wherein the section 29 is divided into the water for eluting after receiving), through the above-mentioned analysis merger, vacuum low-temperature drying in a vacuum drying oven (the 50 [...]) to obtain a total of 6 one stream block, are as follows : (paragraph 1-7 fraction) A, B (section 8-12 flow sub-,), C (paragraph 13-19 flow sub-), D (paragraph 20-26 flow sub-), E (paragraph 27-28 flow sub-), F (section 29 fraction, is primarily water elution, presenting puce). After concentrating the fraction B, with the white crystal is separated out, and continuously collecting white crystal with an organic solvent cleaning and re-crystallization, in order to achieve the purpose of purification, the collected BJ of the white crystal is marked as.

(2) the separation of the fraction B

The processing of the sample in the separation process, the sample and column, solvent system to ethyl acetate-methanol (10:1, 8:1, 6:1, 3:2, etc.), specific step with the former.

B fraction of the separation after the chromatographic column, various parts of the combined classification. Among them are a total of 6 a fraction, are B1, B2, B3, B4, B5, B6, wherein B1-B4 part is a fat-soluble, and B5, B6 is water-soluble. Furthermore, the obtained crystal material BJ and the control group (to B7 expressed), a total of 8 groups of the detection of the active effect.

5. The mouse's immune activity detection

The Kunming MICE are small random packet, each group of 12 only, breeding management under the same conditions. In accordance with the experimental group carry out quantitative of flow, are feeding method for each day, according to the daily dose of 20 mg/kg (body weight of the mouse), every 7d at a time of blood sampling, determining its immunological function. Control group feeding 0.9% physiological saline, breeding management under the same conditions.

(1) SOD activity determination

Specification reference SOD kit, using ultraviolet spectrophotometer SOD activity.

(2) determination of antibody titer of serum agglutionation

Apply 0.4 ml of the blood of MICE is arranged in a centrifuge tube, 2000r/min centrifugal 6 min separation of serum, inactivated for Staphylococcus aureus person source for reaction antigen, the hemagglutination board law serum agglutionation antibody titer determination.

(3) white cell suspension preparation

The eyeball picking picks the blood law , taking 0.5 ml heparin anticoagulant slowly along the tube wall containing tile on 0.8 ml mouse leukocytes in the centrifugal tube nanofiltrated, according to -oddThe method, of a centrifuge, 2500 r/min centrifugal 15 min, slowly pipetted 0.5 ml leukocyte layer into another centrifuge tube, adding 4 times volume (2 ml) of the liquid and its mixing Hanks, for 2000r/min centrifugal 10 min, abandons the fluid 1 ml, adding 1mLHanks mixing, repeat, mixing (cell concentration is about 1 × 10⁷ cfu/mL), situ Zhenstrong and so on and method for secondary detection of leukocyte, live cell not less than 90%.

(4) the determination of the white cells phagocytic activity

Taking the above-mentioned preparation leukocyte suspension, to Escherichia coli for determining the activity of phagocytic leukocytes.

On the basis of the preparation white blood cell fluid , to 4 are added respectively in off the concentric pipe 150 the white cell   L, then adding 0.5mLHanks liquid, each pipe by adding 80 the large intestine   L liquid, respectively in the 37 the incubated in incubator [...] 0, 15, 45, 75 min, to be time to the rear, taking out the centrifugal, 2000r/min, 10 min, discard 300 the flexible   L, fill with the 300  L, repeated once. After the completion of centrifugation, add 0.8mLTween -20, function 2 min, add 1mLMEM, shaking bath 37 °C 14h. Incubation time to, each pipe to the 80  LMTT, shaking, thermostat in the incubation 15 min, every 5 min a shaking, the incubation time, 570 nm measuring OD value and selection of the optimum incubation time.

(5) determination of phagocyte bactericidal activity

Taking the above-mentioned preparation leukocyte suspension, for bactericidal activity of bacillus coli the determination.

