CN106387488A - Puffing carp feed and preparation method thereof - Google Patents

Abstract

The invention discloses a puffing carp feed. The puffing carp feed is prepared from the following components in parts by weight: 5-10 parts of fish meal, 20-40 parts of dehulled soybean meal, 10-30 parts of rapeseed meal, 15-20 parts of flour, 10-15 parts of rice bran, 1-4 parts of soybean oil, 1-3 parts of bentonite, 1-3 parts of calcium dihydrogen phosphate, 0.1-0.5 part of choline chloride, 0.1-0.5 part of composite vitamins, 0.1-0.5 part of composite trace elements, 0.1-0.5 part of a powdery microecological preparation and 0.5-2 parts of a liquid microecological preparation. The microecological preparations contained in the puffing carp feed disclosed by the invention can purify water bodies, can reduce the content of ammonia, nitrogen and nitrite in water bodies, can complement the defects of endogenous digestive enzymes of carps, can improve the feed transformation efficiency, can strengthen the disease resistance of the carps and can increase the survival rate. Compared with a feed in a control group, the puffing carp feed added with the powdery microecological preparation and the liquid microecological preparation disclosed by the invention has the advantages that the growth rate is increased by 8% or above, bait coefficients are obviously reduced, and growth promotion effects are obvious.

Description

A kind of expanded carp feed and preparation method thereof

Technical field

The present invention relates to belonging to field of fodder and in particular to a kind of expanded carp feed and its system Preparation Method.

Background technology

Probiotics is manually separately normal flora under microecology theories instruct, and leads to Cross the biologic product that special process is made, it has supplementary, adjustment and maintains animal intestinal micro- The function of the ecological balance.Thus reaching disease preventing and treating, promoting health and improve production performance Effect.It is a kind of green, environmental protection, pure biologic product, nontoxic, have no side effect, no residual Stay pollution, do not produce resistance, secondary pollution is not produced to water body, therefore receives in recent years Widely pay close attention to.Previous studies report is more common in the interpolation of plain particles feed, and swollen The report changing interpolation probiotics in feed does not also have.Mainly expanded granulating process needs Through the HTHP process of Builking cavity, general Tiny ecosystem bacterial classification survival rate is too low, rises not To useful effect.

Content of the invention

In order to solve the above problems, the present invention provides a kind of expanded carp feed.

A kind of expanded carp feed, the component containing following weight portion：Fish meal 5-10 part, go Skin dregs of beans 20-40 part, rapeseed meal 10-30 part, flour 15-20 part, rice bran 10-15 part, beans Oily 1-4 part, bentonite 1-3 part, calcium dihydrogen phosphate 1-3 part, Choline Chloride 0.1-0.5 part, B B-complex 0.1-0.5 part, composite trace element 0.1-0.5 part, powdery probiotics 0.1-0.5 part and liquid micro-ecological preparations 0.5-2 part.

In a preferred embodiment of the present invention, described expanded carp feed contains following weight The component of part：9 parts of fish meal, 30 parts of dehulled soybean meal, 20 parts of rapeseed meal, flour 18 part, meter 11 parts of chaff, 2 parts of soya-bean oil, 2.1 parts of bentonite, 2 parts of calcium dihydrogen phosphate, Choline Chloride 0.3 Part, 0.3 part of B B-complex, 0.3 part of composite trace element, powdery probiotics 0.1-0.2 part and liquid micro-ecological preparations 1-1.5 part.

Wherein, the viable count in described powdery probiotics is preferably 80-120 hundred million cfu/g.

Wherein, described powdery probiotics contains bacillus subtilis (Bacillus Subtilis), bacillus licheniformis (Bacillus licheniformis) and bacillus pumilus (Bacillus pumilus).Skilled person will appreciate that, powdery probiotics is acceptable Containing one or more of the conventional protective agent in this area, auxiliary material, excipient etc..

Wherein, described bacillus subtilis is preferably bacillus subtilis CGMCC No.9356, described bacillus licheniformis is preferably bacillus licheniformis CGMCC No.7030, Described bacillus pumilus is preferably bacillus pumilus CGMCC No.4756.

Wherein, the viable count in described liquid micro-ecological preparations is preferably 10-20 hundred million cfu/ml.

Wherein, described liquid micro-ecological preparations contain VREF (Enterococcus Faecium), Yue Shi lactobacillus (Lactobacillus johnsonii).Those skilled in the art should When knowing, liquid micro-ecological preparations also can contain other auxiliary materials, such as medium component, fermentation time Raw metabolin etc..

Wherein, described VREF is preferably VREF CGMCC No.9134, described Yue Shi lactobacillus You Xuanweiyueshi lactobacillus CGMCC No.4926.

The bacillus licheniformis (Bacillus licheniformis) of the present invention is applicant from duck intestines Separate in road and obtain.Its vegetative cell is shaft-like, and its gemma is oval or long tubular, in generation Raw oval gemma, sporangiocyst expands.Gram's staining is the positive.In beef extract-peptone training Form white, opaque, irregular bacterium colony, edge is hair-like, corrugationless on foster base.Connect Catalase-positive, can utilize propionate.Various active enzyme can be produced, such as protease, cellulase Deng, raising efficiency of feed utilization, strengthen the activity of animal digestion enzyme, promote the growth of cultivated animals. Bacillus licheniformis (Bacillus licheniformis) applicant of the present invention is in 2012 December 25 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, Abbreviation CGMCC, address：The Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences Institute of microbiology, deposit number is CGMCC No.7030.

