CN106929554A

CN106929554A - A kind of preparation method of globin peptide

Info

Publication number: CN106929554A
Application number: CN201511031024.4A
Authority: CN
Inventors: 成国祥; 江国永; 蒋兴龙; 吕小东
Original assignee: Huaian Genon Biological Products Co Ltd; Shanghai Genius Advanced Materials Group Co Ltd
Current assignee: SHANGHAI TRANSGENIC RESEARCH CENTER
Priority date: 2015-12-31
Filing date: 2015-12-31
Publication date: 2017-07-07

Abstract

The invention provides a kind of preparation method of globin peptide.The inventive method includes：A () provides a haemoglobin liquid I；B () pre-processes to the haemoglobin liquid I；C () carries out the first enzyme digestion reaction with alkali protease；D () pH is adjusted to pH5.5-8.5；E () carries out the second enzyme digestion reaction with neutral proteinase；F () separates and obtains globin peptide liquid VI；(g) is optionally dried, and obtains the globin peptide.The inventive method is adapted to large-scale production, is conducive to the recycling of blood resource, it is to avoid the wasting of resources, effectively reduces environmental pollution.Globin peptide of the invention has that safe, oligopeptides content is high, the low advantage of ash content, and can more effectively improve the immunity of the animals such as domestic animal.

Description

A kind of preparation method of globin peptide

Technical field

The invention belongs to bioengineering and agriculture field, in particular it relates to a kind of system of globin peptide Preparation Method.

Background technology

Modem animal nutrient theory is just theoretical to small peptide nutrient theory mistake from traditional protein, amino acid nutrient Cross, small peptide nutrient turns into after the another focus after proteinaceous nutrient.The research of recent domestic shows, machine Body is not limited to the form of free amino acid to protein utilization, and also quite a few is with 2-3 ammonia The dipeptides of base acid composition absorbs with tripeptides form.With deepening continuously for research, the small peptide with various functions Advantageously, it has been found that such as immune peptide, antibacterial peptide, anti-oxidation peptide, Hormone Peptide, small peptide nutrient is increasingly subject to The concern of people.Currently, peptide feedstuff additive is increasingly paid attention to by domestic and international feedstuff industry, it With improve immunity of organisms, reduce application of the antibiotic in feed, improve food conversion ratio it is strong with livestock and poultry Various works such as health situation, the normal development and growth that promote animal, the reproductive capacity and production performance for improving animal With.

China's livestock and poultry yield is at the forefront in the world, and livestock and poultry blood supply is up to 3,870,000 tons.But current pig blood adds Work amount only accounts for the 23% of total blood volume, and the domestic feeding livestock and poultry blood product mainstream product in the market is dry to spray Based on dry SDPP, spray drying globulin powder.Therefore, animal blood deep processing also has larger opening Hair space.

Currently, ferroheme is separated from hemoglobin preparing the method for the products such as hemoglobin mainly includes：It is molten Agent decoloring method, adsorbent decoloring method, oxidant decoloring method, acidifying oxidative decoloration method, enzymolysis absorption etc., but There is limitation mostly in industrialized production and practical application, such as technique is cumbersome, production cost is high, You Jirong Agent residual, protein recovery are low.

Therefore, this area is in the urgent need to developing a kind of globin peptide that is efficiently convenient, being applicable to large-scale production Preparation method, improves the comprehensive utilization ratio and surcharge of animal blood, reduces environmental pollution.

The content of the invention

The pearl egg obtained it is an object of the invention to provide a kind of preparation method of globin peptide and using the method The purposes of white peptide.

The first aspect of the present invention, there is provided a kind of method for preparing globin peptide, including step：

A () provides a haemoglobin liquid I；

B () pre-processes to the haemoglobin liquid I, the pretreatment is included in pH9-12 and/or temperature A period of time h is processed at 60-85 DEG C, so as to obtain pretreated haemoglobin liquid II；

C () carries out the first enzyme digestion reaction with alkali protease to the haemoglobin liquid II, so as to obtain hemoglobin Enzymolysis liquid III；

D () adjusts to pH5.5-8.5 (alkalescent, neutrality or faintly acid) pH of the haemoglobin liquid III, from And obtain hemoglobin enzymolysis liquid IV；

E () digests liquid IV and carries out the second enzyme digestion reaction with neutral proteinase to the hemoglobin, so as to obtain pearl Protein enzymatic hydrolyzate V；

F () isolates the scopes of W containing molecular weight between 100-2500 dalton from the globin enzymolysis liquid V The solution of peptide, that is, obtain globin peptide liquid VI；With

G () is optionally dried to the globin peptide liquid VI, obtain through dry globin peptide.

