CN108060196B - A kind of Extracted From Oil-tea-cake polypeptide, preparation method and the effect for promoting weary feelings estrus of cow and fertilization - Google Patents
A kind of Extracted From Oil-tea-cake polypeptide, preparation method and the effect for promoting weary feelings estrus of cow and fertilization Download PDFInfo
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 70
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 60
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 60
- 230000012173 estrus Effects 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 230000004720 fertilization Effects 0.000 title claims abstract description 13
- 230000000694 effects Effects 0.000 title abstract description 13
- 230000001737 promoting effect Effects 0.000 title abstract description 4
- 235000007164 Oryza sativa Nutrition 0.000 claims abstract description 51
- 235000009566 rice Nutrition 0.000 claims abstract description 51
- 241001596328 Camellia meiocarpa Species 0.000 claims abstract description 32
- 230000007071 enzymatic hydrolysis Effects 0.000 claims abstract description 30
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims abstract description 30
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 25
- 230000007062 hydrolysis Effects 0.000 claims abstract description 19
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 19
- 241000209094 Oryza Species 0.000 claims description 49
- 239000000843 powder Substances 0.000 claims description 45
- 238000000034 method Methods 0.000 claims description 22
- 239000012043 crude product Substances 0.000 claims description 18
- 239000012528 membrane Substances 0.000 claims description 18
- 239000006228 supernatant Substances 0.000 claims description 18
- 239000000725 suspension Substances 0.000 claims description 18
- 108091005804 Peptidases Proteins 0.000 claims description 12
- 239000004365 Protease Substances 0.000 claims description 12
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 12
- 239000003513 alkali Substances 0.000 claims description 12
- 230000008569 process Effects 0.000 claims description 12
- 102000007079 Peptide Fragments Human genes 0.000 claims description 11
- 108010033276 Peptide Fragments Proteins 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 10
- 239000012498 ultrapure water Substances 0.000 claims description 10
- 229920000742 Cotton Polymers 0.000 claims description 9
- 239000000706 filtrate Substances 0.000 claims description 9
- 238000010438 heat treatment Methods 0.000 claims description 7
- 238000012545 processing Methods 0.000 claims description 7
- 239000012467 final product Substances 0.000 claims description 6
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 claims description 5
- 239000004519 grease Substances 0.000 claims description 5
- 235000013399 edible fruits Nutrition 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 2
- 229940079593 drug Drugs 0.000 claims description 2
- 230000014759 maintenance of location Effects 0.000 claims 1
- 102000004142 Trypsin Human genes 0.000 abstract description 9
- 108090000631 Trypsin Proteins 0.000 abstract description 9
- 238000004108 freeze drying Methods 0.000 abstract description 9
- 239000012588 trypsin Substances 0.000 abstract description 9
- 238000007710 freezing Methods 0.000 abstract description 8
- 230000008014 freezing Effects 0.000 abstract description 8
- 230000003647 oxidation Effects 0.000 abstract description 6
- 238000007254 oxidation reaction Methods 0.000 abstract description 6
- 230000004792 oxidative damage Effects 0.000 abstract description 4
- 239000003223 protective agent Substances 0.000 abstract description 3
- 230000017854 proteolysis Effects 0.000 abstract description 3
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 2
- 230000009467 reduction Effects 0.000 abstract description 2
- 240000007594 Oryza sativa Species 0.000 abstract 2
- 241000283690 Bos taurus Species 0.000 description 27
- 210000000582 semen Anatomy 0.000 description 18
- 210000001215 vagina Anatomy 0.000 description 13
- 244000309464 bull Species 0.000 description 12
- 238000010790 dilution Methods 0.000 description 8
- 239000012895 dilution Substances 0.000 description 8
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 6
- 241001122767 Theaceae Species 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 210000005239 tubule Anatomy 0.000 description 4
- 241000209507 Camellia Species 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 235000018597 common camellia Nutrition 0.000 description 3
- 238000005138 cryopreservation Methods 0.000 description 3
- 238000001938 differential scanning calorimetry curve Methods 0.000 description 3
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- 239000002994 raw material Substances 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- 241000283725 Bos Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
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- 238000007689 inspection Methods 0.000 description 2
- 235000015110 jellies Nutrition 0.000 description 2
- 239000008274 jelly Substances 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
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- 241000894007 species Species 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 241000526900 Camellia oleifera Species 0.000 description 1
- 240000001624 Espostoa lanata Species 0.000 description 1