The bacillus licheniformis CGMCC No.7030 of the present invention, can be produced high by fermentation The cellulase of activity and protease, are added in animal feed and can increase albumen in feed The decomposition of the compositions such as matter, cellulose and hemicellulose, improves feed nutrient utilization rate.

Bacillus subtilis (Bacillus subtilis) H8-1 used by the present invention, its notable area Not in existing bacillus subtilis, it is that applicant separates from the shrimp feed coefficient water of Fujian Arrive, its biological property is as follows：Bacterium colony smooth surface, translucent, in dirty white, bacterium colony Circle, edge becomes zigzag；Colonial morphology is shaft-like, and size is 0.8～1.2 × 1. 5～4.0um, gemma form is that ellipse arrives column, central or slightly inclined positioned at thalline, sporulation Thalline does not expand afterwards.

Bacillus subtilis H8-1 in the present invention is to the ammonia nitrogen for 2.5mg/L for the concentration, nitrous State nitrogen degradation rate respectively 81.72%, 99.70%, the ammonia nitrogen of water body of effectively degrading and/or Asia Nitrate nitrogen level.Being different from existing bacillus subtilis can be 0 ‰, 3 ‰, 15 ‰ Under salt concentration conditions, equal well-grown, can tolerate 10-15 DEG C of low-temperature epitaxy good.The present invention Bacillus subtilis (Bacillus subtilis) H8-1 on June 18th, 2014 protect It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center, referred to as CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences is micro- Biological study institute, deposit number is CGMCC No.9356.

Bacillus subtilis (Bacillus subtilis) H8-1 in the present invention is cultivation water structural reform Good probiotics provides introduces a collection, is reduced harmful in water in the way of pollution-free and noresidue Material such as ammonia nitrogen and nitrite nitrogen, improve cultivation quality, improve fanning economics, have Promotional value.Bacillus subtilis H8-1 in present invention ammonia for 2.5mg/L to concentration Nitrogen, nitrite nitrogen degradation rate are respectively 81.72%, 99.70%.

VREF of the present invention is isolatable from chitling road, and obtains through Uv-induced screening.

The feature of VREF (Enterococcus faecium) CGMCC No.9134 is：

(1) 80 DEG C of VREF (Enterococcus faecium) CGMCC No.9134 bacterium solution Process 5min, survival rate is 0.18%, starting strain relatively improves 22 times.

(2) VREF (Enterococcus faecium) CGMCC No.9134 bacterium solution is used 0.3% pig gall salt treatment 3h, survival rate is 20.80%, and starting strain relatively improves 3.04 Times.

(3) VREF (Enterococcus faecium) CGMCC No.9134 bacterium solution is used 0.6% pig gall salt treatment 3h, survival rate is 21.6%, and starting strain relatively improves 27.69 Times.

The VREF new strains of the present invention are preserved in Chinese micro- life on May 8th, 2014 Thing culture presevation administration committee common micro-organisms center, address：The Chaoyang District, Beijing City North Star West Road 1 No. 3 Institute of Microorganism, Academia Sinica of institute, registration number：CGMCC No.9134.The VREF new strains of the present invention do not move back through the Secondary Culture of more than 10 times Change, there is genetic stability, and resistance is much a lot of than starting strain improves.

The Yue Shi lactobacillus (Lactobacillus johnsonii) of the present invention, is by acidproof in vitro One plant obtaining with bile tolerance screening all has the Yue Shi lactobacillus of potential prebiotic function, is named as Yue Shi lactobacillus (Lactobacillus johnsonii) YS-12, and on June 8th, 2011 in State's Microbiological Culture Collection administration committee common micro-organisms collection has carried out preservation, preservation Number be CGMCC No.4926.

The probiotics energy purifying water body that the expanded carp feed of the present invention contains, reduces water body Ammonia nitrogen and content of nitrite, can supplement the deficiency of the endogenous digestive ferment of carp, improve feed and turn Change efficiency, strengthen carp resistance against diseases, improve survival rate.The present invention preparation interpolation powdery and The expanded carp feed of liquid micro-ecological preparations, compared with control group feed, growth rate increases More than 8%, feed coefficient significantly reduces, and growth-promoting effect is obvious.

Specific embodiment

Following examples are used for the present invention is described, but are not limited to the scope of the present invention.

Embodiment 1 bacillus pumilus (Bacillus pumilus) CGMCC No.4756 bacterium powder system Standby

Bacillus pumilus CGMCC No.4756 (being disclosed in CN201110140925.2) is planted The preparation of sub- liquid：Preservation of bacteria strain is seeded to the conical flask equipped with 100ml MNB culture medium In, cultivate 16h in 30 DEG C of constant-temperature tables, shaking speed is 225rpm；Withered by prepare Careless bacillus seed liquor is seeded to equipped with 100mL MNB culture medium according to 1% inoculum concentration Conical flask in, in 30 DEG C of constant-temperature tables cultivate 16h, shaking speed be 225rpm；

Described MNB culture medium prescription is as follows：Glucose, 5.0g/L；Peptone, 5.0 g/L；Beef extract, 3.0g/L；Yeast extract, 1.0g/L；Epsom salt, 0.5g/L； Manganese sulfate, 0.005g/L；pH 7.0±0.2；

By the centrifugation of zymotic fluid, concentration, add freeze drying protectant, vacuum freeze drying and bacterium The production routines such as powder viable count inspection, finally prepare powdery active bacteria formulation.