In another preference, described haemoglobin liquid I is prepared by the following method：

(a0) rupture of membranes treatment is carried out to separate blood cell, so as to obtain containing cell membrane and the blood cell inclusion being released The first mixture；

(a1) micro-filtration is carried out with aperture 0.01-0.50 microns of microfiltration membranes to first mixture, so as to be gone Except the filtrate of cell membrane, i.e. haemoglobin liquid I.

In another preference, the aperture of described microfiltration membranes is 0.05-0.45 microns, is more preferably 0.10-0.40 Micron.

In another preference, between the step (a) and (b) or step (a1) is and (b) between, methods described is also Including step：The dilution treatment of 1-10 times (preferably 2-5 times) is carried out with water to described haemoglobin liquid I, from And obtain diluted haemoglobin liquid I.

In another preference, in step (b), the pretreatment includes adjusting hemoglobin with pH adjusting agent I The pH to pH9-12 of liquid I, then heats to 60-80 DEG C, and keep a period of time h.

In another preference, in step (b), described a period of time h is 0.1-6 hours, preferably 0.2-3 Hour, more preferably it is 0.5-2 hours.

In another preference, in the step (b), the pH of the haemoglobin liquid I is adjusted with pH adjusting agent I It is worth, and described pH adjusting agent I is selected from the group：NaOH, KOH, sodium carbonate, potassium carbonate or its group Close.

In another preference, in the step (b), the pH scopes of pretreatment are 9.5-11, preferably 9.8-11.

In another preference, in the step (b), pretreatment temperature is 65-80 DEG C, preferably 70-75 DEG C.

In another preference, in the step (c), the hydrolysis temperature is 40-95 DEG C, preferably 50-80 DEG C, more preferably it is 65-85 DEG C.

In another preference, in the step (c), enzymolysis time is 1-30 hours, and preferably 5-25 is small When, more preferably it is 10-20 hours.

In another preference, in the step (c), (the i.e. protease of peptide matters in the haemoglobin liquid II Enzymolysis substrate) concentration (by dry weight) be 2-20%, preferably 4%-18%, be more preferably 6%-10%.

In another preference, described " peptide matters " refer to the peptide material being made up of n amino acid residue, Wherein n is >=2 positive integer, including oligopeptides, polypeptide, protein or its combination.

In another preference, in the step (c), the consumption of the alkali protease is in haemoglobin liquid II The 0.2%-5% of peptide matters gross weight (dry weight), preferably 0.5-4%, are more preferably 1-3%.

In another preference, in the step (d), adjust the haemoglobin liquid II's with pH adjusting agent II PH value, and described pH adjusting agent II is selected from the group：Citric acid, hydrochloric acid or its combination.

In another preference, in the step (d), the pH of the haemoglobin liquid III is adjusted to 6-8, preferably Ground is 7-7.5.

In another preference, in the step (e), the hydrolysis temperature is 35-65 DEG C, preferably 40-55 DEG C, more preferably it is 45-50 DEG C.

In another preference, in the step (e), enzymolysis time is 1-30 hours, and preferably 5-25 is small When, more preferably it is 10-20 hours.

In another preference, in the step (e), (the i.e. protease of peptide matters in the haemoglobin liquid IV Enzymolysis substrate) concentration (by dry weight) be 2-20%, preferably 4%-18%, be more preferably 6%-15%

In another preference, in the step (e), the consumption of the neutral proteinase is in haemoglobin liquid IV The 0.1%-5% of peptide matters gross weight (dry weight), preferably 1-4%.

In another preference, in step (f), the globin peptide liquid VI middle-molecular-weihydroxyethyl W scopes between Content >=90% (preferably >=92%) of the peptide of 100-2500 dalton, sample is accounted for by the peptide of 100-2500 dalton Total protein percentages.

In another preference, in the step (f), described separation includes being filtered with NF membrane.

In another preference, described " being filtered with NF membrane " includes being received with first that cutoff value is d1 Filter membrane carries out the first nanofiltration, and carries out the second nanofiltration with the second NF membrane that cutoff value is d2, and wherein d1 is 100-300 dalton, d2 is 1000-3000 dalton.

In another preference, in the step (g), also including being concentrated to the globin peptide liquid VI.

In another preference, the cycles of concentration of the concentration is 2-5 times.

In another preference, in the step (g), the drying means is selected from the group：Spray drying, freezing Dry or its combination.

In another preference, in step (f) or (g), the described dry pearl eggs of globin peptide liquid VI or described In white peptide, molecular weight 180-1000Da oligopeptides content more than 80%, by the globin peptide liquid VI or The total amount meter of peptide matters in the dry globin peptide.