Taking 4 supporting the test tube, each pipe by adding 0.4 ml of the above-mentioned white cell sap, then each pipe by adding 200 the large intestine   L liquid, respectively for the 37 role [...] water bath to 0, 30, 60, 90 min, the incubating the time, to the tube by adding 0.8mLTween -20, function 2 min the rear, then adding 1.5mLMEM, mixing, into 4 the refrigerator [...] , to be 4 tube is finished 30 min the rear, is the 37 incubating in the shaking bath [...] 14h. Incubation time to, each pipe to the 180  LMTT, shaking, thermostat in the incubation 15 min, every 5 min a shaking, the incubation time, 570 nm measuring OD value, and selecting the optimal incubation time and calculating sterilization index.

According to the KI value, the KI <1.0 to have bactericidal capacity; KI value rise, sterilization capacity reduction, KI > 1.0, no sterilization ability, as the standard of measurement of bactericidal activity.

(6) data processing and analysis

The following SOD activity, the isolated leukocyte phagocytic activity and phagocytic bactericidal activity data using SPSS software analysis, Chapters of the antibody titer in serum agglutinately representing geometric average of the data is.

(B) results and analysis

1. Fermentation product initial active separating

(1) the activity of SOD

In the comparison of the SOD, A stream packet in paragraph 7, 14d, B stream packet in paragraph 7, 14 and 21d, C stream packet in paragraph 7, 14 and 21d, E stream packet in section 7d is compared with the control group (P < 0.01) significant difference, A stream packet in section 21d, E stream packet in section 14 and 21d a significant difference compared with the control group (P < 0.05), but the grouping F G difference does not notably compared with the control group. The SOD value B stream packets in section 14d to reach the peak, the maximum value (106.53 ± 0.32) U/mL, which also is the maximum value in the experimental group. A stream packet maximum of section 14d of the (80.12 ± 0.18) U/mL, the maximum value of the grouping flows C in section 14d of the (93.55 ± 0.16) U/mL, the maximum value of the grouping flows D in section 14d of the (70.69 ± 0.25) U/mL, the maximum value of the packet stream E is section 7d of the (82.48 ± 0.46) U/mL.

Table 1 different SOD activity of the mouse the class minute fills the clothing

(2) serum agglutionation antibody titer

The table 2 can be known, in various different after the mouse the class minute fills the clothing , its serum agglutionation antibody potency compared with the control group, the improvement of a different degree, and when after a certain time later, hemagglutination titer also begin to decline, tends to previous stable level. The grouping flows B the highest potency can reach 1:256, the maximum value of the time range is 7-21d; C, D, grouping flows E the highest titer is 1:64, to the appearing time of the highest titer for 7-21d.

Table 2 mouse the class minute fills the clothing of the different serum agglutionation antibody titer

(3) leukocyte phagocytic activity

White cell activity in vitro mouse maximum OD_{570 nm} value of the packet stream B section 14d, is 0.235 ± 0.008; the maximum value of the grouping flows A in the section 7d, maximum of 0.200 ± 0.016, C stream packet to a maximum value of the section 21d of 0.222 ± 0.009, D stream packets in a maximum value of the section 21d, the maximum value of 0.221 ± 0.017, and grouping flows F E and the maximum value are respectively in the section 7d and 21d, the maximum value are 0.216 ± 0.011 and 0.196 ± 0.009. Wherein B stream packet in section 14 and 21d, C stream packet in section 21d, D stream packet in section 21d, E stream packet in section 7d G the control group has a significant difference compared with (P < 0.01), see table 3.

Table 3 mouse the class minute fills the clothing of different white cell phagocytic activity in vitro

(4) phagocyte bactericidal activity

Mouse phagocytic bactericidal activity compared with the control group, there are also significant (P < 0.05) or is extremely prominent (P < 0.01) difference. A stream packet in section 7d and 21d, B stream packet in section 7d, C stream packet in section 7d and 21d, D stream packet in section 7d and 14d, E stream packet in section 7d and 21d, F stream packet in section 7d and 14d a significant difference compared with the control group (P < 0.05), and grouping B in section 14d and 21d, C stream packet in section 14d and a very significant difference compared with the control group (P < 0.01). Wherein B stream packet in section 14d of the bactericidal activity can achieve the largest 0.510 ± 0.003, specific comparison see table 4.