The mensure of embodiment 2 bacillus licheniformis CGMCC No.7030 zymotic fluid enzyme activity

To be inoculated on beef extract-peptone solid medium for examination bacterium, after 30 DEG C of culture 24h Picking bacterial classification, is suspended from 0.85% physiological saline, and blood counting chamber counts and adjusts concentration about 5.0 ×10⁷Individual/mL accesses bacterium in the 100mL triangular flask equipped with 20mL culture medium and hangs Liquid 2mL, 30 DEG C of natural supply oxygen, fermented and cultured 24h under the conditions of 170r/min, liquid is sent out Zymotic fluid centrifugation 15min under the conditions of 5000r/min, takes supernatant, as crude enzyme liquid.

Cellulase culture medium formula is：Peptone 0.9%, yeast extract 1.0%, CMC-Na 0.5%, pH 8.0.

Protease culture medium formula is：Peptone 1.0%, yeast extract 0.5%, glucose 1.0%, soluble starch 0.5%, KH₂PO40.2%, MgSO₄·7H₂O 0.05%, CaCl₂·2H₂O 0.02%, pH 7.2.

(1) cellulase activity measures

Cellulase activity measures：Draw calibration curve according to GB NY/T 912-2004, adopt Measure the cellulase activity in zymotic fluid with DNS method.1% (w/v) is contained with 1mL PH 8.0 phosphate buffer of CMC-Na is substrate, adds 0.2mL crude enzyme liquid, 50 DEG C of guarantors Warm 30min, is subsequently adding 2.5mL DNS reagent, boiling water bath 5min, after being cooled to room temperature It is settled to 5.0mL, using the absorbance at spectrophotometric determination wavelength 540nm；To boil Boiling inactivation crude enzyme liquid adds above-mentioned substrate reactions liquid as blank, using substrate solution as the moon Property comparison；Generation 1g glucose amount per minute for 1mL enzyme liquid is defined as 1 enzyme activity unit, U/mL.

After measured, the cellulase of the fermentation crude enzyme liquid of bacillus licheniformis CGMCC 7030 Work is：230.5U/mL.

(2) prolease activity measures

The mensure of prolease activity：Measured using GB SB/T 10317-1999 prolease activity Folin's methods in method draws calibration curve.Sample determination：Take 3,15 × 100mm test tube, Numbering 1,2,3, often manage interior addition crude enzyme liquid 1mL, be placed in preheating 2min in 40 DEG C of water-baths, Each 2% casein solution (pH 7.2) 1mL adding through same preheating, is accurately incubated l0 again Min, after the time arrives, respectively adds 0.4mol trichloroacetic acid 2m L, immediately again to terminate Reaction, continues to be placed in insulation 20min in water-bath, centrifugation or mistake after making residual protein precipitate Filter, then separately takes 3,15 × 150mm test tube, numbering l, 2,3, often manage in addition Filtrate 1mL, then plus 0.4mol sodium carbonate 5mL, the Folin reagent 1mL of dilution Shake up, after 40 DEG C of insulation color development 20min, measure OD value under 540nm.Blank test Also take 3, test tube, number (1), (2), (3), assay method ibid, is only adding 2% junket First add 0.4mol trichloroacetic acid 2mL before albumen, so that enzyme is inactivated, add 2% junket egg In vain.

Prolease activity unit definition is：1mL enzyme liquid at 40 DEG C, under conditions of pH 7, often The enzyme amount that minute caseinhydrolysate produces 1 μ g tyrosine is an enzyme activity unit, with (U/ml) Represent.

The compound method of Folin reagent (Folin reagent):In 200mL ground reflux, Add sodium tungstate (Na₂WO₄·2H₂O) 100g, sodium molybdate (Na₂MO₄·2H₂O)25g, Distilled water 700mL, 85% phosphoric acid 50mL, concentrated hydrochloric acid 100mL, slow fire backflow 10h. Remove condenser, add lithium sulfate (Li₂SO₄) 50g, distilled water 50mL, mix, plus People's a few drop of liquid bromine, then boil 15min, to expel residual bromine to remove color, solution should be in Huang Color and non-green.If solution still has green, need Zai Jiajidi Australia liquid, then boil removing. After cooling, it is settled to 1000mL, with (2.2 μm) filtrations of bacterial filter, be placed in brown Preserve in bottle.This solution adds 2 times of distilled water dilutings when using.Dilution forint examination Agent.

After measured, the albumen of every milliliter of bacillus licheniformis CGMCC 7030 fermentation crude enzyme liquid Enzyme activity is 1500U/mL.

Producing enzyme test shows, the bacillus licheniformis CGMCC No.7030 of the present invention can Highly active cellulase and protease are produced by fermentation, is added to permissible in animal feed Increase the decomposition of the composition such as protein, cellulose and hemicellulose in feed, improve feed battalion Form a point utilization rate, reduce the content of nutriment in animal wastes simultaneously, mitigate to environment Pollution, there is very high feeding value.

