CN105254750A - Bioactive polypeptide FGYSGAFKCL as well as preparation and application thereof - Google Patents
Bioactive polypeptide FGYSGAFKCL as well as preparation and application thereof Download PDFInfo
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- CN105254750A CN105254750A CN201510673314.2A CN201510673314A CN105254750A CN 105254750 A CN105254750 A CN 105254750A CN 201510673314 A CN201510673314 A CN 201510673314A CN 105254750 A CN105254750 A CN 105254750A
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- Prior art keywords
- biologically active
- active polypeptides
- fgysgafkcl
- milk
- polypeptide
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Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention relates to the field of protein, in particular to a milk-borne bioactive polypeptide FGYSGAFKCL derived from lactoferrin. Extensive and deep researches show that the bioactive polypeptide has antioxidation bioactivity and cellular immunity promotion activity; results of the researches show that the milk-borne bioactive polypeptide serving as a micromolecular substance has a potential aging delaying function. Additionally, results of simulative digestion experiments show that the bioactive polypeptide still stably exists under a common in-vivo digestion condition of animals, cannot be further degraded, can be directly absorbed and utilized by the animals, performs the bioactive function and has a great significance in development of dairy products, health-care products and medicines with the functions of antioxidation, improvement of immunity and aging delaying.
Description
Technical field
The present invention relates to albumen field, be specifically related to a kind of biologically active polypeptides FGYSGAFKCL and preparation and application thereof.
Background technology
Because biologically active peptides is as sanatory bioactive ingredients, there is transmission physiologic information, the effect of regulation of physiological functions, the maintenance for the normal physiological activity of the system such as nerve, digestion, reproduction, growth, motion, metabolism, circulation of human body is extremely important.They not only have the effects such as antiviral, bacterium, hypertension, decreasing cholesterol, and have immunity moderation reaction, the function such as antitumor as immunomodulator; Can have the effect delayed senility by scavenging free radicals, be the research topic that current international food circle is the most popular and the functional factor having development prospect.Various bioactive peptide Chang Zuowei functional food added ingredients is produced for the practice of food, particularly obtains good result in the application aspect of milky-drinks, foodstuff additive.The function of milk-derived biologically active peptides and on the positive attention of the impact of the health of human consumer.
In recent years, increasing scientific research shows that the little peptide much deriving from bioprotein has various biological activity, as hormonal action, immunomodulatory, antithrombotic, hypertension, decreasing cholesterol, antibacterial, antiviral, antitumous effect etc.Meanwhile, research finds that little peptide can be absorbed better by human body and utilize, and changes traditional protein and can only be degraded to the viewpoint that amino acid just can be absorbed and used.In numerous bioprotein, various cow's milk protein is one of very important biologically active polypeptides source, they are decomposed utilization by milk-acid bacteria, and there occurs a series of biochemical reactions, protein is made to become polypeptide or free amino acid, finally digested or directly entered the blood circulation of human body by the absorption and transport of intestinal epithelial cell, play its biological action.
Therefore, these bioactive micro peptides not only become the natural resource treasure-house of screening of medicaments, are also the main raw material compositions of functional foodstuff industry.At present, the preparation of biologically active peptides and application and development thereof have become the focus of research in world wide.
Immune-active peptides obtains from Ruzhong first after opioid peptides finds and proves a class biologically active peptides of its physiologically active.1981, people's Late Cambrian such as Jolles, utilize trypsin hydrolyzing people lactoprotein, a kind of immune-active peptides section is obtained from hydrolyzate, its aminoacid sequence is Val-Glu-Pro-Ile-Pro-Tyr, this small peptide is proved to be in experiment in vitro can strengthen the phagolysis of Turnover of Mouse Peritoneal Macrophages to sheep erythrocyte, intravenous injection, then can strengthen the resistivity that mouse infects pneumonia kirschner bar.The people such as Elitsur obtain Arg-Tyr-Leu-Gly-Tyr-Leu-Glu and Arg-Tyr-Leu-Gly-Tyr-Leu through pepsin casein, research proves that this two small peptide not only has opioid activity, also there is immunoregulatory activity, lymphopoiesis can be strengthened, improve natural killer cell (NK) ability, promote the movement of biting neutrophilic leukocyte.The people such as Zhang Shaohui utilize lactobacterium helveticus fermented skim milk to obtain biologically active polypeptides QEPVL and degraded product QEPV thereof, experiment is promoted through antioxidation in vitro experiment, ion vitro immunization function, demonstrate polypeptide QEPV and there is the active activity with promoting cellular immunization of good antioxidation biology, the free radical in body can be removed on the one hand, reduce the injury of radical pair human body; On the other hand, biologically active polypeptides QEPVL and QEPV can also enhancing body immunizing power, strengthens the multiplication capacity of lymphocyte, scavenger cell, the scavenger cell nitrogen protoxide amount of inducing is increased, and urge Factor of Macrophage.