In another preference, described blood cell source is selected from the group：Domestic animal, poultry or its combination；Preferably It is selected from the group：Pig, ox, sheep, chicken, duck and goose.

A kind of second aspect present invention, there is provided globin peptide product, the globin peptide product is with the present invention the Prepared by the method described on the one hand.

In another preference, in the globin peptide >=80wt% peptides (preferably >=85wt%, more preferably >= Molecular weight 90wt%) is 150-1000Da.

In another preference, in the globin peptide≤8wt% peptides (preferably≤7wt%, more preferably≤ 6wt%, most preferably≤5wt%) molecular weight is outside 150-2000Da scopes.

In another preference, in the globin peptide≤15wt% peptides (preferably≤14wt%, more preferably≤ 13wt%, most preferably≤10wt%) molecular weight is outside 150-1000Da scopes.

In another preference, the globin peptide is in 150-1000 dalton (more preferably 180-1000 dongles ) between peptide content >=80%, more preferably >=85%, by the total amount of peptide matters in the globin peptide product Meter.

In another preference, the peptide content between 150-500 dalton (more preferably 180-500 dalton) >=40%, more preferably 50-65%, preferably 55-60%, by the globin peptide product peptide matters it is total Gauge.

In another preference, ash content≤8wt%, the preferably≤6wt% of described globin peptide product, more Goodly≤5wt%, most preferably≤4.5wt%.

Third aspect present invention, there is provided a kind of fodder compound, the fodder compound contains second party of the present invention Globin peptide product described in face.

In another preference, the fodder compound contains the globin peptide product described in 0.05-20wt%, presses Fodder compound gross weight meter.

In another preference, the fodder compound contains 0.1-10wt%, preferably the pearl described in 0.2-5wt% Albumen peptide product, by fodder compound gross weight meter.

In another preference, described fodder compound contains acceptable on described globin peptide product and feed Other feedstuffs and/or auxiliary material.

In another preference, described feedstuff includes cereal, hay etc..

Fourth aspect present invention, there is provided a kind of method for preparing fodder compound, including step：

I () prepares described globin peptide liquid VI with the method described in first aspect present invention；Optionally to described Globin peptide liquid VI is dried, and obtains through dry globin peptide；

(ii) by described globin peptide liquid VI or described through on dry globin peptide and feed it is acceptable its Its feedstuff and/or auxiliary material are mixed, so as to obtain fodder compound.

Fifth aspect present invention, there is provided the purposes of a kind of globin peptide as described in the first aspect of the invention, as feeding Feed additives, animal health-care product additive, animal food additive.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and below (such as embodiment) Can be combined with each other between each technical characteristic of middle specific descriptions, so as to constitute new or preferred technical side Case.As space is limited, no longer tire out one by one herein and state.

Brief description of the drawings

Fig. 1 shows that the present invention prepares the schematic flow sheet of the method for globin peptide.

Specific embodiment

The present inventor opens first by in-depth study extensively, the optimization by a large amount of screenings and to technique Send out and a kind of prepared the globin peptide method that oligopeptides content is high, ash content is low on a large scale.It is prepared by the inventive method The oligopeptides of globin peptide middle-molecular-weihydroxyethyl≤1000 dalton account for the overwhelming majority, ash content is extremely low and safe, Therefore the function of immanoprotection action is produced with the preferably animal such as stimulation domestic animal.Additionally, present invention optimization work Skill combine micro-filter technology, pretreatment, sequencing enzymolysis and it is specific receive the technologies such as membrane filtration so that aobvious Write the comprehensive utilization ratio for improving animal blood resource, it is to avoid the wasting of resources, effectively reduce environmental pollution.This Outward, the inventive method also has the advantages that convenient and swift, with low cost.The present invention is completed on this basis.

Term explanation

Unless otherwise defined, whole technologies otherwise used herein are respectively provided with such as institute of the present invention with scientific terminology The identical meanings that the those of ordinary skill in category field is generally understood that.

As used herein, term " blood cell " and " haemocyte " are used interchangeably, and refer to the red blood cell in blood Cell.

As used herein, when being used in mentioning the numerical value specifically enumerated, term " about " means that the value can be from The value enumerated changes and is not more than 1%.For example, as used herein, statement " about 100 " includes 99 and 101 Hes Between whole values (for example, 99.1,99.2,99.3,99.4 etc.).

As used herein, term " containing " or " including (including) " can be it is open, semi-enclosed and It is enclosed.In other words, the term also include " substantially by ... constitute " or " by ... constitute ".

Globin peptide

The invention provides a kind of globin peptide product, in the globin peptide >=80wt% peptides (preferably >= 85wt%, more preferably >=90wt%) molecular weight be 150-1000Da.