The preparation of embodiment 3 bacillus licheniformis CGMCC No.7030 freeze-dried powder

The preparation method of Bacillus licheniformis powder, including the steps

(1) flat board culture rejuvenation：Bacillus licheniformis strain is inoculated on plating medium, Cultivate 18h in 30 DEG C, make bacillus licheniformis rejuvenation, and form single bacterium colony, picking single bacterium colony On inoculation plating medium, 30 DEG C of culture 24h；

Described plating medium formula is：Tryptone 1%, yeast extract 0.5%, chlorination Sodium 1%, agar 2%, pH 7.0 ± 0.2.

(2) preparation of primary seed solution：The Bacillus licheniformis strain that step (1) is cultivated It is forwarded in the test tube equipped with 10mL culture medium, 30 DEG C of shaking table cultures 16h, rotating speed is 170rpm, Make to be in late log phase, obtain primary seed solution；

(3) preparation of secondary seed solution：Primary seed solution prepared by step (2) is by 1% The capacity that inoculum concentration is inoculated in equipped with 75ml culture medium is 30 DEG C of perseverances in the conical flask of 250ml Warm shaking table culture 16h, rotating speed is 170rpm, obtains secondary seed solution；

Seed culture based formulas are：Tryptone 1%, yeast extract 0.5%, sodium chloride 1%, pH 7.0 ± 0.2.

(4) preparation of the lichen bacillus ferments liquid：By the seed liquor in step (3) by connecing The amount of kind is inoculated in the fermentation tank of 100L for 1%, sample-loading amount 20%, 30 DEG C of temperature, rotating speed 250rpm is aerobic to be cultivated to gemma production rate more than 80%, and viable count is 10¹⁰More than CFU/ml, Then put tank and terminate fermentation, obtain the lichen bacillus ferments liquid；

Described fermentation medium is：Wheat bran 2%, corn flour 1%, dregs of beans 2%, ammonium sulfate 2%, magnesium sulfate 0.03%；Ammonium citrate 0.5%, pH is 7.0-7.4.

(5) bacillus licheniformis is lyophilized the preparation of bacterium powder：Zymotic fluid prepared by step (4) After centrifugation, (frozen-dried protective agent prescription is to add 10% freeze drying protectant in bacterium mud：Degreasing Milk powder 10%, lactose 10%, glycerine 0.5%, vitamin C 0.2%, distilled water 79.3%), Freeze-drying after mixing, obtains the Bacillus licheniformis powder of moisture ＜ 5%, every gram of jelly In dry bacterium powder, viable count is about 40,000,000,000.

The screening of embodiment 4 bacillus subtilis and identification

Take and come from Tianjin Wuqing, the water sample in Fujian, Jiangsu fish pond and the shrimp pool and mud sample 124 Individual, take 1mL water sample or mud sample 1g in the 9mL LB fluid nutrient medium of 18ml test tube, 85 DEG C of water bath processing 10min.

It is 30 DEG C that test tube after high-temperature process is placed on temperature, permanent under the aerobic condition of 180rpm Enrichment culture liquid is obtained after warm shaken cultivation 16h.

Enrichment culture liquid is carried out by gradient dilution using coubling dilution, coats the training of LB solid On foster base, it is positioned in 30 DEG C of insulating boxs and carries out cultivating 20h, picking flat board is grown Different single bacterium colonies are rule, and purify twice, obtain single bacterium colony alive, obtain 275 altogether Strain bacterium.

By the single bacterium colony after purifying twice, with toothpick dibbling on fresh haemolysis plate, place Observe 24h, 48h, 72h in 30 DEG C of insulating boxs, if haemolysis circle occurs, according to making blood The Capability Categories of cell agar haemolysis：1) α haemolysis：Grass green haemolysis, periphery of bacterial colonies is cultivated The grass green colour circle of 1～2mm in base, caused by being ferrihemoglobin；2) β haemolysis：Completely Haemolysis, periphery of bacterial colonies forms a completely Clear ＆ Transparent zone of hemolysis, is bacteriogenic molten Caused by sanguinin makes red blood cell be completely dissolved；3) γ haemolysis：Not haemolysis.To there is haemolysis circle bacterial strain Eliminate.

No hemolytic strain is rule on Selective solid culture medium, well-grown bacterial strain Use LB Liquid Culture, in 30 DEG C of constant temperature Amplification Cultures 16h.

Solid selective medium is two kinds：With NH₄ ⁺- N is that the ammonia nitrogen of only nitrogen source is selective Culture medium and with NO₂ ^-- N is the nitrite nitrogen culture medium of only nitrogen source；

Ammonia nitrogen Selective solid culture medium is every L by (NH₄)₂SO₄0.5g, sodium succinate 2.17g, Vickers salting liquid 50mL, the water composition of agar 20g and surplus, pH 7.0-7.2, 115 DEG C of autoclaving 20min, described Vickers salting liquid is every L by K₂HPO₄5.0g, MgSO₄·7H₂O 2.5g, NaCl 2.5g, FeSO₄·7H₂O 0.05g, MnSO₄·4H₂O The water composition of 0.05g and surplus；

Nitrite nitrogen Selective solid culture medium is every L by NaNO₂0.5g, sodium succinate 2.17g, Vickers salting liquid 50mL, the water composition of agar 20g and surplus, pH 7.0-7.2, 115 DEG C of autoclaving 20min, described Vickers salting liquid is every L by K₂HPO₄5.0g, MgSO₄·7H₂O 2.5g, NaCl 2.5g, FeSO₄·7H₂O 0.05g, MnSO₄·4H₂O The water composition of 0.05g and surplus.

Final acquisition no haemolysis circle and life on ammonia nitrogen and nitrite nitrogen Selective solid culture medium 63 good bacillus of length.