Table 4 different mouse the class minute fills the clothing the phagocytic cell after bactericidal activity

Comprehensive the above-mentioned analysis, we can see that: the fraction A-F the effect of enhancing the immune function of the mouse are different, in the above-mentioned separation of the 6 in a stream, the activity of the fraction with the most powerful B, C of fraction, with the other stream hours and more significant or very significant difference, that fraction B as the main active material. Flow separation is therefore next B the focus of further active separation.

2. B fraction of the separation and the active determination results

(1) the activity of SOD

The table 5 can know, B3 and the BJ section 7d, 14d, 21d with SOD activity of the B7 a very significant difference compared (P < 0.01), B2 group in section 14d, B4 group in section 7d and 14d, B6 group in section 14d and 21d a control group B7 there is significant difference (P < 0.05), no significant difference other the group compares the control group.

Table 5 after the different grouping drenched when cool mouse SOD activity

From table 4 can be known, the SOD activity of the maximum group BJ section 14d, the maximum peak for (112.03 ± 0.26) U/mL, B2 group in section 14d reach peak (82.16 ± 0.32) U/mL, B3 group in section 14 d reaches peak (103.40 ± 0.43) U/mL, B4 and B6 group are also the peak of section 14d of the (83.25 ± 0.20) U/mL and (82.16 ± 0.33) U/mL.

(2) serum agglutionation antibody titer

From table 6 it can be seen: B2, B5, B6 and BJ group in section 14d to reach the peak, B1, B3 and B4 the section 21d to reach the peak, then the antibody level with the passage of time, the trend of a steady decrease. In 7 in a group, the highest titer of serum agglutionation antibody BJ group of section 14d, the highest potency can reach 1:256; and B3 group can also reach the highest titer of 1:256, B4 group of highest can be up to 1:64.

Table 6 after different grouping drenched when cool mouse the serum agglutionation antibody titer

(3) leukocyte phagocytic activity

After different group material drenched when cool mouse isolated leukocyte phagocytic activity see table 7, can know after analysis B1, B2, B3, B4, B5 and BJ a control group B7 leukocyte phagocytic activity enhancement of a certain range, while in 28d gradually restored to the normal level. Mouse isolated white phagocytic activity in the largest value of O.D B3 group of section 14d, to 0.379 ± 0.004, BJ set to the maximum value of section 14d of the 0.346 ± 0.006, and B2 and B4 respectively set to the maximum value of 7d of 0.274 ± 0.005 and 0.278 ± 0.022, and its difference comparison see table 7.

Table 7 after different grouping drenched when cool mouse isolated leukocyte phagocytic activity

(4) each group of phagocyte bactericidal activity

Each group of mouse phagocytic activity as compared to the control group (P < 0.05) or is extremely prominent (P < 0.01) difference. B3 group in section 14d and 21d, B4 in section 14d, BJ the 7d-21 d and compared with the control group has a significant difference (P < 0.01), B1 group in section 14d and 21d, B3 group in section 7d, B4 group in section 7d and 21d, B5 group in section 14d and 21d, B6 group in section 7d there is significant difference compared with the control group (P < 0.05). BJ group can achieve sterilization index the strongest 0.433 ± 0.011, and B3 and B4 group of the most powerful sterilizing index can be achieved the most intensity value 0.506 ± 0.038 and 0.696 ± 0.033, each group of comparison are shown in table 8.

Table 8 after the different grouping drenched when cool mouse phagocyte bactericidal activity

Test measure the fat-soluble B3 flow still exists in the active material has a part of the function of using, to B3 display TLC of fraction 4 a spot, note B3 at least flow separation in 4 or 4 or more kinds of substance that exists, but activity is low, is not the main active material.