Manyly have bioactive peptide sequence although be implied with in cow's milk protein, these bio-active peptide sequence only have and are discharged competence exertion effect by certain means.The Main Means obtaining lactoprotein bioactive peptide has two kinds: fermentation, utilizes microorganism (milk-acid bacteria) fermented-milk source protein to obtain; Enzymolysis, comprises the digestive enzyme from animal and the proteolytic enzyme from plant and microorganism.
Result of study shows that milk-derived biologically active peptides is as small molecule material, and the unique advantage low with its molecular weight, activity is strong, consumption is few, more and more causes the attention of people.So they are not only used for the manufacture of various functional foodstuff as functional food ingredient, and as immunomodulator, there is immunity moderation reaction, the function such as antitumor; The effect delayed senility can be had by scavenging free radicals.In addition, most milk-derived biologically active peptides can resist GI digestion, is absorbed, and need not be broken down into single amino acids in small intestine with complete form by body, and have absorption easy to digest, edible safety is high.Therefore milk-derived biologically active peptides can improve body resist extraneous undesirable element stimulation, reduce the functional performance attention such as body sickness rate, be expected to become the non-medication raw material of substance that there is scavenging free radicals, reduce trouble cancer probability and delay senility, at functional foodstuff and field of medicaments, there is boundless application prospect, the most popular research topic of international food circle and biopharmaceutical production industry will be become from now on, for improving level of human health, do not fall ill fall ill less in other words, delay senility, prolongs life plays an important role.
Summary of the invention
The object of the present invention is to provide a kind of biologically active polypeptides FGYSGAFKCL and preparation and application thereof of separation.
For achieving the above object and other relevant objects, the present invention adopts following technical scheme:
A first aspect of the present invention, provide a kind of biologically active polypeptides FGYSGAFKCL of separation, it comprises
A polypeptide that () is made up of the amino acid shown in Phe-Gly-Tyr-Ser-Gly-Ala-Phe-Lys-Cys-Leu (SEQIDNO:1);
(b) or in the aminoacid sequence shown in SEQIDNO:1, be substituted, lack or add one or several amino acid and there is the polypeptide derivative by (a) of same isoreactivity.
Preferably, the source of described biologically active polypeptides is milk-derived.
Biologically active polypeptides PheGlyTyrSerGlyAlaPheLysCysLeu of the present invention is milk-derived, specifically derives from lactoferrin, and is the amino-acid residue of lactoferrin (SEQIDNO:2) 190th ~ 199.
The aminoacid sequence of lactoferrin is existing technology, and sequence is as shown in SEQIDNO:2:
AlaProArgLysAsnValArgTrpCysThrIleSerGlnProGluTrpPheLysCysArg
ArgTrpGlnTrpArgMetLysLysleuGlyAlaProSerIleThrCysValArgArgAla
PheAlaLeuGluCysIleArgAlaIleAlaGluLysLysAlaAspAlaValThrLeuAsp
GlyGlyMetValPheGluAlaGlyArgAspProTyrLysLeuArgProValAlaAla
GluIleTyrGlyThrLysGluSerProGlnThrHisTyrTyrAlaValAlaValValLys
LysGlySerAsnPheGlnLeuAspGlnLeuGlnGlyArgLysSerCysHisThrGlyLeu
GlyArgSerAlaGlyTrpIleIleProMetGlyIleLeuArgProTyrLeuSerTrpThr
GluSerLeuGluProLeuGlnGlyAlaValAlaLysPhePheSerAlaSerCysValPro
CysIleAspArgGlnAlaTyrProAsnLeuCysGlnLeuCysLysGlyGluGlyGluAsn
GlnCysAlaCysSerSerArgGluProTyrPheGlyTyrSerGlyAlaPheLysCysLeu
GlnAspGlyAlaGlyAspValAlaPheValLysGluThrThrValPheGluAsnLeuPro
GluLysAlaAspArgAspGlnTyrGluLeuLeuCysLeuAsnAsnSerArgAlaProVal
AspAlaPheLysGluCysHisLeuAlaGlnValProSerHisAlaValValAlaArgSer
ValAspGlyLysGluAspLeuIleTrpLysLeuLeuSerLysAlaGlnGluLysPheGly
LysAsnLysSerArgSerPheGlnLeuPheGlySerProProGlyGlnArgAspLeuLeu
PheLysAspSerAlaLeuGlyPheLeuArgIleProSerLysValAspSerAlaLeuTyr
LeuGlySerArgTyrLeuThrThrLeuLysAsnLeuArgGluThrAlaGluGluValLys
AlaArgTyrThrArgValValTrpCysAlaValGlyProGluGluGlnLysLysCysGln
GlnTrpSerGlnGlnSerGlyGlnAsnValThrCysAlaThrAlaSerThrThrAspAsp