In super-clean bench, 63 plants of test strains bacterium solution are accessed in corresponding screening and culturing medium, Inoculate final concentration of 5 × 10⁵CFU/mL, repeat number is 3；Put in shaking table and cultivated 30 DEG C, 180r/min, cultivates 48h；Uniform sampling in 1.5mL centrifuge tube, 12000 Rpm is centrifuged 3min, takes supernatant 200 μ L to add 96 orifice plates；

When measuring nitrite nitrogen, add in 96 orifice plates after test sample supernatant 200 μ L, often Hole successively adds each 20 μ L of Griess reagent A, B, in 550nm colorimetric estimation；Measure ammonium During state nitrogen, every hole adds nessler reagent 20 μ L, in 450nm colorimetric estimation；

Screening and culturing medium is two kinds：Ammonia nitrogen screening and culturing medium and nitrite nitrogen screening and culturing medium；

Ammonia nitrogen screening and culturing medium is every 50mL by (NH₄)₂SO₄Mother liquor 50 μ l, commercially available pulverizing shrimp The water of material 0.05g and surplus forms, and ammonium nitrogen concentration is：2.5mg/l；

Nitrite nitrogen screening and culturing medium is every 50mL by NaNO₂Mother liquor 50 μ l, commercially available pulverizing The water of shrimp material 0.05g and surplus forms, and nitrite nitrogen concentration is：2.5mg/l.

Ammonium sulfate liquor is to weigh 9g (NH₄)₂SO₄Add 1L simulation shrimp pool water, now ammonium state Nitrogen concentration is about：2.5g/l；

Natrium nitrosum mother liquor is to weigh 3.75g NaNO₂Add 1L simulation shrimp pool water, now sub- Nitrate is about：2.5g/l.

Choose 10 plants of bacterium (numbering is A1-A10) or the nitrous acid that ammonia nitrogen degradation rate is higher than 30% Nitrogen degradation rate is higher than 70% 10 plants of bacterium (numbering is Y1-Y10), not inoculate any bacterial strain For negative control (CK), carry out replication.

Assay method：In super-clean bench, test strains bacterium solution is accessed corresponding screening and culturing medium In, inoculate final concentration of 5 × 10⁵CFU/mL, repeat number is 3；Put in shaking table and trained Support 30 DEG C, 180r/min, cultivates 24h, 48h；Uniform sampling in 1.5mL centrifuge tube, 12000r/min is centrifuged 3min, takes supernatant 200 μ L to add 96 orifice plates.

When measuring nitrite nitrogen, add in 96 orifice plates after test sample supernatant 200 μ L, often Hole successively adds each 20 μ L of Griess reagent A, B, in 550nm colorimetric estimation；Measure ammonia During nitrogen, every hole adds nessler reagent 20 μ L, in 450nm colorimetric estimation.

Qualitative determination：When measuring nitrite nitrogen, solution is changed into pink, rose, orange Color or brown etc. indicate nitrate reductase, react for the positive, i.e. color more elementary introduction publicly price-reduction Solution effect is better；When measuring ammonium nitrogen, it is positive reaction that solution is changed into orange, yellow etc., Shallow it is preferred.

Quantitative determination：Formulate nitrite nitrogen concentration (Y₁) and measure OD value (X₁) between join Examine calibration curve and ammonia nitrogen concentration (Y₂) and measure OD value (X₂) between normative reference curve.

Screening and culturing medium includes ammonia nitrogen screening and culturing medium and nitrite nitrogen screening and culturing medium；

Ammonia nitrogen screening and culturing medium：(NH₄)₂SO₄Mother liquor 50 μ l, shrimp material 0.05g, distilled water 50mL, ammonia nitrogen concentration is about：2.5mg/L, 121 DEG C of sterilizings.Ammonium sulfate liquor：Weigh 9 g(NH₄)₂SO₄Add 1L distilled water, now ammonium nitrogen concentration is about：2.5g/l.

Nitrite nitrogen screening and culturing medium：NaNO₂Mother liquor 50 μ l, shrimp material 0.05g, distilled water 50mL, nitrous nitrogen concentration is about：2.5mg/l, 121 DEG C of sterilizings.Natrium nitrosum mother liquor：Claim Take 3.75g NaNO₂Add 1L distilled water, now nitrite nitrogen concentration is about：2.5g/l.

Ge Lisishi reagent (Griess) A：P-aminobenzene sulfonic acid 0.5g, (10% is left for spirit of vinegar Right) 150mL, brown bottle lucifuge, 4 DEG C of preservations.Ge Lisishi reagent B：αnaphthylamine 0.1g, Distilled water 20mL, spirit of vinegar (10% about) 150mL, the 4 DEG C of preservations of brown bottle lucifuge.

Nessler reagent：Cash purchase, 4 DEG C of preservations.

As shown in Table 1 and Table 2, the average ammonia nitrogen degradation rate of H8-1 is 81.72% to result, puts down All nitrous degradation rate is 99.70%, drops nitrogen ability highest.