- 235000009161 Espostoa lanata Nutrition 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000005662 Paraffin oil Substances 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000002361 compost Substances 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 210000003953 foreskin Anatomy 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
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- 150000002632 lipids Chemical class 0.000 description 1
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- 210000003899 penis Anatomy 0.000 description 1
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- 238000003860 storage Methods 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Engineering & Computer Science (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
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- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a kind of Extracted From Oil-tea-cake polypeptide, preparation method and the effects for promoting weary feelings estrus of cow and fertilization.The hydrolyzed peptide of Extracted From Oil-tea-cake of the present invention has various active, and the activity of hydrolyzed peptide is influenced by proteolysis sites in Extracted From Oil-tea-cake：Through trypsin hydrolysis generate molecular weight be (5000u, 3000u] polypeptide have the function of improve ox freeze sperm oxidation resistance, can be used as ox sperm freezing protective agent reduction oxidative damage；Through hydrolysis by novo generate molecular weight be (5000u, 3000u] polypeptide can promote weary feelings estrus of cow and fertilization.Moreover, the preparation method of hydrolyzed peptide of the present invention is simple, simple crushing, enzymatic hydrolysis, ultrafiltration, freeze-drying is used only, these steps are all that can realize large-scale production in the factory, easy to implement.The present invention realizes the refuse reclamation of the camellia meiocarpa dregs of rice, and the economic value added of the camellia meiocarpa dregs of rice can be improved.
Description
Technical field
The invention belongs to herding field, it is related to Extracted From Oil-tea-cake polypeptide, preparation method and promotes weary feelings estrus of cow and fertilization
Effect.
Background technique
Camellia meiocarpa (Camellia meiocarpa Hu.) is that cultivated area and annual output are only second in China Camellia
The main cultivation species of C. olelfera (Camellia oleiferaAbel.), be distributed mainly on China Huaihe River, the Fujian on the south the Changjiang river,
The ground such as Jiangxi, Guangxi, Hunan, Guizhou.Camellia meiocarpa also known as Jiangxi, small tea, heart, Camellia yuansienensis, sheep dung etc..Fruit
It is small, leaf is small, bud is small, it is its notable feature that bud, bract, which do not have hair,.
The camellia meiocarpa dregs of rice are that camellia meiocarpa extracts the byproduct obtained after grease.Although existing tea plantation is oily by these at present
Be used as animal feed after tea seed cake detoxification, still, since technique is immature, most Extracted From Oil-tea-cakes be still when waste material is thrown away, or
It is used as the fertilizer on tea plantation after person's compost, availability is low, and added value is low.In order to improve the availability of dell Extracted From Oil-tea-cake, its warp is improved
Ji added value, applicant have carried out Depth Study and exploitation to tea seed cake.
Currently, there is not yet the report of the hydrolyzed peptide deep development of the camellia meiocarpa dregs of rice.
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of Extracted From Oil-tea-cake polypeptide, preparation method and promotions
The effect of weary feelings estrus of cow and fertilization.
Above-mentioned purpose of the invention is achieved by following technical solution：
Trypsin hydrolysis prepares ox semen cryopreservation additive：
A kind of Extracted From Oil-tea-cake polypeptide after trypsin hydrolysis, collects molecular weight and exists using the camellia meiocarpa dregs of rice as raw material
(5000u, 3000u] peptide fragment in range, it is freeze-dried to obtain the final product.
Preferably, the camellia meiocarpa dregs of rice refer to the fruit of camellia meiocarpa by squeezing processing or lixiviation process processing removal grease
The solid residue obtained afterwards.
The preparation method of above-mentioned Extracted From Oil-tea-cake polypeptide, includes the following steps：
The camellia meiocarpa dregs of rice are crushed to 80-100 mesh, soaked in water by step S1, are eventually fabricated dregs of rice powder suspension；
Step S2, is added trypsin digestion, and enzymatic hydrolysis condition is：PH value, 7.8-8.2；Hydrolysis temperature, 43-47 DEG C；Enzymatic hydrolysis
Time, 10-12 hours；After enzymatic hydrolysis, heating inactivates trypsase, is cooled to room temperature, and standing takes supernatant；
Step S3 filters supernatant with absorbent cotton, collects filtrate, crude product freeze-dried powder is lyophilized to obtain；
Step S4, crude product freeze-dried powder are dissolved with ultrapure water, are separated with the ultrafiltration membrane of different molecular weight cut off, are collected and are divided
Son amount (5000u, 3000u] peptide fragment in range, be freeze-dried to obtain the final product.