CysIleValLeuValLeuLysGlyGluAlaAspAlaLeuAsnLeuAspGlyGlyTyrIle
TyrThrAlaGlyLysCysGlyLeuValproValLeuAlaGluAsnArgLysSerSerLys
HisSerSerLeuAspCysValLeuArgProThrGluGlyTyrLeuAlaValAlaValVal
LysLysAlaAsnGluGlyLeuThrTrpAsnSerLeuLysAspLysLysSerCysHisThr
AlaValAspArgThrAlaGlyTrpAsnIleProMetGlyLeuIleValAsnGlnThrGly
SerCysAlaPheAspGluPhePheSerGlnSerCysAlaProGlyAlaAspproLysSer
ArgLeuCysAlaLeuCysAlaGlyAspAspGlnGlyLeuAspLysCysValproAsnSer
LysGluLysTyrTyrGlyTryThrGlyAlaPheArgCysLeuAlaGluAspValGlyAsp
ValAlaPheValLysAsnAspThrValTrpGluAsnThrAsnGlyGluSerThrAlaAsp
TrpAlaLysAsnLeuAsnArgGluAspPheArgLeuLeuCysLeuAspGlyThrArgLys
ProValThrGluAlaGlnSerCysHisLeuAlaValAlaProAsnHisAlaValValSer
ArgSerAspArgAlaAlaHisValLysGlnValLeuLeuHisGlnGlnAlaLeuPheGly
LysAsnGlyLysAsnCysProAspLysPheCysLeuPheLysSerGluThrLysAsnLeu
LeuPheAsnAspAsnThrGluCysLeuAlaLysLeuGlyGlyArgProThrTyrGluGlu
TyrLeuGlyThrGluTyrValThrAlaIleAlaAsnLeuLysLysCysSerThrSerPro
LeuLeuGluAlaCysAlaPheLeuThrArg。
Preferably, described biologically active polypeptides FGYSGAFKCL has the function of antioxidation activity in vitro and enhancing body immunizing power.
Biologically active polypeptides FGYSGAFKCL of the present invention by engineered method and chemical process synthetic, also can be obtained by the method for separation and purification, enzyme liberating from milk-product.
The invention also discloses the nucleotide fragments of coding aforementioned biological active polypeptide FGYSGAFKCL.
The aminoacid sequence of lactoferrin and nucleotides sequence are classified as existing technology, the biologically active polypeptides FGYSGAFKCL of the nucleotide fragments energy encoding mature of the amino-acid residue of coding lactoferrin (SEQIDNO:2) 190th ~ 199.
Second aspect present invention, provide the preparation method of aforementioned biological active polypeptide, step is as follows:
1) ferment: lactobacterium helveticus is added in skimming milk and carry out anaerobically fermenting, obtain lactobacterium helveticus fermented-milk;
2) broken wall: the thalline obtained after centrifugal to fermented-milk carries out ultrasonic, broken bacterium wall makes peptide in born of the same parents dissociate, and obtains bacterium peptide mixed solution;
3) slightly the carrying of polypeptide: to step 2) in bacterium peptide mixed solution carry out low-temperature centrifugation separation, get supernatant liquor;
4) purifying of polypeptide:
A. to step 3) supernatant liquor carry out uf processing, collect filtrate;
B. solid-phase extraction column is separated: the filtrate of collection adopts WatersSep-pakC18 Solid-Phase Extraction column extracting, collection of biological active polypeptide mixture;
5) digestion of polypeptide and stability: adopt two step enzymolysis process enzymolysis step 4) biologically active polypeptides mixture, obtain digested after biologically active polypeptides mixture; The enzyme that the first step enzymolysis adopts is stomach en-, and the enzyme that second step enzymolysis adopts is pancreatin.
Skimming milk of the present invention is the cow's milk through skimming treatment, and in usual skimming milk, lipid content is less than 0.1%.
Preferably, step 1) in, lactobacterium helveticus is lactobacterium helveticus (Lactobacillushelveticus, CICC6024).
Preferably, step 1) in, the condition of described anaerobically fermenting is: leavening temperature 36 ~ 38 DEG C, fermentation culture 6 ~ 8h; More preferably leavening temperature 37 DEG C, fermentation culture 7h.
Preferably, step 2) described centrifugal condition is 20 DEG C, 4000 ~ 5000rpm, centrifugal 15min..
Preferably, step 2) condition of described broken wall is cell concentration 0.0238g/ml, broken power 300W, broken total time 26min (ultrasonic 3s, interval 5s).
Preferably, step 3) in, the condition of described low-temperature centrifugation is: 4 DEG C, 8000 ~ 10000rpm, centrifugal 15 ~ 30min.
Preferably, step 4) in a, what described uf processing adopted is the super filter tube that molecular weight cut-off is respectively 3kDa.
More excellent, step 4) in a, in described process of ultrafiltration treatment, rotating speed is 4800r/min, and the time is 30min, and centrifuging temperature is 4 DEG C.