1 10 plants of preferred strain ammonia nitrogen degradation rate comparative measurements of table (No. A10 is H8-1)

Bacterial strain number	A1	A2	A3	A4	A5	A6	A7	A8	A9	A10	ck
												24h ammonia nitrogen	34.096	30.000	19.789	45.702	37.728	39.985	59.574	21.277	75.487	79.572	0.011
48h ammonia nitrogen	45.897	35.786	38.931	56.971	40.302	44.818	31.405	40.626	61.079	83.873	0.441

2 10 plants of preferred strain nitrite nitrogen degradation rate comparative measurements of table (No. Y10 is H8-1)

Bacterial strain number	Y1	Y2	Y3	Y4	Y5	Y6	Y7	Y8	Y9	Y10	ck
												24h nitrous	83.121	74.374	90.671	92.332	75.629	74.506	90.467	95.714	92.196	99.603	0.002
48h nitrous	83.018	74.508	90.128	93.671	75.457	70.165	92.467	96.541	94.467	99.806	2.443

The bacillus subtilis screening H8-1 is preserved in China on June 18th, 2014 Microbiological Culture Collection administration committee common micro-organisms center, abbreviation CGMCC, ground Location：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, Deposit number is CGMCC No.9356.

Under the different salt concentration conditions of embodiment 5, H8-1 growing state compares

Prepare LB solid medium using the water that salinity is respectively 0 ‰, 3 ‰, 15 ‰, with going out Toothpick dibbling H8-1 after bacterium, on the flat board of different salinity, is positioned over training in 30 DEG C of incubators Foster 16h, observes colony growth situation on flat board.Find after culture, dibbling is in three kinds of salinity not Colony growth on same flat board is all good, and the bacterium colony size after the completion of growth is more consistent, says Bright H8-1 salt resistant character is strong.

LB culture medium：Tryptone：10g/L；Yeast extract 5g/L；Sodium chloride 10g/L；Agar powder 15g/L.Prepare salinity with salinometer and be respectively 0 ‰, 3 ‰, 15 ‰ Water, carries out the preparation of culture medium.

H8-1 fall nitrogen performance measurement under the different salt concentration conditions of embodiment 6

The results are shown in Table 3, it is as shown in the table, under 0 ‰, 3 ‰, 15 ‰ salinity, the fall of H8-1 Nitrogen performance difference, but affected very little by salinity.

Table 3 measures the fall nitrogen performance of H8-1 under different salinity

Ammonia nitrogen screening and culturing medium：(NH₄)₂SO₄Mother liquor 50 μ l, shrimp material 0.05mg, make respectively Water (the water of 0 ‰ salinity with different salinity：Distilled water；The water of 3 ‰ and 15 ‰ salinity:Add Sea salt, in distilled water, is measured with salinometer, reaches salinity and is respectively 3 ‰ and 15 ‰) 50mL, ammonia nitrogen concentration is about：2.5mg/L, 121 DEG C of sterilizings.Ammonium sulfate liquor：Weigh 9 g(NH₄)₂SO₄Add 1L distilled water, now ammonium nitrogen concentration is about：2.5g/l.

Nitrite nitrogen screening and culturing medium：NaNO₂Mother liquor 50 μ l, shrimp material 0.05mg, different salinity Water 50mL, nitrous nitrogen concentration is about：2.5mg/l, 121 DEG C of sterilizings.Natrium nitrosum is female Liquid：Weigh 3.75g NaNO₂Add 1L distilled water, now nitrite nitrogen concentration is about： 2.5g/l.

Embodiment 7 H8-1 growing state under cryogenic

With the toothpick dibbling H8-1 single bacterium colony after sterilizing on LB solid plate, place respectively Temperature be 10 DEG C, 15 DEG C, 24h in 30 DEG C of incubators, observe colony growth feelings on flat board Condition.Find after culture, bacterium colony size phase on the flat board of growth in 10 DEG C and 15 DEG C of incubators Seemingly, it is and is grown in 1/2 of bacterium colony size on 30 DEG C of incubator middle plateforms, this illustrates H8-1 In the relatively low breeding environment of temperature also can normal growth and play fall nitrogen function.

LB solid medium：Tryptone：10g/L；Yeast extract 5g/L；Sodium chloride 10g/L；Agar powder 15g/L.

The preparation of embodiment 8 bacillus subtilis CGMCC No.9356 bacterium powder：

The preparation of bacillus subtilis seed liquor：Preservation of bacteria strain is seeded to equipped with 100ml In the conical flask of MNB culture medium, in 30 DEG C of constant-temperature tables, cultivate 16h, shaking speed is 225rpm；The bacillus subtilis preparing seed liquor is seeded to according to the inoculum concentration of 1-3% In conical flask equipped with 100mL MNB culture medium, cultivate 16h in 30 DEG C of constant-temperature tables, shake Bed rotating speed is 225rpm；

Described MNB culture medium prescription is as follows：Glucose, 5.0g/L；Peptone, 5.0 g/L；Beef extract, 3.0g/L；Yeast extract, 1.0g/L；Epsom salt, 0.5g/L； Manganese sulfate, 0.005g/L；pH 7.0±0.2；

By the centrifugation of zymotic fluid, concentration, add freeze drying protectant, vacuum freeze drying and bacterium The production routines such as powder viable count inspection, finally prepare powdery active bacteria formulation.

Prepared by embodiment 9 powdery probiotics

By the bacterium powder of the bacillus subtilis CGMCC No.9356 of preparation, bacillus licheniformis The bacterium powder of CGMCC No.7030, the bacterium powder of bacillus pumilus CGMCC No.4756 is pressed Weight compares 1:1:After 1 mixing, obtain final product the packing bacillus of the present invention, powdery probiotics In viable count be about 11,000,000,000 cfu/g.