Analysis obtained in the test B3 fraction and BJ material can effectively enhance the mouse's immune function, the control group and has an obvious and very significant difference. On the B3 and BJ in the active comparison, except B3 stream packet the phagocytic activity is stronger group BJ, other immune indicators BJ group to be more ideal. Various data analysis, can infer BJ as the main active material.

Embodiment 3: bacteriophagic leech vibrionales S0416 product active ingredient of the metabolism of structure identification

To the obtained crystal substance measuring BJ of an ultraviolet (UV), infrared spectrum (IR), proton nuclear magnetic resonance spectrum (¹ H-NMR),¹³ C nuclear magnetic resonance spectrum (¹³ C-NMR), time-of-flight mass spectrometry (TOFMS), elemental analysis, and structure analysis.

1. BJ physicochemical nature of the active substance

From the organic solvent in crystal BJ natural precipitation of white transparent of the granular crystal, and water after dissolving crystalline change is an irregular. In water and soluble in dimethyl sulfoxide, methanol, acetone, can be dissolved in chloroform, insoluble in petroleum ether. The material under the flame completely burned into ashes can be very fast, the melting point of the 202.9-203.5 the [...][...]. When detecting TLC, in the three kinds of the above-mentioned developing solvent (ethyl acetate: methanol, chloroform: methanol, ethyl acetate: isopropanol) to the single-point.

Aqueous solution and BJ AgNO₃ and CaCl₂ chemical reaction of the solution has no obvious phenomenon, and under the same condition with the NaCl AgNO₃ solution, KH₂ PO₄ and CaCl₂ chemical reaction of the solution to a white flocculent precipitate produce, can be eliminated from this the possibility of the substance is salt.

2. Active substance poipu analysis and structural description

(1) mass spectrometry

Time-of-flight mass spectrometry (fig. 3) in the display material has a molecular weight of 860.40 molecule-ion of (MH⁺), so as to obtain compound M1 is the molecular weight of 860.40.

(2) nuclear magnetic resonance spectrum

Compounds of structure BJ according to their nuclear magnetic resonance is determined to finish. Compounds from nuclear magnetic resonance spectrum can be seen with BJ 10 amino residues and 5 a a crystal are, which belongs to the cyclic peptide family.

¹ H-NMR   DMSO   TMS     399.357MHz

δ_H 1.978-2.046 (2H, m), δ_H 2.078-2.132, 2 . 343-2.404 (2H, m), δ_H 3.583-3.588 (2H, s), δ_H 3.616-3.663, 4 . 339-4.376 (2H, m), δ_H 3.717-3.770 (1H, dd), δ_H 4.209-4.250 (1H, s), δ_H 8.076 (1H, s)

¹³ C-NMR   DMSO   TMS   399.357MHz

δ_C 163.353 (C-(1)), δ_C 168.790 (C-(7)), δ_C 21.560 (C-(4)), δ_C 27.366 (C-(5)), δ_C 44.141 (C-(3)), δ_C 45.393 (C-(6)), δ_C 57.497 (C-(2))

(3) the results of elemental analysis

By elemental analysis, containing BJ C: 54.47%, N: 17.96%, H: 6.286%, O: 21.28%.

(4) structure analysis

Through the time-of-flight mass spectrometry, nuclear magnetic resonance carbon spectrum , nuclear magnetic resonance analysis of hydrogen spectrum BJ that the preliminary structure of the compound. It is provided with 5 a crystal are the cyclic decapeptide compounds. By the 5 structure units, each unit is a chest--Ganong amino acid residues, each residue and are respectively connected with a crystal water, so the water-soluble is very good. This 5 unit structure is connected with the first end, to form a cyclic structure. The cyclic decapeptide molecular formula to C₃₅ H₅₀ N₁₀ O₁₀ · 5H₂ O, of the molecular structure containing no crystal water as shown in Figure 4.