Preferably, step 4) in b, solid-phase extraction column is separated, and concrete grammar is: activation and balance WatersSep-pakC18 solid-phase extraction column; By step 4) filtrate the carrying out of collecting in a dilute rear loading, and adopt elution, collect the elutriant obtained, namely comprise biologically active polypeptides mixture.
Preferably, step 4) in b, in solid-phase extraction column partition method, the elutriant adopted is methyl alcohol and ddH
2the mixed solution of O, described methyl alcohol and ddH
2methyl alcohol and ddH in the mixed solution of O
2the volume ratio of O is 80:20, described methyl alcohol and ddH
2formic acid containing 0.1% (v/v) in the mixed solution of O.
Preferably, step 4) in b, in solid-phase extraction column partition method, adopt methyl alcohol activation WatersSep-pakC18 solid-phase extraction column; Preferred 2ml methyl alcohol.
Preferably, step 4) in b, in solid-phase extraction column partition method, adopt ddH
2o balances WatersSep-pakC18 solid-phase extraction column; Preferred 1mlddH
2o.
Preferably, step 4) in b, in solid-phase extraction column partition method, by step 4) the ultrafiltrated loading of collecting in a, adopt 400ul elution.
Preferably, step 4) in b solid-phase extraction column partition method, first adopt 2mL methyl alcohol activation WatersSep-pakC18 solid-phase extraction column, adopt 1mLddH
2o balances WatersSep-pakC18 solid-phase extraction column; Loading sample is 2mL; By step 4) collect ultrafiltrated loading in a, adopt 400 μ L elution.
Preferably, step 5) described two step enzymolysis process concrete steps are for by step 4) mixture containing polypeptide that obtains is dissolved in sterilizing deionized water, adjust ph is 2.0 ± 0.1, add stomach en-again, obtain reaction solution, insulation reaction 90min in the water bath with thermostatic control of 37 ± 0.5 DEG C, obtains the first step enzymolysis solution; The pH value of the first step enzymolysis solution is adjusted to 7.5 ± 0.1, and adds pancreatin, insulation reaction 150min in the water bath with thermostatic control of 37 ± 0.5 DEG C, obtain second step enzymolysis solution; Adopt Boiling bath method to make enzyme deactivation second step enzymolysis solution, the time is 5min, obtains enzymolysis product; Lyophilize is adopted to obtain powdery enzymolysis product.
Preferred, described Boiling bath method is 95 DEG C of immersion methods.
Preferably, step 5) described pepsic addition is stomach en-10 ~ 30mg/g substrate; The addition of described pancreatin is pancreatin 30 ~ 50mg/g substrate.
More excellent, step 5) described pepsic addition is stomach en-20mg/g substrate; The addition of described pancreatin is pancreatin 40mg/g substrate.
Preferably, extract the elution peak that molecular weight is the polypeptide of 1092.52Da, be biologically active polypeptides FGYSGAFKCL.
In the present invention, the molecular weight of known FGYSGAFKCL, extracting molecular size is the elution peak of 1092.52Da, is biologically active polypeptides FGYSGAFKCL of the present invention.Concrete, FGYSGAFKCL molecular size of the present invention is its retention time of elution peak of 1092.52Da is 13.89min.
Third aspect present invention, provides the application of aforementioned biological active polypeptide FGYSGAFKCL or derivatives thereof in the food of the anti-oxidant and/or enhancing body immunizing power of preparation, healthcare products and medicine.
Biologically active polypeptides FGYSGAFKCL or derivatives thereof of the present invention may be used for the milk-product such as Yoghourt and various food, reduces the makeup of radical pair skin damage; And be not degraded because biologically active polypeptides FGYSGAFKCL of the present invention directly can be absorbed by gi tract, therefore may be used for preparing the healthcare products improving immunizing power, or for the preparation of having medicine that is anti-oxidant and/or enhancing body immunizing power.
Fourth aspect present invention, provides a kind of anti-oxidation medicine, comprises the derivative of aforementioned biological active polypeptide FGYSGAFKCL or aforementioned biological active polypeptide.
Fifth aspect present invention, provides a kind of enhancing body immunizing power medicine, comprises the derivative of aforementioned biological active polypeptide FGYSGAFKCL or aforementioned biological active polypeptide.
The derivative of described polypeptide, refer on the amino acid side groups of polypeptide, aminoterminal or carboxyl terminal carry out hydroxylation, carboxylated, carbonylation, methylate, acetylize, phosphorylation, the modification such as esterification or glycosylation, the polypeptide derivative obtained.
Compared with prior art, the beneficial effect of biologically active polypeptides of the present invention is:
Milk-derived biologically active polypeptides FGYSGAFKCL of the present invention has good anti-oxidant activity and promotes that immunity of organisms is active; The free radical in body can be removed on the one hand, reduce the injury of radical pair human body; On the other hand, biologically active polypeptides of the present invention can also enhancing body immunizing power, strengthen the phagocytic function of scavenger cell, improve the ability that body resists extraneous pathogenic infection, reduce body sickness rate, and directly to absorb through pipe intestinal digesting simulated experiment and be not degraded, enter the immunological rejection that can not cause body in body.In addition, simulation digestion experiment result shows that this biologically active polypeptides found is stable under the digested condition of common animal body, can not be degraded further, directly can be absorbed by animal body and play its bioactive functions had, to exploitation, there is anti-oxidant function, enhancing immunologic function, the food delayed senility, healthcare products and medicine tool are of great significance and using value.