Preferably, it is respectively the super of 5000u, 3000u that the ultrafiltration membrane of the different molecular weight cut off, which includes molecular cut off,
Filter membrane.
It preferably, further include the ultrafiltration membrane of molecular cut off 10000u.
Preferably, the method that heating inactivates trypsase：90 DEG C high-temperature process 10 minutes.
Preferably, the preferred 130-170g/L of dregs of rice powder suspension.
Preferably, the addition weight of the trypsase is calculated as 2.5-3.5g/100g with dregs of rice powder weight.
Preferably, the enzymatic hydrolysis condition preferable ph 8.0 of the trypsase, temperature 45 C, the time 11 hours.
Application of the above-mentioned Extracted From Oil-tea-cake polypeptide in terms of ox semen cryopreservation.
Hydrolysis by novo preparation promotes the polypeptide of weary feelings estrus of cow and fertilization：
A kind of Extracted From Oil-tea-cake polypeptide after hydrolysis by novo, collects molecular weight and exists using the camellia meiocarpa dregs of rice as raw material
(5000u, 3000u] peptide fragment in range, it is freeze-dried to obtain the final product.
Preferably, the camellia meiocarpa dregs of rice refer to the fruit of camellia meiocarpa by squeezing processing or lixiviation process processing removal grease
The solid residue obtained afterwards.
The preparation method of above-mentioned Extracted From Oil-tea-cake polypeptide, includes the following steps：
The camellia meiocarpa dregs of rice are crushed to 80-100 mesh, soaked in water by step S1, are eventually fabricated dregs of rice powder suspension；
Step S2, is added alkali protease enzymatic hydrolysis, and enzymatic hydrolysis condition is：PH value, 8.3-8.7；Hydrolysis temperature, 53-57 DEG C；Enzyme
The solution time, 9-11 hours；After enzymatic hydrolysis, heating makes basic protein enzyme-deactivating, is cooled to room temperature, and standing takes supernatant；
Step S3 filters supernatant with absorbent cotton, collects filtrate, crude product freeze-dried powder is lyophilized to obtain；
Step S4, crude product freeze-dried powder are dissolved with ultrapure water, are separated with the ultrafiltration membrane of different molecular weight cut off, are collected and are divided
Son amount (5000u, 3000u] peptide fragment in range, be freeze-dried to obtain the final product.
Preferably, it is respectively the super of 5000u, 3000u that the ultrafiltration membrane of the different molecular weight cut off, which includes molecular cut off,
Filter membrane.
It preferably, further include the ultrafiltration membrane of molecular cut off 10000u.
Preferably, heating makes the method for basic protein enzyme-deactivating：90 DEG C high-temperature process 10 minutes.
Preferably, the preferred 130-170g/L of dregs of rice powder suspension.
Preferably, the addition weight of the alkali protease is calculated as 2.5-3.5g/100g with dregs of rice powder weight.
Preferably, 55 DEG C of the enzymatic hydrolysis condition preferable ph 8.5, temperature of the alkali protease, 10 hours time.
Application of the above-mentioned Extracted From Oil-tea-cake polypeptide in terms of the feed or drug that preparation promotes weary feelings estrus of cow and fertilization.
Advantages of the present invention：
1, it is a discovery of the invention that the hydrolyzed peptide of Extracted From Oil-tea-cake has various active, and the activity of hydrolyzed peptide is by Extracted From Oil-tea-cake
Middle proteolysis sites influence：Through trypsin hydrolysis generate molecular weight be (5000u, 3000u] polypeptide have improve ox
The effect for freezing sperm oxidation resistance, can be used as ox sperm freezing protective agent reduces oxidative damage, and through alkali protease
The polypeptide of generation is then unobvious；Through hydrolysis by novo generate molecular weight be (5000u, 3000u] polypeptide can promote
Weary feelings estrus of cow and fertilization, and the polypeptide generated through trypsase is then unobvious；
2, the preparation method of hydrolyzed peptide of the present invention is simple, and simple crushing, enzymatic hydrolysis, ultrafiltration, freeze-drying is used only, this
A little steps are all that can realize large-scale production in the factory, easy to implement；
3, the present invention realizes the refuse reclamation of the camellia meiocarpa dregs of rice, and the economic value added of the camellia meiocarpa dregs of rice can be improved.