Embodiment 4: cyclic decapeptide (C₃₅ H₅₀ N₁₀ O₁₀ · 5H₂ O) increase the effect of strong fish immune function

A material and method

The reagent (a) material

Staphylococcus aureus (Staphylococcus   aurevs), Micrococcus dissolves the wall (Micrococcus lysoleikticus), the north-west of agriculture and forestry and aquatic laboratory of the University of science and technology provide; Aeromonad (  hydrophila Aeromonas), eel vibrionales (Vibrio   anguillarum), the Chinese Academy of Sciences research Institute for the aquatic organisms to fish; remora (Carassius   auratus), provided the fishing grounds Shaanxi aquatic Institute, average weight (62 ± 3) g.

Lysozyme (lysozyme): Sigma Company. Other reagent with the former.

(B) method

1. For test fish packet and breeding

Health disease is selected, the weight of the remora, through 30d in order to eliminate temporary the interference factors. Divided into 5 experimental groups, each group of 10 tail, the same in the aquarium is, the control water temperature is 25 ± 1 the [...]. Wherein the section 1-4 group is a group, the oral dose of 0.5 mg/kg.d, 1 mg/kg·d, 2 mg/kg ·d, 4 mg/kg ·d (per kilogram of body weight per day of dose), section 5 as a blank group group.

First, the cyclic decapeptide (C₃₅ H₅ 0N₁₀ O₁₀ · 5H₂ O, fermentation to separate to obtain a, content of 98.5%)in accordance with the consumption are respectively dissolved in water, is added to a base feed in accordance with the each group of feeding, feeding every day 2 times, for throws the bait rate 1% / d, daily heat a certain amount of new water, continuous feeding 25d.

2. Bacterial suspension preparation

The test uses the Aeromonad, Vibrio, Staphylococcus aureus inoculate the ordinary broth culture, respectively, plate count method for counting after the physiological saline water diluted to the concentration of 1 × 10⁸ cfu/mL. For Staphylococcus aureus, Vibrio bacterial suspension by adding 0.5% formalin inactivated 24h. Both are provided 4 the storing [...].

3. Blood sampling

In oral section 5, 10, 15, 20, 25d sampling, sampling at random in the group 3 tail, the tail venous blood sampling, each tail fish heating haematoid 1.5 ml.

4. The determination of the immune-enhancing activity

Serum agglutionation antibody titer determination, leukocyte phagocytic activity, phagocyte bactericidal activity measuring method with the former.

(1) lysozyme activity determination

The two activated dissolves the wall micrococcal in liquid culture medium in the culture cradle 48h, after taking out 2000r/min, centrifugal 10 min, collect the thalli. Using 0.1mol/L   pH6.4 potassium phosphate buffer solution (PBS) into the substrate suspension (OD₅₇₀ ≈ 0.3). Take 3 ml of the suspension, the 50   L mixed in the test serum to be measured, measuring the A₀ value. Then the test solution into the 37 in the water bath [...] thermal insulation 30 min. Immediately after being taken out in the ice 10 min, in order to terminate the reaction, measuring the A value. Lysozyme activity is calculated by the following formula:

U= (A₀-A)/ A

U---Lyz vitality

A₀---reaction front optical density

A---reaction the light density,

According to small and large value of U as lysozyme activity the detection of the size of the standard, the larger U value the stronger the activity.