In another preference, the globin peptide has following functional characteristic：

(1) biological safety is high；

(2) oligopeptides content is high；

(3) ash content is low.

Blood cell raw material

Blood cell of the present invention derives from animal, including domestic animals and birds etc..From the blood of animal blood Ball can be used as the raw material that globin peptide is produced in the present invention.Described domestic animals and birds are such as (but not limited to)： Pig, ox, sheep, chicken, duck etc..It is preferred that using the blood cell of fresh and healthy as the original for producing globin peptide Material.

It is technology well known to those skilled in the art that blood cell is isolated from blood, and the present invention does not make especially to this Limitation.By blood anticoagulant, separate, can obtain blood plasma and blood cell, will be rear as life after the treatment of blood cell rupture of membranes Produce the raw material of globin peptide.

Preparation method

The method that the present invention prepares globin peptide, including step：

A () provides a haemoglobin liquid I；

Detection method

For globin peptide product of the invention or the other products or sample of preparation process, conventional method can be used Determine the content and molecular weight distribution of its peptide matters.

For small peptide content detection, typically pearl is determined using conventional method (including but not limited to TCA factures) Peptide content in protein peptides or other samples.Typically, protein precipitation is made first with trichloroacetic acid (TCA) Agent, by the peptide precipitation more long of the protein and peptide chain in sample protein, and uses acid molten short chain small peptide therein Solution out, by filtering, centrifugation, digestion, distillation, determines its protein content, and it is thick to account for sample with it The percentage of protein represents content.

Determined for total protein content, representational assay method includes (but being not limited to)：Kjeldahl's method.

For globin peptide or the molecular weight distribution of other samples, representational assay method includes (but not limiting In)：Gel filtration chromatography.

If determined using high performance gel filtration chromatography, typically with porous filler as fixing phase, according to sample The difference of product component molecular volume size is separated, under the conditions of 220 nanometers of the UV absorption wavelength of peptide bond Detection, the exclusive data for determining molecular weight distribution using gel chromatography processes software (i.e. GPC softwares), checks colors Spectrogram and its data are processed, and are calculated relative molecular weight size and the distribution (reference of globin peptide High performance gel filtration chromatography is utilized specified in standard GB/T/T 22729-2008 oceans fish oligopeptide powder Determine peptide molecular weight distribution).

Globin peptide purposes

Globin peptide of the invention can be used as feedstuff, animal health-care product raw material and carrier.

Main advantages of the present invention are：

(1) oligopeptides of globin peptide middle-molecular-weihydroxyethyl≤1000 dalton prepared by the inventive method accounts for portion big absolutely Point, ash content is extremely low and safe, therefore produces immanoprotection action with the preferably animal such as stimulation domestic animal Function.

(2) globin peptide product of the invention has highly-water-soluble, high fluidity, can be used for many animals and exists In the feed of different developmental phases, it is also possible to make animal health product raw material and carrier.

(3) optimize technique of the invention combine micro-filter technology, pretreatment, sequencing enzymolysis and it is specific Receive the technologies such as membrane filtration, so as to significantly improve the comprehensive utilization ratio of animal blood resource, it is to avoid the wasting of resources, Effectively reduce environmental pollution.

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are only used for The bright present invention rather than limitation the scope of the present invention.The experiment side of unreceipted actual conditions in the following example Method, generally according to normal condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise Percentage and number are percentage by weight and parts by weight.

The unit in quality percent by volume in the present invention be it is well-known to those skilled in the art, e.g. Refer to the quality of the solute in 100 milliliters of solution.

Experiment material used and reagent can be obtained from commercially available channel unless otherwise instructed in following examples.

Universal method

1. the crude protein content that Kjeldahl's method is detected

Detecting step：Sample 6g accurately is weighed, it is accurate to add 15% trichloroacetic acid 50mL, it is well mixed, Stand 5min.Filtered with middling speed qualitative filter paper, discard a little initial filtrate, filtrate is transferred to centrifuge tube, 4000 revs/min of centrifugation 10min.The accurate centrifugation supernatant (suggestion 2mL or 4mL) for pipetting certain volume V, Carry out crude protein measure.Determination sample total crude protein.

Computational methods and repeatability：Small peptide %=is centrifuged the supernatant crude protein content %*50/V/ thick eggs of sample total protein Permission relative deviation is the 10% of its arithmetic mean of instantaneous value between Bai Hanliang * 100%, two Duplicate Samples of each sample, With its formula average value as reported result.Acquired results refer to the percentage composition that small peptide accounts for crude protein.

