CN103232527B

CN103232527B - Bioactive polypeptide SLPQ and its preparation method and use

Info

Publication number: CN103232527B
Application number: CN201310047985.9A
Authority: CN
Inventors: 张少辉; 卢姗姗; 孙冠华; 马鎏镠; 周婕慧; 郭海滨; 胡迪
Original assignee: Shanghai Jiaotong University
Current assignee: Zhang Shaohui; Zhejiang Huitai Life Health Technology Co ltd
Priority date: 2013-02-06
Filing date: 2013-02-06
Publication date: 2015-06-10
Anticipated expiration: 2033-02-06
Also published as: CN103232527A; WO2014121725A1

Abstract

The invention relates to the field of proteins and especially relates to a bioactive polypeptide SLPQ and its preparation method and use. The bioactive polypeptide SLPQ is a milk-derived bioactive polypeptide having the effect of improving immunity and has an amino acid sequence of Ser-Leu-Pro-Gln. An external immunity promotion experiment proves that the bioactive polypeptide SLPQ can improve immunity, external propagation capability of lymphocyte and macrophage, a nitric oxide induction amount of macrophage, and external pathogen infection resistance, reduce incidence of diseases, avoid immunological rejection and stably exist in the digestive tract, is not decomposed, is directly absorbed by the human body and has bioactive effects. The bioactive polypeptide SLPQ lays a good foundation for development of SLPQ-containing health products and functional dairy products.

Description

A kind of biologically active polypeptides SLPQ and Synthesis and applications thereof

Technical field

The present invention relates to albumen field, be specifically related to a kind of milk-derived biologically active polypeptides SLPQ and Synthesis and applications thereof.

Background technology

At cow's milk in the process of lactobacillus-fermented, a part of protein in cow's milk is utilized by milk-acid bacteria metabolism, and there occurs a series of biochemical reactions, protein is made to become polypeptide or free amino acid, digested or directly entered the blood circulation of human body by the absorption and transport of intestinal epithelial cell.In these polypeptide, some has special physiological function, is called as " biologically active peptides ".

Immune-active peptides obtains from Ruzhong first after opioid peptides finds and proves a class biologically active polypeptides of its physiologically active.People's Late Cambrian such as Jolles in 1981, utilize trypsin hydrolyzing people lactoprotein, can obtain six peptides that an aminoacid sequence is Val-Glu-Pro-Ile-Pro-Tyr, experiment in vitro proves that this peptide can strengthen the phagolysis of Turnover of Mouse Peritoneal Macrophages to sheep red blood cell (SRBC).The people such as Migliore-Samour finds sheep erythrocyte can be stimulated the phagolysis of mouse peritoneal macrophages and the opposing that strengthens for Klebsiella Pneumoniae from caseic six peptide Thr-Thr-Met-Pro-Leu-Trp.Rat of feeding finds that the immunoloregulation function that the phagolysis of rat peritoneal macrophages is relevant with red corpuscle has significant enhancing for newborn source immunomodulatory peptides (PGPIPN) of the people such as Li Suping synthesis.

Research shows, immune-active peptides can not only enhancing body immunizing power, stimulate the lymphocytic propagation of body, strengthen the phagocytic function of scavenger cell, promote the release of cytokine, improve the ability that body resists extraneous pathogenic infection, reduce body sickness rate, and the immunological rejection of body can not be caused.

No matter immune-active peptides is as functional food or medicine, and it finally all will be taken in human body and be absorbed by the body, and only has and arrives corresponding its physiological function of target organ competence exertion.Under human gastrointestinal tract complex environment, peptide is as easy as rolling off a log to be subject to the impact of a series of enzyme such as stomach en-, trypsinase and to degrade, thus loses physiologically active in vivo, cannot accomplish the end in view.Therefore, the ability studying the anti-gastrointestinal tract enzyme liberating of immunomodulatory peptides is significant for its application from now on.

Summary of the invention

The object of the present invention is to provide a kind of biologically active polypeptides, its aminoacid sequence is Ser-Leu-Pro-Gln(SLPQ, SEQ ID NO:1).

Preferably, the source of described biologically active polypeptides is milk-derived.

Biologically active polypeptides SLPQ of the present invention is milk-derived, specifically derives from beta-casein, and is the amino-acid residue of beta-casein (SEQ IDNO:3) 84th ~ 87.

Preferably, described biologically active polypeptides has the function of enhancing body immunizing power.

Biologically active polypeptides of the present invention by engineered method and chemical process synthetic, also can the method by separation and purification from milk-product directly can obtain.

The invention also discloses the nucleotide fragments of coding aforementioned biological active polypeptide.

The aminoacid sequence of beta-casein and nucleotides sequence are classified as existing technology, the biologically active polypeptides SLPQ of the nucleotide fragments energy encoding mature of encoding ss-casein 84th ~ 87 amino acids residue.

Further, the nucleotide fragments of coding aforementioned biological active polypeptide, its sequence is: agc ctc cca cag(SEQ IDNO:2).