Detailed description of the invention
Fig. 1 is the DSC curve of Extracted From Oil-tea-cake polypeptide 1-6；
Fig. 2 is the influence that Extracted From Oil-tea-cake polypeptide freezes rear oxidation product MDA content to sperm；
Fig. 3 is influence of the Extracted From Oil-tea-cake polypeptide to weary feelings estrus of cow rate and conception rate.
Specific embodiment
It is specific with reference to the accompanying drawings and examples to introduce essentiality content of the present invention, but guarantor of the invention is not limited with this
Protect range.
In following embodiments, it is colonized with the 1982-1983 spring in Fujian Province Minhou Tong Kou state-owned forest farms Camellia oleifera Germplasms
The camellia meiocarpa (Camellia meiocarpa Hu.) for collecting garden is raw material, obtains camellia meiocarpa through squeezing process removal grease
The dregs of rice, it is spare.
Embodiment 1：Trypsase prepares Extracted From Oil-tea-cake polypeptide 1
Include the following steps：
The camellia meiocarpa dregs of rice are crushed to 80-100 mesh by step S1, are soaked in water overnight, are eventually fabricated dregs of rice powder suspension, the dregs of rice
The mass concentration of powder suspension is 150g/L；
Trypsin digestion (addition weight is calculated as 3.0g/100g with dregs of rice powder weight) is added in step S2, and enzymatic hydrolysis condition is：
PH value, 8.0；Hydrolysis temperature, 45 DEG C；Enzymolysis time, 11 hours；After enzymatic hydrolysis, 90 DEG C make tryptose in high-temperature process 10 minutes
Enzyme-deactivating is cooled to room temperature, and standing takes supernatant；
Supernatant absorbent cotton filter 23 is collected merging filtrate, crude product freeze-dried powder is lyophilized to obtain by step S3；
Step S4, crude product freeze-dried powder are dissolved with ultrapure water, successively with molecular cut off be respectively 10000u, 5000u,
The ultrafiltration membrane of 3000u (5000u, 3000u can also be used only, but increase 10000u to be conducive to improve subsequent ultrafiltration speed) carries out
Separation, collect molecular weight (5000u, 3000u] peptide fragment in range, freeze-drying.
Embodiment 2：Trypsase prepares Extracted From Oil-tea-cake polypeptide 2
Include the following steps：
The camellia meiocarpa dregs of rice are crushed to 80-100 mesh by step S1, are soaked in water overnight, are eventually fabricated dregs of rice powder suspension, the dregs of rice
The mass concentration of powder suspension is 130g/L；
Trypsin digestion (addition weight is calculated as 2.5g/100g with dregs of rice powder weight) is added in step S2, and enzymatic hydrolysis condition is：
PH value, 7.8；Hydrolysis temperature, 43 DEG C；Enzymolysis time, 12 hours；After enzymatic hydrolysis, 90 DEG C make tryptose in high-temperature process 10 minutes
Enzyme-deactivating is cooled to room temperature, and standing takes supernatant；
Supernatant absorbent cotton filter 23 is collected merging filtrate, crude product freeze-dried powder is lyophilized to obtain by step S3；
Step S4, crude product freeze-dried powder are dissolved with ultrapure water, successively with molecular cut off be respectively 10000u, 5000u,
The ultrafiltration membrane of 3000u (5000u, 3000u can also be used only, but increase 10000u to be conducive to improve subsequent ultrafiltration speed) carries out
Separation, collect molecular weight (5000u, 3000u] peptide fragment in range, freeze-drying.