(2) immune protection test

In order to prove that the separated active monomer ring gastritis (C₃₅ H₅ 0N₁₀ O₁₀ · 5H₂ O) the immune protection effect, in oral dose of 3 mg/kg · d section after 7 days to Aeromonad for brachydanio. Hygrophilous cell cultivating rice seedlings 24h, pre-test results show that, brachydanio concentration is 10⁸ ml^-1, intraperitoneal brachydanio, dose the 100  l/tail, continuous observation of two weeks, the test recording after crusta 14d the cumulative mortality of each group, to calculate relative immune protection ratio and (  Survival   Percent Relative, RPS):

RPS= (1-group mortality/control group mortality) × 100%

B results and analysis

(A) serum agglutionation antibody titer

Table 9 remora oral different dosage of the antibody titer of the serum agglutionation

J: Staphylococcus aureus Staphylococcus   M   aurevs: eel vibrionales Vibrio   anguillarum

The table 9 can be seen, the dose of 2 mg/kg and 4 mg/kg group of serum agglutionation antibody titer from the section 5d which is improved compared with the control group a, and 0.5 mg/kg, 1 mg/kg there is no obvious change in the high dose group. Wherein 4 mg/kg group of up to 1:256, in the article 10d, started to decline after; 2 mg/kg group of a maximum of 1:64, a long time but sustained.

Lysozyme activity (b)

From table 10 it can be seen, from the section 5d begin to 4 mg/kg dose group started to strengthen the lysozyme activity, significant differences in the control group (P < 0.05), to the section 20d to reach the peak, very significant difference in the control group (P < 0.01), other groups without significant change compared with the control group.

Table 10 remora oral administration of lysozyme activity after different dosage

(Three) leukocyte phagocytic activity

Table 11 remora oral different dose leukocyte phagocytic activity after

The table 11 can be seen, the leukocyte phagocytic activity dose group compared with the control group have different elevated degree. Wherein 4 mg/kg dose group of leukocyte phagocytic activity in section 15d and a very significant difference compared with the control group (P < 0.01), and its maximum value as the article 10d of 0.232 ± 0.008.

(Four) phagocyte bactericidal activity

Table 12 remora oral different dosage of the phagocyte bactericidal activity

The table 12 can be seen, each group compared with the control group phagocyte bactericidal activity are improved. 0.5 mg/kg dose group section 15d, 1 mg/kg section of the high dose group 20d, there is significant difference compared with the control group (P < 0.05); 2 mg/kg, the dose group 4 mg/kg section 15d, section 20d and a very significant difference compared with the control group (P < 0.01).

(Five) immune protection test

Through oral active monomer, the remora brachydanio test results (see table 13) display, it has prominent immune protection function.

Table 13 remora orally active monomer brachydanio immune protection test results after

Embodiment 5: cyclic decapeptide (C₃₅ H₅₀ N₁₀ O₁₀ · 5H₂ O) to the effect of promoting the growth of animals

Solid-state fermentation crude product after drying, crushing, measuring cyclic decapeptide (C₃₅ H₅₀ N₁₀ O₁₀ · 5H₂ O) ≥ 2.0 the content [...].

Bdellovibrio (a) the solid-state fermentation growth-promoting action

1 material and method

(1) test material

Health not gets sick the carp (  arpio Cyprinusc) for the indoor, feed for feeding each day, and to the prevention and treatment of conventional fish diseases. 14d rear, random packets according to their weight, average weight 16.0g left and right, divided into 6 groups, each set 3 parallel, each parallel 30 tail. Test 1 group as a blank control does not add the solid-state fermentation product, test 2 in the feed group basis by adding 2% solid-state fermentation product, test 3 group to feed in 4% solid-state fermentation product, test 4 in the feed group basis by adding 6% solid-state fermentation product, test 5 to feed in group 8% solid-state fermentation product, test 6 in the feed group basis by adding 10% solid-state fermentation product. The diameter of the respectively that 0.6m, deep 0.8m the round plastic water tank, of fully aerated for flowing water tap water to pneumatic feeding. Feeding each day 2 times, of the weight of the fish scattering bait amount is 3%-5%, after the feeding 1h suction remnant bait.

(2) feed

Diet formulation design reference carp nutrition standard, feed composition and nutrition levels in table 14.

Table 14 a feed formulation and nutrition

2 growth index measurement results

After the beginning of the test section 0, 14, 28, 42, 56, 70d, each parallel random weighing 15 tail, recording weight, formula calculates the correlation indicators.