2.TCA factures determine peptide content (as determined the peptide content in globin peptide)

Make protein precipitant using trichloroacetic acid (TCA), the protein and peptide chain in sample protein is more long Peptide precipitation, and short chain small peptide therein is dissolved out with acid, by filtering, centrifugation, digestion, distillation, Determine its protein content, and content is represented with percentage that it accounts for sample thick protein.

The reagent and instrument for using：100mL beakers；50mL, 10mL pipette；Filter；Survey Determine crude protein equipment, 15% trichloroacetic acid, 4000 revs/min of centrifuge.

3. peptide molecular weight measure of spread

Specify with reference in standard GB/T/T 22729-2008 oceans fish oligopeptide powder, using efficient gel mistake The peptide molecular weight distribution of filter chromatography determination.

Embodiment 1

Prepare globin peptide No.1

Preparation method is comprised the following steps：

(a0) rupture of membranes treatment：(separated with blood plasma) for fresh blood cell, processed using conventional rupture of membranes, So as to discharge hemoglobin from blood cell, (fresh blood cell has complete eucaryotic cell structure because of it, makes hemoglobin It is wrapped in the cell membrane with lipid as Main Ingredients and Appearance, therefore rupture of membranes treatment is favorably improved globin peptide most Whole yield)；

(a1) demoulding：The solution of the hemoglobin containing the release after rupture of membranes is processed as raw material, through 0.3 Micron membrane filter micro-filtration, filters off cell membrane, obtains the haemoglobin liquid I of removal membrane component；

B () pre-processes：PH adjusting agent NaOH, the pH to about 10 of regulation haemoglobin liquid I are used, is heated up To about 75 DEG C, 1 hour is incubated, (after measured, peptide matters contain to obtain pretreated haemoglobin liquid II It is wt% to measure.)；

C () first digests：Alkali protease (addition 2% (enzyme/substrate protein mass percent)) is added, to institute Stating haemoglobin liquid II carries out the first enzyme digestion reaction (58 DEG C of temperature, 6 hours time), so as to obtain hemoglobin Enzymolysis liquid III；

D () adjusts pH value to alkalescent, neutral or faintly acid：PH adjusting agent citric acid is used, will be described blood red The pH regulation pH to about 7 of protein liquid III, so as to obtain hemoglobin enzymolysis liquid IV (after measured, peptide matters Content be wt%.)；

E () second digests：With neutral proteinase (addition 2% (enzyme/substrate protein mass percent)), to described Hemoglobin enzymolysis liquid IV carries out the second enzyme digestion reaction (temperature 45 C, enzymolysis time 8 hours), so as to obtain pearl Protein enzymatic hydrolyzate V；

F () separates globin peptide：Nanofiltration is combined using the NF membrane (1kd and 180 dalton) of different cutoff values, Nanofiltration is carried out to the globin enzymolysis liquid V, so as to be isolated containing molecular weight from the globin enzymolysis liquid V W scopes are the solution of the peptide of 180-1000 dalton, as obtain globin peptide liquid VI；With

G () dries：The globin peptide liquid VI is spray-dried, so as to obtain through dry globin peptide.

Embodiment 2

Prepare globin peptide No.2

Embodiment 1 is repeated, difference is, in step (a1), 0.3 micron is replaced with 0.45 micron of micro-filtration Filter membrane micro-filtration.

Comparative example 1

Prepare globin peptide No.C1

Repeat embodiment 1, difference is, in step (a1), with 4000 turns/min be centrifuged 15 minutes from And remove cell membrane and other impurity.(replacing " 0.3 micron membrane filter micro-filtration ")

The globin peptide product prepared to embodiment 1-2 and comparative example 1, carries out thick egg in globin peptide product The assay of white and peptide, is as a result listed in the table below A：

Table A

Embodiment/comparative example	Production code member	Treatment conditions	Crude protein	Peptide content
					Embodiment 1	No.1	0.3 micron of micro-filtration	91.2%	82.1%
Embodiment 2	No.2	0.45 micron of micro-filtration	90.9%	81.3%
					Comparative example 1	No.C1	4000 turns/min is centrifuged	85.3%	73.9%

Result shows, although micro-filtration and centrifugation all can effectively remove film component, but micro-filtration can be reduced and separated The loss of protein substance in journey, protein content, peptide content are higher in the globin peptide of preparation.

Comparative example 2

Prepare globin peptide No.C2

Embodiment 1 is repeated, difference is only to omit step (d).

Comparative example 3

Prepare globin peptide No.C3

Embodiment 1 is repeated, difference is only to omit step (c).

Comparative example 4

Prepare globin peptide No.C4

Embodiment 1 is repeated, and difference is to omit step (c) and pH is adjusted to 3 in step (d), And (consumption is 2% (enzyme/substrate protein mass percent), carries out second enzyme in the middle acid protease of step (e) Solution (temperature, time are identical).