Embodiment 3：Trypsase prepares Extracted From Oil-tea-cake polypeptide 3
Include the following steps：
The camellia meiocarpa dregs of rice are crushed to 80-100 mesh by step S1, are soaked in water overnight, are eventually fabricated dregs of rice powder suspension, the dregs of rice
The mass concentration of powder suspension is 170g/L；
Trypsin digestion (addition weight is calculated as 3.5g/100g with dregs of rice powder weight) is added in step S2, and enzymatic hydrolysis condition is：
PH value, 8.2；Hydrolysis temperature, 47 DEG C；Enzymolysis time, 10 hours；After enzymatic hydrolysis, 90 DEG C make tryptose in high-temperature process 10 minutes
Enzyme-deactivating is cooled to room temperature, and standing takes supernatant；
Supernatant absorbent cotton filter 23 is collected merging filtrate, crude product freeze-dried powder is lyophilized to obtain by step S3；
Step S4, crude product freeze-dried powder are dissolved with ultrapure water, successively with molecular cut off be respectively 10000u, 5000u,
The ultrafiltration membrane of 3000u (5000u, 3000u can also be used only, but increase 10000u to be conducive to improve subsequent ultrafiltration speed) carries out
Separation, collect molecular weight (5000u, 3000u] peptide fragment in range, freeze-drying.
Embodiment 4：Alkali protease prepares Extracted From Oil-tea-cake polypeptide 4
Include the following steps：
The camellia meiocarpa dregs of rice are crushed to 80-100 mesh by step S1, are soaked in water overnight, are eventually fabricated dregs of rice powder suspension, the dregs of rice
The mass concentration of powder suspension is 150g/L；
Alkali protease enzymatic hydrolysis (addition weight is calculated as 3.0g/100g with dregs of rice powder weight), enzymatic hydrolysis condition is added in step S2
For：PH value, 8.5；Hydrolysis temperature, 55 DEG C；Enzymolysis time, 10 hours；After enzymatic hydrolysis, 90 DEG C make alkalinity in high-temperature process 10 minutes
Albumen enzyme-deactivating, is cooled to room temperature, and standing takes supernatant；
Supernatant absorbent cotton filter 23 is collected merging filtrate, crude product freeze-dried powder is lyophilized to obtain by step S3；
Step S4, crude product freeze-dried powder are dissolved with ultrapure water, successively with molecular cut off be respectively 10000u, 5000u,
The ultrafiltration membrane of 3000u (5000u, 3000u can also be used only, but increase 10000u to be conducive to improve subsequent ultrafiltration speed) carries out
Separation, collect molecular weight (5000u, 3000u] peptide fragment in range, freeze-drying.
Embodiment 5：Alkali protease prepares Extracted From Oil-tea-cake polypeptide 5
Include the following steps：
The camellia meiocarpa dregs of rice are crushed to 80-100 mesh by step S1, are soaked in water overnight, are eventually fabricated dregs of rice powder suspension, the dregs of rice
The mass concentration of powder suspension is 130g/L；
Alkali protease enzymatic hydrolysis (addition weight is calculated as 2.5g/100g with dregs of rice powder weight), enzymatic hydrolysis condition is added in step S2
For：PH value, 8.3；Hydrolysis temperature, 53 DEG C；Enzymolysis time, 11 hours；After enzymatic hydrolysis, 90 DEG C make alkalinity in high-temperature process 10 minutes
Albumen enzyme-deactivating, is cooled to room temperature, and standing takes supernatant；
Supernatant absorbent cotton filter 23 is collected merging filtrate, crude product freeze-dried powder is lyophilized to obtain by step S3；
Step S4, crude product freeze-dried powder are dissolved with ultrapure water, successively with molecular cut off be respectively 10000u, 5000u,
The ultrafiltration membrane of 3000u (5000u, 3000u can also be used only, but increase 10000u to be conducive to improve subsequent ultrafiltration speed) carries out
Separation, collect molecular weight (5000u, 3000u] peptide fragment in range, freeze-drying.