The preparation of embodiment 10 Yue Shi lactobacillus CGMCC No.4926 zymotic fluid

1) by the Yue Shi lactobacillus CGMCC No.4926 of freezen protective in MRS agar plate Activation three times, picking single bacterium colony is inoculated in 10mL deep layer MRS liquid tube, 37 DEG C of cultures After 10 hours, transferred in 250mL MRS fluid nutrient medium with 1% inoculum concentration, cultivate 10 As seed liquor after hour；

2) with 5% inoculum concentration by step 1) seed liquor of gained is inoculated in the fermentation tank of 5L Middle culture；

3) zymotic fluid pH value is maintained to be 6.0 in sweat, when pH exceedes setting value, Add feed liquid；When pH is less than setting value, auto-feeding antalkali ammoniacal liquor；In temperature 37 DEG C, rotating speed 100rpm condition bottom fermentation 14 hours, sweat is passed through N₂, maintain molten Oxygen is less than 1%；

4) no longer reduce or light absorption value under 600nm wavelength for the zymotic fluid when adding material liquid pH When (i.e. OD600) is not further added by, terminates fermentation, obtain final product Yue Shi living preparation of lactobacillus number and reach 10¹¹The zymotic fluid of more than cfu/ml.

Prepared by embodiment 11 VREF CGMCC No.9134 bacterium solution

1) medium sterilization：

Culture medium prescription：10g/L fructose, 50g/L beef extract, 10g/L yeast extract, 15g/L Magnesium sulfate, 15g/L NaCl, 15%CaCO₃.

Prepare culture medium, water filling (running water), sterilising temp is 115 DEG C, and the time is 20min, regulation pH value to 6.8；

2) stablize physical and chemical index：

Fermentation tank rotating speed 50rpm, ventilation 1:0.1vvm, tank pressure maintains 0.05MPa, liquid After stable physical-chemical indexes, demarcate dissolved oxygen.

3) inoculation fermentation：

According to 1% ratio inoculation, condition of culture：20 DEG C of temperature, rotating speed 50rpm, tank pressure For 0.01 MPa, ventilation is 1:0.1vvm.Under this state, tank under culture 10h, lower tank When viable count be 1 × 10⁹More than cfu/ml.

Prepared by embodiment 12 liquid micro-ecological preparations

By the zymotic fluid of VREF CGMCC No.9134 and Yue Shi lactobacillus CGMCC No.4926 by volume 1:1 ratio is mixed, and obtains final product the liquid micro-ecological system of the present invention Agent, wherein, viable count is about 2,000,000,000 cfu/ml.

Prepared by embodiment 13 carp expanded pellet diet

10 parts of fish meal, 40 parts of dehulled soybean meal, 10 parts of rapeseed meal, 20 parts of flour, rice bran 15 Part, 4 parts of soya-bean oil, 1 part of bentonite, 3 parts of calcium dihydrogen phosphate, 0.1 part of Choline Chloride, multiple Close 0.5 part of vitamin, 0.5 part of composite trace element, 0.5 part of powdery probiotics and liquid 0.5 part of body probiotics.

Weigh fish meal, dehulled soybean meal, cotton dregs, rapeseed meal and rice bran by weight, first carry out first Pulverize, after then mixing with other raw materials and powdery probiotics, put into superfine In pulverizer, it is ground into 90% mash feed crossing 80 mesh, then carries out expanded granulation；

After expanded granulation, liquid micro-ecological preparations are added to by particle surface by rear spraying, dry Dry.

Prepared by embodiment 14 carp expanded pellet diet

5 parts of fish meal, 20 parts of dehulled soybean meal, 30 parts of rapeseed meal, 15 parts of flour, rice bran 10 Part, 1 part of soya-bean oil, 3 parts of bentonite, 1 part of calcium dihydrogen phosphate, 0.5 part of Choline Chloride, multiple Close 0.1 part of vitamin, 0.1 part of composite trace element, 0.1 part of powdery probiotics and liquid 2 parts of body probiotics.

Weigh fish meal, dehulled soybean meal, cotton dregs, rapeseed meal and rice bran by weight, first carry out first Pulverize, after then mixing with other raw materials and powdery probiotics, put into superfine In pulverizer, it is ground into 90% mash feed crossing 80 mesh, then carries out expanded granulation；

After expanded granulation, liquid micro-ecological preparations are added to by particle surface by rear spraying, dry Dry.

Prepared by embodiment 15 carp expanded pellet diet

9 parts of fish meal, 30 parts of dehulled soybean meal, 20 parts of rapeseed meal, 18 parts of flour, rice bran 11 Part, 2 parts of soya-bean oil, 2.1 parts of bentonite, 2 parts of calcium dihydrogen phosphate, 0.3 part of Choline Chloride, 0.3 part of B B-complex, 0.3 part of composite trace element, 0.2 part of powdery probiotics and 1.5 parts of liquid micro-ecological preparations.

Weigh fish meal, dehulled soybean meal, cotton dregs, rapeseed meal and rice bran by weight, first carry out first Pulverize, after then mixing with other raw materials and powdery probiotics, put into superfine In pulverizer, it is ground into 90% mash feed crossing 80 mesh, then carries out expanded granulation；

After expanded granulation, liquid micro-ecological preparations are added to by particle surface by rear spraying, dry Dry.