Comparative example 5

Prepare globin peptide No.C5

Embodiment 1 is repeated, and difference is to omit step (c) and pH is adjusted to 7 in step (d), And in the middle papain of step (e) (consumption is 2% (enzyme/substrate protein mass percent)), carry out second enzyme Solution (temperature, time are identical).

Embodiment 3

Prepare globin peptide No.3

Embodiment 1 is repeated, and difference is that pH is adjusted to 3, and in step (e) in step (d) With acid protease (consumption be 2% (enzyme/substrate protein mass percent)), carry out the second enzymolysis (temperature, when Between it is identical).

Embodiment 4

Prepare globin peptide No.4

Embodiment 1 is repeated, and difference is that pH is adjusted to 7, and in step (e) in step (d) With papain (consumption be 2% (enzyme/substrate protein mass percent)), carry out the second enzymolysis (temperature, when Between it is identical).

The globin peptide product prepared to above-described embodiment 1 and 3-4 and comparative example 2-5, carries out peptide content survey It is fixed, as a result it is listed in the table below B：

Table B

Embodiment/comparative example	Production code member	Enzyme class	Peptide content
				Comparative example 2	C2	Alkali protease	61.1%
Comparative example 3	C3	Neutral proteinase	61.3%
				Comparative example 4	C4	Acid protease	56.8%
Comparative example 5	C5	Papain	69.5%
				Embodiment 1	No.1	Alkali protease+neutral proteinase	82.1%
Embodiment 3	No.3	Alkali protease+acid protease	71.3%
				Embodiment 4	No.4	Alkali protease+papain	73.2%

Table B results show, using the extraction of alkali protease+neutral proteinase sequencing enzymolysis (secondary enzymolysis) Efficiency is greatly improved, and reaches more than 80%.

Comparative example 6

Prepare globin peptide No.C6

Embodiment 1 is repeated, difference is to omit step (b).

Result shows, compared with Example 1, omits pre-treatment step, although help to save certain hour, But the viscosity of hemoglobin solutions is higher, and liquid fluidity is poor, is unfavorable for subsequent operation.

The globin peptide product prepared to above-described embodiment 1 and comparative example 6, carries out peptide content measure, as a result It is listed in the table below：

Table C

Embodiment/comparative example	Production code member	Peptide content
			Embodiment 1	No.1	82.1%
Comparative example 6	C6	50.9%

This shows that it (is probably because contributing to that pretreatment not only facilitates the oligopeptides content improved in final products So that enzymolysis is more abundant), and mobility through above-mentioned pretreated hemoglobin solutions has unexpected Significantly improve, digested beneficial to down-streamization and operated.

Embodiment 5-10

Prepare globin peptide No.5 to No.10

Embodiment 1 is repeated, is technology stability experiment.The measurement result of gained globin peptide product such as table D It is shown.

Table D sequencing hydrolysis results

Result shows that preparation method technology stability of the invention is good, is suitable to commercial Application.

Embodiment 11-14

Prepare globin peptide No.11 to No.14

Embodiment 1 is repeated, difference is globin peptide to be separated, using the different cutoff values shown in table E NF membrane, carry out independent nanofiltration or combination nanofiltration.

Result is as shown in table E.

Table E enzymolysis liquid membrane filtrations

Embodiment	No.	NF membrane species	Peptide content	Ash content
					11	No.11	2K molecular films	81.20%	10.10%
12	No.12	1K molecular films	82.10%	10.20%
					13	No.13	180 molecular films	83.10%	4.30%
14	No.14	180-1K is combined	84.70%	4.10%

Small peptide %=centrifugation supernatant crude protein content %*50/V/ sample total protein crude protein contents * 100%, each Permission relative deviation is the 10% of its arithmetic mean of instantaneous value between two Duplicate Samples of sample, is with its formula average value Reported result.Acquired results refer to the percentage composition that small peptide accounts for crude protein.

In above-described embodiment 11-14, membrane filtration isolation technics is employed, molecule is necessarily blocked by selection The filter membrane of amount is processed enzymolysis liquid, is obtained target peptide solution and is spray-dried.Result shows, cuts When value (cut-off) of breaking is excessive, ash content is higher in causing product, and cutoff value (cut-off) is too small to cause product Middle high molecular weight protein increases.

Result shows, nanofiltration is combined using such as 180Da and 1000Da, not only can effectively reduce product Product ash content, also can further improve oligopeptides content.

Embodiment 15：Pilot plant test

In the present embodiment, blood cell pre-treatment, sequencing enzymolysis and combination of membrane filtration carried out integrated and carried out Have a try and test, globin peptide is obtained eventually through spray drying, pilot scale parameter is as shown in table F.