Embodiment 6：Alkali protease prepares Extracted From Oil-tea-cake polypeptide 6
Include the following steps：
The camellia meiocarpa dregs of rice are crushed to 80-100 mesh by step S1, are soaked in water overnight, are eventually fabricated dregs of rice powder suspension, the dregs of rice
The mass concentration of powder suspension is 170g/L；
Alkali protease enzymatic hydrolysis (addition weight is calculated as 3.5g/100g with dregs of rice powder weight), enzymatic hydrolysis condition is added in step S2
For：PH value, 8.7；Hydrolysis temperature, 57 DEG C；Enzymolysis time, 9 hours；After enzymatic hydrolysis, 90 DEG C make alkalinity in high-temperature process 10 minutes
Albumen enzyme-deactivating, is cooled to room temperature, and standing takes supernatant；
Supernatant absorbent cotton filter 23 is collected merging filtrate, crude product freeze-dried powder is lyophilized to obtain by step S3；
Step S4, crude product freeze-dried powder are dissolved with ultrapure water, successively with molecular cut off be respectively 10000u, 5000u,
The ultrafiltration membrane of 3000u (5000u, 3000u can also be used only, but increase 10000u to be conducive to improve subsequent ultrafiltration speed) carries out
Separation, collect molecular weight (5000u, 3000u] peptide fragment in range, freeze-drying.
Measure the DSC curve of above-mentioned Extracted From Oil-tea-cake polypeptide 1-6 respectively, it is seen that the property of Extracted From Oil-tea-cake polypeptide 1-3 as ingredient,
The property of Extracted From Oil-tea-cake polypeptide 4-6 is as ingredient, and the property of Extracted From Oil-tea-cake polypeptide 1-3 and Extracted From Oil-tea-cake polypeptide 4-6 and ingredient are distinguished
It is larger.The DSC curve of Extracted From Oil-tea-cake polypeptide 1-6 (NETZSCH STA449C type synchronous solving, sample quality as shown in Figure 1
5mg, 30~900 DEG C of measuring temperature range, argon gas flow velocity 30mL/min, 10 DEG C/min of heating rate).
Effect example 1：Antioxidation of the Extracted From Oil-tea-cake polypeptide to ox semen cryopreservation
One, experimental material
Ox species are Holstein cow；Bull semen dilution uses TCM-199.
Malonaldehyde (MDA) testing cassete is purchased from Nanjing and builds up Bioengineering Research Institute.
The polypeptide 1-6 for accurately weighing embodiment 1-6 preparation respectively is dissolved with sterile ultrapure water, and the storage of concentration 5mg/mL is made
Standby liquid.
Two, experimental method
1, acquisition and inspection (acquisition and the inspection method bibliography of sperm：Aftosa vaccine is injected to bull semen
The influence of quality is studied, Chinese Cattle industry science, 2014)
Instrument used will be rinsed in advance, disinfection；Glassware is sent into drying box after cleaning dry, artificial vagina and its
The 75% cotton ball soaked in alcohol cleaning disinfection such as his instrument, artificial vagina, which installs, puts on protective case, and the artificial vagina after disinfection can only use
Once.Water filling temperature is at 40 DEG C or so in artificial vagina, and about in 300mL, constant temperature the temperature inside the box is controlled at 40 DEG C -42 DEG C water injection rate,
Inner tube of a tyre temperature when semen collection is maintained between 38 DEG C -40 DEG C, and inner tube of a tyre temperature can do adjustment appropriate according to different oxen, and highest is not
It can be more than 43 DEG C.Lubricant is uniformly applied to preceding the 2/3 of the artificial vagina inner tube of a tyre, and lubricant albolene and liquid paraffin oil press 1：
The modulation of 1 ratio.The interior tire pressure (inflating before semen collection from artificial vagina piston hole) of artificial vagina is advisable so that artificial vagina mouth is triangular in shape.Platform
Ox wants healthy and strong, disposition is docile, without epidemic disease, and vulva, huckle cleaning are clean.It takes and lures, by mounting, sky is climbed repeatedly when bull semen collection
The methods of stimulation bull it is excited.Also replaceable semen collection place, the methods of replacement dummy cow increase the excitement levels of breeding oxen.Semen collection place
It should keep quite, non-slip mat is cleaned and is laid on ground, in order to avoid slip when bull semen collection.The semen collection when bull is highly sexed, semen collection
Member's right hand holds artificial vagina, stands in bull right back, and when bull take-off, forelimb is climbed up after dummy cow rapidly to forestock bull foreskin,
The right hand holds artificial vagina and dummy cow into 40 degree of angles, and the oblique lower section of artificial vagina mouth, right-hand man, which cooperates, is naturally introduced into bull penis
In artificial vagina, bull completes ejaculation movement to previous punching, lands to bull forelimb, slowly artificial vagina is deviate from.It opens and lives
Plug is deflated, and is carefully removed collection spermaduct, is transferred quickly to sperm sensing chamber and is detected.Choosing fresh semen (should be in milky white slightly band
Yellow or cream-colored opaque liquid), the constant temperature (37 DEG C) after taking a drop original sperm to cover slide at 400 times can be under stereomicroscope
The percentage of linear motile is observed, the sperm of sperm of the motility rate not less than 80% is used for freezing of semen.