The average weight gain = wt-Wo

RGR = wt-Wo/Wo × 100%

In the formula: Wo: initial weight; wt: terminal weight

From table 15 analysis found, leavening crude product from 2% to 10% addition level, the relative growthrates compare carp growth difference is extremely prominent performance.

Table 15 to carp different crude product fermented product of the level of the impact of the added growth

^** said that the difference is extremely prominent (p < 0.01)

(II) the solid-state fermentation of the shrimps bdellovibrio bacteriovorus growth-promoting action

1. Material and method

Prawn selecting health test, weight-average is 1.0g left and right. 7d teaches the food temporary the testing. Random is divided into 6 groups, each group of 3 parallel, each parallel 30 tail. Test 1 group as a blank control does not add the solid-state fermentation product, test 2 in the feed group basis by adding 2% solid-state fermentation product, test 3 group to feed in 4% solid-state fermentation product, test 4 in the feed group basis by adding 6% solid-state fermentation product, test 5 to feed in group 8% solid-state fermentation product, test 6 in the feed group basis by adding 10% solid-state fermentation product. Test for the volume is solving severe pollution 0.2m² of the plastic water tank. Feeding the frequency is 5 times/d, is thrown is thrown sooner or later of the total 60%. Prawn growth conditions according to the per week, throws raises the quantity corresponding increase. Experiment carried out 30d, said weight roller 24h cessation of feeding. Experiment uninterrupted inflatable oxygenation of the whole process.

2. Basic daily grain

Nutrient composition formulation and ration basis of the results of the analysis are shown in table 16.

Table 16. Daily basis of the formula

3 growth index measurement results

At the end of the trial, weighing recording, and according to the following formula calculates the correlation indicators.

The average weight gain = wt-Wo

RGR = wt-Wo/Wo × 100%

Bait coefficient = setsu appetite / (terminally weight + death individual weight-initial weight)

In the formula: Wo: initial weight; wt: terminal weight

Table 17 fermented product of the addition level of crude different prawn the influence of growth and bait coefficient

^* the difference is prominent (p < 0.05);^** the difference is extremely prominent (p < 0.01)

The table 17 found that, in the feed of crude fermentation feeding prawn in addition, in the 4% more with a significant growth-promoting action, and it has been found that the advantages are obvious.

Bdellovibrio (c) solid-state fermentation to poulard growth-promoting action

1 material and method

(1) test animals and design

450 Dewa 1 date age Ivy cushion poulard random is divided into 6 groups, each group of 5 repeats, each repeat 15 only. Test 1 group is the control group (without adding solid-state fermentation product), other groups are respectively addition of the solid-state fermentation product is 4%, 8%, and 12% to all tries the chicken 1-20 day old, 21-42 day old two-stage feeding, feeding each of the with a particle feed (table 18).

(2) for breeding management

Raising the semi-stepped in cage throughout the period, test chicken 1-4 to ages 24h illumination, later drops gradually, to 21 days old illumination after 16h. Test chicken daily feeding two, free drinking water. 4 kidney infectious bronchitis vaccine on immune age drop nose ; 7 ages Newcastle vaccines first-free drinking water; 14 day old water immunoradioassay pouch vaccine; 25 day old Newcastle disease 2 secondary immunization.

2 measuring indicators and method

(1) performance of production

Recording test abyn initial weight, and end is heavyconsumes estimated , phased (1-21d; 22-42d) respectively calculating the energy consumption quantity average, the daily weight gain and material anharmonic ratio (see table 19, table 20), the record at the same time the situation dies washes. The calculation formula of the material to-weight ratio: average weight ratio (%) = weight consumes estimated /day × 100%

Table 18 composition of the experimental ration

Raw materials   1-21 day old   21-42 day old nutrient   1-21 day old   21-42 ages