The blood cell material quantity of each pilot scale treatment is 2 tons.

Table F pilot scale parameter lists

Scheme	Sequencing is digested
		Concentration of substrate	10%
Enzyme concentration	2%
		First step pH	10
The first step time	6
		Second step pH	7.5
The second step time	8
		Acid adjustment is filtered	Hydrochloric acid acid adjustment, filtering
Separate	180-1K molecular weight combination of membrane filtration
		Dry	Spray drying

The molecular weight distribution of product is tested using exclusion chromatography, finds the peptide overwhelming majority in product 2 to 8 amino acid molecular amount levels are concentrated on, therefore, prepared using sequencing zymolysis technique of the invention Product in the peptide overwhelming majority be oligopeptides, globin peptide molecular weight distribution tests result is listed in table G.

Table G globin Gly-His-Lys molecular weight distribution is tested

Embodiment 16：Influence of the globin peptide to hog cholera antibody potency

Select " long × big " binary castration boar 40 that 35 ages in days, body weight parity are close.

Basal diet is produced by Shanghai Gao get feed corporation,Ltds, and trophic level is：Metabolizable energy 13.6MJ/kg, slightly Protein 19 .3%, calcium 0.7%, phosphorus 0.6%, lysine 1.2%.

0.5% globin peptide is added based on test group daily ration in daily ration, by the outstanding grand limited public affairs of biological products in Shanghai Department provides.

Experiment is randomly divided into 2 groups with piglet by single factor test completely randomized design：

Control group (n=20)：Basal diet

Test group (n=20)：Basal diet+0.5wt% globins peptide (such as prepared by embodiment 15),

Every group of 2 repetitions, each repeats 10 piglets, 30 days experimental periods.

Experimental animal is used uniformly across high bed flat foster Double row type weaning drylot feeding and supports, respectively feeding 1 time of daily morning, noon and afternoon, Free choice feeding and drinking-water, once, prevention from suffering from the diseases and feeding and management are by the conventional journey of pig factory for cleaning swinery daily Sequence is carried out.Antibody titer after the vaccine inoculation of tracing detection experiment pig.Using hog cholera antibody detection kit (Classical Swine Fever Virus Antibody Test Kit (CSFV Ab)) is detected,

Assay method mechanism

Antibody against swine fever virus ELISA detection kit is that CSFV resists in detecting Swine serum or blood plasma The detection kit of body.The kit is with the coated micro-reaction plate of CSFV antigen, using blocking ELISA principles are come antibody against swine fever virus in detecting Swine serum or blood plasma.If there is hog cholera in test sample Malicious antibody, they will block peppery

The monoclonal antibody of the swine fever virus resistant of root peroxidase labelling (HRPO).Monoclonal antibody and swine fever The combination of viral antigen can be judged by horseradish peroxidase with the colour developing degree of substrate, that is, use enzyme Mark instrument determines the absorbance of the reaction system in Single wavelength 450nm or dual wavelength 450nm and 620nm.When In test sample contain antibody against swine fever virus (positive findings) when, colour developing will shoal, when in test sample not During containing swine fever virus resistant antibody (negative findings), colour developing will be deepened.The blocking rate of sample can pass through 450nm wavelength sample absorbances determine with the ratio of negative control absorbance.

Reagent prepares

Coated micro-reaction plate, sample diluting liquid, positive control, negative control, ELIAS secondary antibody, substrate Solution and terminate liquid are using preceding without treatment.

The cleaning solution of cleaning solution (10 ×) concentration must carry out 10 times of dilutions before with ultra-pure water.It is diluted Cleaning solution can be preserved 3 days under conditions of 4 DEG C, or preserved 1 year under freezing conditions.Such as：250ml Cleaning solution need to be sufficiently mixed to be made into the ultra-pure water of the concentrated cleaning solution of 25ml and 225ml and form.Note： If it must be melted before the use containing crystallization in concentrate, should be by the liquid temperature water-bath 30 minutes More than.

Preparation of samples

Fresh, refrigerating (4 DEG C are less than 8 days), the serum of frost or blood plasma may be used to detection.

Operating procedure

1. when in use, all of reagent constituents must all return to 18-25 DEG C of room temperature.Should be by before Each component is positioned over room temperature at least one hour.

2. respectively by 50 μ l sample diluting liquids add each detection hole and control wells in.

3. in the positive control of 50 μ l and the corresponding control wells of negative control addition, will note different right respectively According to suction nozzle to change, with anti-pollution.

4. notice that the suction nozzle of different samples will by the remaining detection hole of test sample addition of 50 μ l respectively Separate, with anti-pollution.