According to the method described above, the sperm of random acquisition 10 healthy Holstein cows.
2, semen dilution
TCM-199 dilution is put into 37 DEG C of water-baths and is preheated, it is 10 μ g/ that polypeptide stock solution, which is added, and is mixed and made into concentration
The ox sperm freezing dilution liquid of mL, then by ox sperm and the dilution according to volume ratio 1：4 are mixed to get dilution sperm.
Control group is diluted ox sperm using pure TCM-199 dilution.
3, freezing of semen and defrosting
(1) freezing of semen.First dilution sperm is fitted into the tubule of 0.25mL and is pre-chilled, is then placed in -5 DEG C of refrigerator and puts down
Weigh 2.5h.After balance, tubule is put into imprecise bubble chamber, liquid nitrogen is added into bubble chamber, fumigates tubule
30min, -120 DEG C of temperature or so.Tubule is finally put into freezen protective in liquid nitrogen.
(2) it thaws.Jelly fine tube after preservation 35d is taken out, the 20s that thaws is immediately placed in 37 DEG C of water-baths.
4, determination oxidative
MDA content in sperm after measurement control group and each polypeptide group are thawed respectively, it is cold which can embody each group sperm
The antioxidant effect of degree of oxidation and each polypeptide during jelly.MDA measurement is carried out according to malonaldehyde testing cassete specification.
5, statistical method
It is analyzed using 19.0 software statistics of SPSS, data are indicated with mean value ± deviation, and comparison among groups use single factor test variance
Analysis.
Three, experimental result
Malonaldehyde (MDA) is the final product of lipid oxidation, and the generation of MDA can aggravate the damage of film and have cytotoxicity.
Compared with the control group, Extracted From Oil-tea-cake polypeptide 1-3 group MDA content significantly reduces, and the reduction of Extracted From Oil-tea-cake polypeptide 4-6 group MDA content is unobvious,
Show that Extracted From Oil-tea-cake polypeptide 1-3 can significantly inhibit the oxidative damage frozen to ox sperm.As a result as shown in table 1 and Fig. 2.
1 each group sperm of table freezes rear MDA comparision contents (n=10)
Effect example 2：Extracted From Oil-tea-cake polypeptide influences weary feelings estrus of cow, fecundation
One, experimental material
Cow kind is Simmental.Judge the standard of the weary feelings of cow：Non-reproductive system inflammation and deformity, but postpartum 3
Still do not occur heat after a month, influence the cow of breeding fertilization to be weary feelings cow.
Two, experimental method
1, experimental group and raising
The weary feelings XIMEN Qin at monthly age almost the same (between the 3.5-4.0 month) is randomly divided into control group, Extracted From Oil-tea-cake
Polypeptide 1-6 group, every group 20.Control group gives conventional feed nursing, and Extracted From Oil-tea-cake polypeptide 1-6 group is added respectively in conventional feed
The Extracted From Oil-tea-cake polypeptide 1-6 of 5g/kg.Each group cow is continuously fed 75 days, and heat is showed in observation 75 days and receives to breed and be impregnated
Situation.
2, statistical method
It is analyzed using 19.0 software statistics of SPSS, data are indicated with mean value ± deviation, and comparison among groups use single factor test variance
Analysis.
Three, experimental result
1, influence of the Extracted From Oil-tea-cake polypeptide to weary feelings estrus of cow rate
Compared with the control group, the weary feelings estrus of cow rate of Extracted From Oil-tea-cake polypeptide 4-6 group significantly improves, and Extracted From Oil-tea-cake polypeptide 1-3 group
It is unobvious, show that Extracted From Oil-tea-cake polypeptide 4-6 has the function of promoting weary feelings estrus of cow.Concrete outcome is shown in Table 2 and Fig. 3.