Accompanying drawing explanation
Fig. 1: the wash-out collection of illustrative plates of peptide digestion product in thalline born of the same parents in lactobacterium helveticus fermented-milk
Fig. 2: mass chromatography extracts figure (m/z=1092.52)
Fig. 3: mass-to-charge ratio is the first mass spectrometric figure of the fragment of 1092.52
Fig. 4: mass-to-charge ratio is the second order ms figure of the fragment 1 of 1092.52
Fig. 5: mass-to-charge ratio is the az of the sequence that 1092.52 predictions obtain, by crack conditions (FGYSGAFKCL)
Fig. 6: Trolox typical curve
Embodiment
Before further describing the specific embodiment of the invention, should be understood that protection scope of the present invention is not limited to following specific specific embodiments; It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments, instead of in order to limit the scope of the invention.
When embodiment provides numerical range, should be understood that except non-invention is otherwise noted, between two end points of each numerical range and two end points, any one numerical value all can be selected.Unless otherwise defined, the same meaning that all technology used in the present invention and scientific terminology and those skilled in the art of the present technique understand usually.Except the concrete grammar used in embodiment, equipment, material, according to those skilled in the art to the grasp of prior art and record of the present invention, any method of prior art that is similar with the method described in the embodiment of the present invention, equipment, material or that be equal to, equipment and material can also be used to realize the present invention.
Unless otherwise indicated, disclosed in the present invention experimental technique, detection method, preparation method all adopt the routine techniques of the molecular biology of the art routine, biological chemistry, chromatin Structure and analysis, analytical chemistry, cell cultures, recombinant DNA technology and association area.These technology are existing in existing document improves explanation, specifically can see the MOLECULARCLONING:ALABORATORYMANUAL such as Sambrook, Secondedition, ColdSpringHarborLaboratoryPress, 1989andThirdedition, 2001; Ausubel etc., CURRENTPROTOCOLSINMOLECULARBIOLOGY, JohnWiley & Sons, NewYork, 1987andperiodicupdates; TheseriesMETHODSINENZYMOLOGY, AcademicPress, SanDiego; Wolffe, CHROMATINSTRUCTUREANDFUNCTION, Thirdedition, AcademicPress, SanDiego, 1998; METHODSINENZYMOLOGY, Vol.304, Chromatin (P.M.WassarmanandA.P.Wolffe, eds.), AcademicPress, SanDiego, 1999; And METHODSINMOLECULARBIOLOGY, Vol.119, ChromatinProtocols (P.B.Becker, ed.) HumanaPress, Totowa, 1999 etc.
The discovery of embodiment 1 bioactive peptide FGYSGAFKCL
One, the preparation of lactobacterium helveticus fermented-milk
Skim-milk (New Zealand NZMP board skim-milk) and water is adopted to configure the skimming milk (12g skim-milk joins in 88g water by being prepared as of skimming milk of 12wt%, lower same) of 12wt%.Subsequently, by the bacterial classification (Lactobacillushelveticus bought, CICC6024) activation namely with 2% amount be inoculated into 12% (W/V) sterilized non-fat Ruzhong cultivate, temperature 37 DEG C, incubation time 7h, namely completes the activation of lactobacterium helveticus, continuously activation 2 times, obtained fermented-milk, for subsequent use as lactobacterium helveticus fermented-milk starter.
Get the lactobacterium helveticus starter that 10mL prepared and be inoculated into (rate of vaccination is 2v/v%) in the sterilized 12wt% skimming milk of 500mL, 37 DEG C of fermentations, after 7 hours, after stirring out curdled milk, are preserved, are obtained lactobacterium helveticus fermented-milk under 4 DEG C of conditions.
Two, the obtained and confirmation of biologically active polypeptides mixture
1. experimental technique
1) sample preparation
Carry out centrifugal collection thalline in lactobacterium helveticus fermented-milk loading centrifuge tube previous step prepared, centrifugal condition is 4000rpm/min, 20 DEG C, 15min.Abandon supernatant liquor after centrifugal, taking precipitate, be required lactobacterium helveticus thalline.
By obtained thalline with 4000,4500,5000rpm/min, 20 DEG C, the centrifugal condition of 15min carries out three subgradient centrifuge washings, abandons supernatant liquor, taking precipitate after centrifugal.
Thalline after washing is added deionized water and is made into the phage solution that concentration is 0.0238g/ml, after carry out supersonic wave wall breaking, ultrasound condition is broken power 300W, broken total time 26min (ultrasonic 3s, interval 5s).