Test example 1

This is tested on July 5th, 2015 to September 5 days in great Bei agriculture group Tangshan Yu Tianshui Produce proving ground to carry out, totally 4 groups of tests, 1 control group and 3 test group.Test group 1-3 It is respectively the expanded carp feed of embodiment 13-15 preparation, the same embodiment of control group other compositions 15, difference is without probiotics.Each test group sets 4 repetitions, totally 16 Breeding barrel (800L/ bucket), puts 30 tail carps (50.13 ± 0.15 grams) for every barrel.During cultivation Throw rate of raising day for the 3% of body weight, morning and afternoon respectively feeds once, adjustment of weighing every two weeks is once thrown Feed amount.Change water 1/3 every afternoon, wanted with ensureing that breeding water body meets fishery cultivating water standard Ask, after off-test, every barrel takes 5 tail fishes to measure the growth indexes such as body length, body weight at random.

After off-test, the survival rate of each group carp is 100%, its growth data measurement result It is shown in Table 4：

The each test group growth indexes of table 4

Group	Fish body initial weight (g)	Fish body end weight (g)	Rate of body weight gain %	Feed coefficient	Coefficient of condition %
						Control group	50.28±0.07	77.67±2.28	54.48±3.60a	2.15±0.18a	2.56±0.05a
Test group 1	50.10±0.25	81.48±3.15	62.63±4.25b	2.03±0.21b	2.48±0.11a
						Test group 2	50.08±0.15	82.38±3.07	64.49±4.19b	1.98±0.25b	2.53±0.13a
Test group 3	50.06±0.12	83.17±2.12	66.14±3.59b	1.86±0.21b	2.64±0.12b

Note：Have different Superscript letters in same row and represent significant difference (P<0.05).

From the point of view of rate of body weight gain, the rate of body weight gain of 3 test group of the present invention is all remarkably higher than comparison Group, wherein test group 1 are higher than control group 8.15%, and test group 2 is higher than control group 10.01%, Test group 3 is higher than control group 11.66%；Feed coefficient aspect, 3 test group are also significantly lower than Control group；From the point of view of coefficient of condition, test group 3 is significantly higher than control group.By above analysis knot Fruit understands, after adding probiotics, can significantly improve the growth of carp in expanded pellet diet Speed, reduces feed coefficient.

The above is only the preferred embodiment of the present invention it is noted that leading for this technology For the those of ordinary skill in domain, on the premise of without departing from the technology of the present invention principle, acceptable Make some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention.

Claims (9)

1. a kind of expanded carp feed is it is characterised in that contain the component of following weight portion： Fish meal 5-10 part, dehulled soybean meal 20-40 part, rapeseed meal 10-30 part, flour 15-20 part, rice Chaff 10-15 part, soya-bean oil 1-4 part, bentonite 1-3 part, calcium dihydrogen phosphate 1-3 part, chlorination Choline 0.1-0.5 part, B B-complex 0.1-0.5 part, composite trace element 0.1-0.5 part, powder Shape probiotics 0.1-0.5 part and liquid micro-ecological preparations 0.5-2 part.

2. as claimed in claim 1 expanded carp feed it is characterised in that containing as follows The component of weight portion：9 parts of fish meal, 30 parts of dehulled soybean meal, 20 parts of rapeseed meal, 18 parts of flour, 11 parts of rice bran, 2 parts of soya-bean oil, 2.1 parts of bentonite, 2 parts of calcium dihydrogen phosphate, Choline Chloride 0.3 part, 0.3 part of B B-complex, 0.3 part of composite trace element, powdery probiotics 0.1-0.2 part and liquid micro-ecological preparations 1-1.5 part.

3. as claimed in claim 1 or 2 expanded carp feed it is characterised in that described Powdery probiotics in viable count be 80-120 hundred million cfu/g.

4. as claimed in claim 3 expanded carp feed it is characterised in that described powder Shape probiotics contains bacillus subtilis (Bacillus subtilis), bacillus licheniformis (Bacillus licheniformis) and bacillus pumilus (Bacillus pumilus).

5. as claimed in claim 4 expanded carp feed it is characterised in that described is withered Careless bacillus is bacillus subtilis CGMCC No.9356, and described bacillus licheniformis is Bacillus licheniformis CGMCC No.7030, described bacillus pumilus is short and small gemma bar Bacterium CGMCC No.4756.

6. as claimed in claim 1 or 2 expanded carp feed it is characterised in that described Liquid micro-ecological preparations in viable count be 10-20 hundred million cfu/ml.

7. as claimed in claim 6 expanded carp feed it is characterised in that described liquid Body probiotics contains VREF (Enterococcus faecium), Yue Shi lactobacillus (Lactobacillus johnsonii).

8. as claimed in claim 7 expanded carp feed it is characterised in that described dung Enterococcus is VREF CGMCC No.9134, and described Yue Shi lactobacillus is Yue Shi breast bar Bacterium CGMCC No.4926.

9. the preparation method of the expanded carp feed described in any one of claim 1-8, its bag Include following steps：

1) weigh fish meal, dehulled soybean meal, cotton dregs, rapeseed meal and rice bran by weight, first carry out First pulverizing, puts into super after then mixing with other raw materials and powdery probiotics In trickle pulverizer, it is ground into 90% mash feed crossing 80 mesh, then carries out expanded granulation；

2) after expanded granulation, liquid micro-ecological preparations are added to by particle surface by rear spraying, Dry.