2, influence of the Extracted From Oil-tea-cake polypeptide to weary feelings cow conception rate
Compared with the control group, the weary feelings cow conception rate of Extracted From Oil-tea-cake polypeptide 4-6 group significantly improves, and Extracted From Oil-tea-cake polypeptide 1-3 group
It is unobvious, show that Extracted From Oil-tea-cake polypeptide 4-6 has the function of promoting weary feelings cow fertilization.Concrete outcome is shown in Table 2 and Fig. 3.
Influence of the 2 Extracted From Oil-tea-cake polypeptide of table to weary feelings estrus of cow rate and conception rate
To sum up, the hydrolyzed peptide of Extracted From Oil-tea-cake of the present invention has various active, and the activity of hydrolyzed peptide is by oil tea
Proteolysis sites influence in the dregs of rice：Through trypsin hydrolysis generate molecular weight be (5000u, 3000u] polypeptide have improve
Ox freezes the effect of sperm oxidation resistance, and can be used as ox sperm freezing protective agent reduces oxidative damage, and through basic protein
The polypeptide that enzyme generates is then unobvious；Through hydrolysis by novo generate molecular weight be (5000u, 3000u] polypeptide can promote
Into weary feelings estrus of cow and fertilization, and the polypeptide generated through trypsase is then unobvious.Moreover, the preparation of hydrolyzed peptide of the present invention
Method is simple, simple crushing, enzymatic hydrolysis, ultrafiltration, freeze-drying is used only, these steps are all that can realize big life in the factory
Productionization, it is easy to implement.The present invention realizes the refuse reclamation of the camellia meiocarpa dregs of rice, and the economy that the camellia meiocarpa dregs of rice can be improved is additional
Value.
The effect of above-described embodiment is specifically to introduce essentiality content of the invention, but those skilled in the art should know
Protection scope of the present invention should not be confined to the specific embodiment by road.
Claims (7)
1. a kind of application of Extracted From Oil-tea-cake polypeptide in terms of the feed or drug that preparation promotes weary feelings estrus of cow and fertilization, feature
It is：The Extracted From Oil-tea-cake polypeptide is prepared by following steps：
The camellia meiocarpa dregs of rice are crushed to 80-100 mesh, soaked in water by step S1, are eventually fabricated dregs of rice powder suspension；
Step S2, is added alkali protease enzymatic hydrolysis, and enzymatic hydrolysis condition is：PH value, 8.3-8.7；Hydrolysis temperature, 53-57 DEG C；When enzymatic hydrolysis
Between, 9-11 hours；After enzymatic hydrolysis, heating makes basic protein enzyme-deactivating, is cooled to room temperature, and standing takes supernatant；
Step S3 filters supernatant with absorbent cotton, collects filtrate, crude product freeze-dried powder is lyophilized to obtain；
Step S4, crude product freeze-dried powder are dissolved with ultrapure water, are separated with the ultrafiltration membrane of different molecular weight cut off, and molecular weight is collected
(5000u, 3000u] peptide fragment in range, it is freeze-dried to obtain the final product.
2. application according to claim 1, it is characterised in that：The camellia meiocarpa dregs of rice refer to the fruit of camellia meiocarpa through over-voltage
Squeeze the solid residue obtained after processing or lixiviation process processing removal grease.
3. application according to claim 1, it is characterised in that：The ultrafiltration membrane of the different molecular weight cut off includes retention point
Son amount is respectively the ultrafiltration membrane of 5000u, 3000u.
4. application according to claim 3, it is characterised in that：It further include the ultrafiltration membrane of molecular cut off 10000u.
5. application according to claim 1, which is characterized in that heating makes the method for basic protein enzyme-deactivating：90 DEG C of height
Temperature processing 10 minutes.
6. application according to claim 1, it is characterised in that：Dregs of rice powder suspension is 130-170g/L.
7. application according to claim 1, which is characterized in that the addition weight of the alkali protease is in terms of dregs of rice powder weight
For 2.5-3.5g/100g.
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| PCT/CN2018/082479 WO2019114159A1 (en) | 2017-12-11 | 2018-04-10 | Camellia oleifera meal polypeptide and polysaccharide as well as preparation method and application thereof, and camellia oleifera meal detoxification method |
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