Bacterium peptide mixed solution after ultrasonic is carried out low-temperature centrifugation, and centrifugal condition is 9000rpm/min, 4 DEG C, 20min.Abandon precipitation after centrifugal, get supernatant liquor.
Poured into respectively in super filter tube by supernatant liquor, the molecular weight cut-off of filter membrane is 3kDa, and in ultra-filtration process, rotating speed is 4800r/min, and the time is 30min, and centrifuging temperature is 4 DEG C.Collect the filtrate of lactobacterium helveticus fermented-milk, in-4 DEG C of freezen protective.
Ultrafiltrated is carried out Solid-Phase Extraction, uses 1mLC18 pillar, 2mL methyl alcohol activation pillar, 1mLddH
2o balances pillar, and loading sample is the ultrafiltrated of 2mL, and last 400 μ L elution, wherein elutriant is the formic acid of 80:20v/v methanol/water and 0.1%v/v.
Dry powder is lyophilized into after being blown by the wash-out liquid nitrogen obtained, rear simulation pipe intestinal digesting experiment: first, adopt sterilizing deionized water dissolving dry powder, add stomach en-(available from Sigma) in the solution, ratio is that every gram of sample adds stomach en-20mg, regulate the pH value to 2.0 of reaction solution, in 37 DEG C of waters bath with thermostatic control, be incubated 90min; Then the pH value of reaction solution is adjusted to 7.5, add pancreatin (CorolasePP, purchased from German AB company), ratio is that every gram of sample adds pancreatin 40mg, in 37 DEG C of waters bath with thermostatic control, be incubated 150min; Finally be placed in 95 DEG C of water-baths to heat 5min and make enzyme deactivation, reaction solution freeze concentration is dry, make dry powder, under being stored in-20 DEG C of conditions, for subsequent use.
2) liquid matter is analyzed
Liquid phase chromatogram condition:
Instrument: WatersACQUITYUPLC Ultra Performance Liquid Chromatography instrument
Chromatographic column specification: CSHC18 chromatographic column
Flow velocity: 0.4mL/min
Temperature: 45 DEG C
Ultraviolet detection wavelength: 220nm
Sample size: 5 μ L
Mobile phase A liquid: ddH
2o
Mobile phase B liquid: acetonitrile solution
Gradient condition: 0min-2.5min keeps 99%A liquid, 1%B liquid; 2.5min-5minB liquid becomes 5%, A liquid from 1% and becomes 95% from 99%; 5min-10minB liquid becomes 10%, A liquid from 5% and becomes 90% from 95%; 10min-17min%B liquid becomes 25%, A liquid from 10% and becomes 75% from 90%; 17min-22min, B liquid becomes 40%, A liquid from 25% and becomes 60% from 75%; 22min-27min, B liquid becomes 80%, A liquid from 40% and becomes 20% from 60%; It is 0% that 27min-29min, B liquid becomes 100%, A liquid from 80%, keeps 2min; 31min-31.5min, B liquid becomes 5%, A liquid from 100% and becomes 95% from 0%; 31.5min-32min, B liquid becomes 1%, A liquid from 5% and becomes 99% from 95%; 32min-34min, keeps 99%A liquid, 1%B liquid.
Mass Spectrometry Conditions:
Ionic means: ES+
Mass range (m/z): 50-2000
Capillary voltage (Capillary) (kV): 3.0
Sampling spiroid (V): 35.0
Ion source temperature (DEG C)): 105
Desolventizing temperature (DEG C): 350
Taper hole airshed (L/Hr): 50.0
Desolventizing air-flow (L/hr): 600.0
Collision energy (eV): 6.0
Impinging air flows (ml/min): 0.6
Sweep time (sec): 0.26
The interscan time (sec): 0.02
According to above-mentioned experiment condition, utilize Masslynx software analysis, obtain peptide material retention time and molecular weight in lactobacterium helveticus fermented-milk digestion product, as shown in table 1, the mass chromatography extracting polypeptide with Masslynx software extracts figure, first mass spectrometric figure, sees Fig. 2 and Fig. 3.
Polypeptide nuclear-cytoplasmic ratio in table 1 lactobacterium helveticus fermented-milk digestion product
Three, the aminoacid sequence defining method of biologically active polypeptides
1. experimental technique
(1) foundation in lactoferrin aminoacid sequence storehouse, cow's milk source
Lactoferrin aminoacid sequence is obtained by BIOPEP search, builds up a database by searching for the milk proteins aminoacid sequence obtained.
2) peptide masses is right
UPLC-MS is analyzed to the quality obtained, search in milk serum albumin aminoacid sequence storehouse, obtain the peptide sequence of being correlated with.This step is realized by JAVA program and MySQL database, and concrete requirement is as follows: input the quality obtained by UPLC-MS, the quality of the peptide sequence of output quality error in ± 0.01, this sequence, the concrete dietary protein origin of this sequence.