5. flick micro-reaction plate or vibrated with oscillator, the solution in reaction plate is mixed.

6. by micro-reaction plate with strip of paper used for sealing close or in wet tank (18-25 DEG C) be incubated 2 hours, it is also possible to will Micro-reaction plate is closed or the overnight incubation in wet tank with strip of paper used for sealing.

7. the liquid in reacting hole is suctioned out, and is washed 3 times with the cleaning solution for having diluted, note washing every time When cleaning solution will be filled it up with reacting hole, discard the cleaning solution in reacting hole and pat dry reaction plate.Can also use Board-washing machine washing is washed 3 times, and each reacting hole should add the cleaning solution of 300 μ l or so.Note being careful during board-washing, In order to avoid the cross pollution between sample.

8. strip of paper used for sealing is used by anti-CSFV ELIAS secondary antibodies (take and use) the addition reacting hole of 100 μ l respectively Capping plate is simultaneously incubated 30 minutes at room temperature or in wet tank.

9. after board-washing (see 6), during the substrate solution of 100 μ l added into reacting hole respectively, and in lucifuge, room Placed 10 minutes under the conditions of temperature.Add behind the first hole i.e. Timing.

10. the terminate liquid terminating reaction of 100 μ l is added in each reacting hole.Note will by plus ELIAS secondary antibody Order add terminate liquid.

11. absorbances that sample and control are determined at 450nm, it is also possible to dual wavelength (450nm and 620nm) determine the absorbance of sample and control, air zeroing.

12. mean absorbance values for calculating sample and control (see calculating).

Computational methods

Calculate the average value OD of tested sample₄₅₀(=OD_TEST), the average value (=OD of positive control_POS)、 The average value (=OD of negative control_NEG)。

The blocking rate of tested sample and positive control is calculated according to below equation；(positive control blocking rate is with same Method is calculated)

Blocking rate=(OD_NEG-OD_TEST)/OD_NEG× 100%

Test validity

The average OD of negative control₄₅₀Should be greater than 0.50.The blocking rate of positive control should be greater than 50%.

Result judgement

If the blocking rate of tested sample is more than or equal to 40%, the sample can just be judged to the positive (to be had CSFV antibody presence).If the blocking rate of tested sample is less than or equal to 30%, the sample can just be judged to It is negative (presence of nonreactive CSFV antibody).If the blocking rate of tested sample is between 30-40%, just should be The animal is resurveyed again after a few days, testing result is shown in Table H.

Table H result judgements

Note：A-1 to A-20 is 20 numberings of pig of control group.

B-1 to B-20 is 20 numberings of pig of test group.

Antibody titer tracking after to the vaccine inoculation of test group pig, the antibody of globin peptide group is than control Group at least 2 titres scoring high.

The all documents referred in the present invention are all incorporated as reference in this application, just as each document It is individually recited as with reference to such.In addition, it is to be understood that after above-mentioned instruction content of the invention has been read, Those skilled in the art can make various changes or modifications to the present invention, and these equivalent form of values equally fall within this Shen Please appended claims limited range.

Claims

1. a kind of method for preparing globin peptide, including step：

A () provides a haemoglobin liquid I；

2. the method for claim 1, it is characterised in that in step (b), the pretreatment includes use The pH of pH adjusting agent I regulation haemoglobin liquids I then heats to 60-80 DEG C, and kept for one section to pH9-12 Time h.

3. the method for claim 1, it is characterised in that in the step (c), the hydrolysis temperature is 40-95 DEG C, preferably 50-80 DEG C, be more preferably 65-85 DEG C.

4. the method for claim 1, it is characterised in that in the step (d), by the hemoglobin The pH of liquid III is adjusted to 6-8, preferably 7-7.5.

5. the method for claim 1, it is characterised in that in the step (f), the separation includes use NF membrane is filtered.

6. the method for claim 1, it is characterised in that in step (f) or (g), described globin In the dry globin peptides of peptide liquid VI or described, molecular weight 180-1000Da oligopeptides content more than 80%, Based on the total amount of peptide matters in the dry globin peptides of the globin peptide liquid VI or described.

7. a kind of globin peptide product, it is characterised in that the globin peptide product is with claim 1 institute Prepared by the method stated.

8. a kind of fodder compound, it is characterised in that the fodder compound contains the pearl described in claim 2 Albumen peptide product.

9. a kind of method for preparing fodder compound, it is characterised in that including step：

I () prepares described globin peptide liquid VI with the method described in claim 1；Optionally to the pearl egg White peptide liquid VI is dried, and obtains through dry globin peptide；

10. the purposes of globin peptide as claimed in claim 1, it is characterised in that as feed addictive, dynamic Thing products and health products additive agent, animal food additive.