Corn   56.20     58.50     AME     12.55     12.76

Bran   2.00     2.50     CP     20.70     18.50

Wheat sign powder   3.00     3.50     Ca     0.94     1.35

Corn oil   0.50     1.50     TP     0.72     0.70

Soymeal   18.30     8.00     AP     0.48     0.44

Seed meal   2.00     2.50     Lys     1.17     0.97

LEEs (DDGS)     2.00     5.00     Met     0.45     0.35

Carp   2.00     4.00     M+C     0.80     0.70

meat bonedust  -    2.00     Thr     0.68     0.68

Meat powder Meatmeal    -    2.00Il     e     0.69     0.63

Corn protein powder   2.00     4.00     Leu     1.52     1.45

Cotton seed protein   5.00    -    Arg     1.36     1.04

Soybean phospholipid   2.00     2.50

Composite premix   5.00     4.00

Note : (1) two-stage composite premix containing a plurality of different levels of the vitamin, composite trace element, the enzyme, choline chloride, lysine, threonine, methionine, calcium hydrogen phosphate, stone dust and salt ; (2) diet AME, AP content is a calculated value.

(2) test results

Table 19 fermented product of the addition level of the meat product different increasing weight and the impact of the weight (g)

Note: the same row data shoulder sign different small Letters are expressed significant difference (P < 0.05), the same difference does not significantly expressed letter (P> 0.05).

Table 20 fermented product of the addition level of crude different broiler heating intake and material than the impact of the meat

Note: the same row data shoulder sign different small Letters are expressed significant difference (P < 0.05), the same difference does not significantly expressed letter (P> 0.05).

Bdellovibrio (four) solid-state fermentation on the swine growth-promoting action

1 material and method

(1) test animals and design

Test is the average ablactation ages 21d, close to the body weight of approximately × Changbai × du Locker for polyculture of the summer, by gender and weight random is divided into 4 processing, each processing 2 with a, each repeat 10 Pigs, male and female mixed breed, trial 28d, respectively feeding including leech vibrionales solid-state fermentation is 0%, 3%, 6%, and 9% of the diet.

(2) for breeding management

Into the child care that give up immediately after weaning piglets, semi-enclosed Pig house, in order to exhaust fan and window ventilation, libitum and nipple type bubbler drinking water. Other day-to-day management according to the conventional process.

(3) test diet

Test diet the corn-soybean meal- fish meal daily basis, the NRC (1998) Pig nutrition standards and at home and abroad the latest pigling daily grain preparation progress of the study (see table 21), powder on the grain type.

Table 21 formulation and basic feed nutritional level

 The content (%) (%) raw materials of   nutrient levels

Corn   63.0     ME (MJ/kg)     13.4

Soymeal   21.5     CP (%)     18.0

Fish meal   2.0     Lys (%)     1.1

Silkworm PUPA   2.5     Met (%)     0.38

Tikitiki   5.0     M+C (%)     0.65

Phosphoric acid fat   2.0     Ca (%)     0.8

Premix   4.0     P (%)     0.67

2 measuring index

At the beginning of the test to determine the initial average head heavy, weighing final at the end of the trial, clearing residual material, in order to repeatedly for units statistical average daily weight gain, the feed conversion and walking indicators. Results are shown in table 22.

Table 22 leavening crude different add the level of the production performance of the test Pig

Groups   at the end of initial heavy kg g-  increasing weight kg   meat than walking

Control group   18.7 ± 0.8     35.9 ± 1.6     617.7 ± 28.6     2.49 ± 0.09     2.80 ± 2.00

3% disposal   18.9 ± 0.4     38.0 ± 0.6     680.7 ± 28.8^**   2.27 ± 0.06^**   2.83 ± 2.18

6% disposal   18.7 ± 1.7     38.2 ± 1.2     695.1 ± 34.8^**   2.20 ± 0.05^**   1.41 ± 1.18^**

9% disposal   18.8 ± 0.7     38.9 ± 0.7     717.5 ± 3.8^**   2.13 ± 0.04^**   1.84 ± 0.90^**

^** the difference is extremely prominent (p < 0.01)