3) checking of peptide sequence
The aminoacid sequence obtained is verified by the Biolynx in Masslynx software, prediction is obtained the actual second order ms figure that peptide sequence theoretic second order ms figure and MS/MS obtains contrast, software by peak main in second order ms figure whether to success, provide score value, confirm to predict the polypeptide obtained.Second order ms is to figure and the az predicting the sequence obtained, and by crack conditions figure is shown in Fig. 4 and Fig. 5.
2. experimental result
Obtaining biologically active polypeptides FGYSGAFKCL through checking is milk-derived, specifically derives from Cow Milk ferritin, for the amino-acid residue of lactoferrin 190-199 position is designated as SEQIDNO:1.
In addition, described biologically active polypeptides FGYSGAFKCL obtains through gi tract simulation digestion degraded, is not be originally present in lactobacterium helveticus fermented-milk.It can exist after the effect of digestive ferment, illustrates that polypeptide has stability, is not easily degraded after gi tract simulation digestion trial.
The anti-oxidant activity experiment of embodiment 2 biologically active peptides
Adopt scavenging free radicals method (DPPH method) and total antioxidant capacity method (ABTS method), the anti-oxidant activity of the biologically active polypeptides that embodiment 1 obtains is tested.
1, [DPPH] method measures the antioxidation activity in vitro of biologically active peptides
1) experiment reagent and instrument
Reagent: 1,1-phenylbenzene-2-trinitrophenyl-hydrazine (1,1-Diphenyl-2-picrylhydrazyl [DPPH]), Japanese Wako company produces; Methyl alcohol, Shanghai traditional Chinese medicines company provides; The milk-derived biologically active polypeptides obtained in synthetic example 1.
Key instrument: Pro200 microplate reader, Austrian Tecan Products; 96 porocyte culture plates, Millipore company of the U.S. manufactures; Analytical balance, Meitelei-tolido Products.
2) experimental technique
(1) 0.1mmol/L [DPPH] methanol solution
Taking 19.72mg [DPPH] with analytical balance is dissolved in 500mL methanol solution, and prepare 0.1mmol/L [DPPH] methanol solution obtained, tinfoil keeps in Dark Place, and namely joins and namely uses.
(2) [DPPH] method measures the anti-oxidant activity of biologically active peptides
In 1.5mLEP pipe, add 500 μ L concentration be 0.1mmol/L [DPPH] methanol solution, add the testing sample of 500 μ L different concns and deionized water respectively by table 2 as blank.
After detected sample application of sample, mix, room temperature is got in 200 microlitre to 96 orifice plates after leaving standstill 30min, detects light absorption value by microplate reader at 517nm place.Calculate free radical scavenging activity according to the following formula, experimental result is in table 2.
Formula: [DPPH] free radical scavenging activity=(A
0-A
s)/A
0× 100%
Wherein A
0represent the light absorption value of blank group, A
srepresent the light absorption value of sample sets.
Table 2DPPH method measures the total antioxidant capacity result of biologically active polypeptides
By [DPPH] method, the external total antioxidant activity of biologically active polypeptides FGYSGAFKCL is measured, find that it has Scavenging ability, concrete IC
50 (mg/mL)value is in table 2.Can assert that the biologically active polypeptides FGYSGAFKCL of invention has good resistance of oxidation according to the standard of relative antioxidant ability.
2, ABTS method measures the antioxidation activity in vitro of biologically active peptides
1) experiment reagent and instrument
Reagent: antioxidative activities test kit (ABTS method), green skies company produces; Methyl alcohol, Shanghai traditional Chinese medicines company provides; The milk-derived biologically active polypeptides obtained in synthetic example 1.
Key instrument: Pro200 microplate reader, Austrian Tecan Products; 96 porocyte culture plates, Millipore company of the U.S. manufactures.
2) experimental technique
(1) configuration of ABTS working fluid
Get 40 microlitre ABTS solution and 40 microlitre oxidizing agent solutions are mixed with ABTS working stocks, after preparation, lucifuge is deposited after 12-16 hour and is used, and the ABTS working stocks prepared at room temperature lucifuge is deposited, stable in 2-3 days.Before using, ABTS working stocks PBS is diluted to ABTS working fluid, about 40 times, after requiring that the absorbancy of ABTS working fluid deducts corresponding PBS blank, A734 is 0.7 ± 0.05.
(2) making of Trolox typical curve
With sample preparation solution dilution standard substance, 10mMTrolox standardized solution is diluted to 0.005,0.01,0.03,0.05,0.1,0.25,0.5 and 1.0mM.In each detect aperture of 96 orifice plates, add 200 microlitre ABTS working fluids, add the Trolox standardized solution of the various concentration of 10 microlitre in typical curve detect aperture, mix gently.Incubated at room, after 4 minutes, measures light absorption value by microplate reader at 734nm place.
Trolox concentration and light absorption value are good proportional relation, and concentration is higher, and light absorption value is lower.Trolox typical curve of the present invention the results are shown in Figure 6, and the linear relationship of typical curve is good, and relation conefficient is that the preci-sion and accuracy of 0.9995, Trolox typical curve all meets testing requirement, can be used for subsequent calculations.
(3) ABTS method measures the anti-oxidant activity of biologically active peptides
In each detect aperture of 96 orifice plates, add 200 microlitre ABTS working fluids, in blank control wells, add 10 microlitre PBS solution, add the various sample of 10 microlitre in sample detection hole, mix gently.Incubated at room, after 4 minutes, measures light absorption value by microplate reader at 734nm place.Measure the total antioxidant capacity calculating sample according to typical curve.Total antioxidant capacity representation represents with the concentration of Trolox standardized solution.Calculate total antioxidant capacity according to the following formula, experimental result is in table 3:
Table 3ABTS method measures the total antioxidant capacity result of biologically active polypeptides
By total antioxidant capacity method (ABTS method), the external total antioxidant activity of biologically active polypeptides FGYSGAFKCL is measured, find that biologically active polypeptides FGYSGAFKCL has resistance of oxidation: under peptide concentration is 1mg/mL situation, the Trolox concentration corresponding to it is 0.9398mmol/L.Therefore, the biologically active polypeptides FGYSGAFKCL of invention can be assert
There is good resistance of oxidation.
The above; be only preferred embodiment of the present invention; above-described embodiment is illustrative principle of the present invention and effect thereof only; and not to any formal and substantial restriction of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; also can make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.All those skilled in the art, without departing from the spirit and scope of the present invention, a little change made when utilizing disclosed above technology contents, the equivalent variations of modifying and developing, be Equivalent embodiments of the present invention; Meanwhile, all according to substantial technological of the present invention to the change of any equivalent variations that above-described embodiment is done, modify and differentiation, all still belong in the scope of technical scheme of the present invention.
Claims (10)
1. the biologically active polypeptides FGYSGAFKCL be separated, it comprises
A polypeptide that () is made up of the amino acid shown in SEQIDNO:1;
(b) or in the aminoacid sequence shown in SEQIDNO:1, be substituted, lack or add one or several amino acid and there is the polypeptide derivative by (a) of same isoreactivity.
2. the nucleotide fragments of biologically active polypeptides FGYSGAFKCL described in coding claim 1.
3. the preparation method of biologically active polypeptides described in claim 1, comprises the steps:
1) ferment: lactobacterium helveticus is added in skimming milk and carry out anaerobically fermenting, obtain lactobacterium helveticus fermented-milk;
2) broken wall: the thalline obtained after centrifugal to fermented-milk carries out ultrasonic, broken bacterium wall makes peptide in born of the same parents dissociate, and obtains bacterium peptide mixed solution;
3) slightly the carrying of polypeptide: to step 2) in bacterium peptide mixed solution carry out low-temperature centrifugation separation, get supernatant liquor;
4) purifying of polypeptide:
A. to step 3) supernatant liquor carry out uf processing, collect filtrate;
B. solid-phase extraction column is separated: the filtrate of collection adopts WatersSep-pakC18 Solid-Phase Extraction column extracting, collection of biological active polypeptide mixture;
5) digestion of polypeptide and stability: adopt two step enzymolysis process enzymolysis step 4) biologically active polypeptides mixture, obtain biologically active polypeptides mixture; The enzyme that the first step enzymolysis adopts is stomach en-, and the enzyme that second step enzymolysis adopts is pancreatin.
4. preparation method as claimed in claim 3, is characterized in that, step 1) condition of described anaerobically fermenting is: leavening temperature 36 ~ 38 DEG C, fermentation time 6 ~ 8h.
5. preparation method as claimed in claim 3, is characterized in that, step 2) described centrifugal condition is 20 DEG C, 4000 ~ 5000rpm, centrifugal 15min.; Step 2) condition of described broken wall is cell concentration 0.0238g/ml, broken power 300W, broken total time 26min.
6. preparation method as claimed in claim 3, is characterized in that, step 3) in, the condition of described low-temperature centrifugation is: 4 DEG C, 8000 ~ 10000rpm, centrifugal 15 ~ 30min.
7. preparation method as claimed in claim 3, is characterized in that, step 3) uf processing described in a adopts is the super filter tube that molecular weight cut-off is respectively 3kDa; In described process of ultrafiltration treatment, rotating speed is 4800r/min, and the time is 30min, and centrifuging temperature is 4 DEG C.
8. the application of derivative in the food of the anti-oxidant and/or enhancing body immunizing power of preparation, healthcare products and medicine of biologically active polypeptides FGYSGAFKCL or described biologically active polypeptides described in claim 1.
9. an anti-oxidation medicine, comprises the derivative of biologically active polypeptides FGYSGAFKCL or described biologically active polypeptides as claimed in claim 1.
10. an enhancing body immunizing power medicine, comprises the derivative of biologically active polypeptides FGYSGAFKCL or described biologically active polypeptides as claimed in claim 1.
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