Second aspect present invention discloses the preparation method of aforementioned biological active polypeptide, and step is as follows:

1) ferment: lactobacterium helveticus (Lactobacillus helveticus) is added in skimming milk and carries out anaerobically fermenting, obtain lactobacterium helveticus fermented-milk;

2) slightly the carrying of polypeptide: low-temperature centrifugation separation is carried out to the lactobacterium helveticus fermented-milk of step 1), gets supernatant liquor;

3) purifying of polypeptide:

A. to step 2) supernatant liquor carry out uf processing, collect filtrate;

The filtrate of b. collecting adopts reverse chromatography column SOURSE5RPC ST(4.6 × 150mm) carry out RPLC separation, collecting elution time is the polypeptide mixture of 21min;

C. adopt ultra-high efficiency liquid phase-electron spray(ES)-level Four bar-flight time matter instrument to be separated and obtain polypeptide SLPQ.

Skimming milk of the present invention is the milk-product through skimming treatment, and in usual skimming milk, lipid content is less than 0.1%.

Preferably, the condition of anaerobically fermenting described in step 1) is: leavening temperature 36 ~ 38 DEG C, fermentation culture 15 ~ 20h; Preferred fermentation culture 19h.

Preferably, step 2) condition of described low-temperature centrifugation is: 4 DEG C, 8000 ~ 10000rpm, centrifugal 15 ~ 30min.

Preferably, the molecular weight cut-off of filter membrane that ultrafiltration process described in step 3) a adopts is respectively 10kDa and 3kDa.The present invention adopts molecular weight cut-off to be respectively the filter membrane of 10kDa, 3kDa, makes sample carry out ultrafiltration by two filter membranes successively.

More excellent, in ultra-filtration process described in step 3) a, pressure range is 0.1 ~ 0.3MPa, and filtrate flow velocity is 0.8 ~ 1.2mL/min.

Preferably, in step 3) b RPLC partition method, mobile phase A is the ddH containing 2% acetonitrile and 0.05%TFA ₂o; Mobile phase B is 100% acetonitrile.

The molecular weight of biologically active polypeptides SLPQ of the present invention is 444.24Da.

Third aspect present invention discloses the application of aforementioned biological active polypeptide in the food of preparation enhancing body immunizing power, healthcare products and medicine.

The invention also discloses the application in the food of nucleotide fragments in preparation enhancing body immunizing power of coding aforementioned biological active polypeptide, healthcare products and medicine.

Biologically active polypeptides SLPQ of the present invention may be used for the dairy foodstuff such as Yoghourt, and be not degraded because biologically active polypeptides SLPQ of the present invention directly can be absorbed by gi tract, therefore may be used for preparing the healthcare products improving immunizing power, or the medicine of enhancing body immunizing power.Encode the nucleotide fragments of described biologically active polypeptides SLPQ owing to may be used for preparing SLPQ, therefore, also may be used for preparing food, improve the healthcare products of immunizing power or the medicine of enhancing body immunizing power.

Fourth aspect present invention discloses a kind of enhancing body immunizing power medicine, comprises the derivative of aforementioned biological active polypeptide SLPQ or aforementioned biological active polypeptide SLPQ.

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.

Biologically active polypeptides SLPQ of the present invention can enhancing body immunizing power, strengthen the in-vitro multiplication ability of lymphocyte and scavenger cell, promote that the scavenger cell nitrogen protoxide amount of inducing increases, improve the ability that body resists extraneous pathogenic infection, reduce body sickness rate, and the immunological rejection of body can not be caused, have exploitation and strengthen the milk-product of immunologic function and healthcare products tool is of great significance.

Accompanying drawing explanation

Fig. 1: lactobacterium helveticus fermented-milk and the mass spectrum comparison diagram (A:3000Da unleavened skimming milk crude extract mass spectrum, B:3000Da lactobacterium helveticus fermented-milk crude extract mass spectrum) without crude extract after the skim milk ultra of fermentative processing

Fig. 2: 3000Da without fermented skim milk crude extract and 3000Da lactobacterium helveticus fermented skim milk crude extract molecular weight difference and relative abundance comparatively

Fig. 3: RPLC is separated (a curve: the wash-out collection of illustrative plates of control fermentation breast RPLC 215nm of biologically active polypeptides comparison diagram in control fermentation breast and lactobacterium helveticus fermented-milk; B curve: the wash-out collection of illustrative plates of lactobacterium helveticus fermented-milk 3000Da supernatant liquor RPLC 215nm)

The first mass spectrometric at Fig. 4: B peak and second order ms original graph

Fig. 5: through the second order ms figure (m/z=444) at the B peak that Masslynx software processes is crossed

The aminoacid sequence of Fig. 6: the B little peptide in peak and az, by crack conditions

Fig. 7: biologically active polypeptides SLPQ total ion current figure before and after digestive ferment process

Fig. 8: biologically active polypeptides SLPQ through the first mass spectrometric figure of stomach en--pancreatin process gained 444.24 leading peak

Fig. 9: biologically active polypeptides SLPQ through the first mass spectrometric figure of stomach en--pancreatin process gained 444.24 postpeak

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 MOLECULAR CLONING:A LABORATORY MANUAL such as Sambrook, Second edition, Cold SpringHarbor Laboratory Press, 1989and Third edition, 2001; Ausubel etc., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987andperiodic updates; The series METHODS IN ENZYMOLOGY, Academic Press, SanDiego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, AcademicPress, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc.

The preparation of embodiment 1 bioactive peptide

One, the preparation of fermented-milk

1) 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, lower same) of 12wt%.Aseptically, picking lactobacterium helveticus (Lactobacillus helveticus, CICC6024) bacterium colony three ring, is added in the skimming milk of sterilized 12wt%, is stirred.After having inoculated, with aluminium foil sealing, with preventing pollution.Be placed in incubator 37 DEG C to cultivate 19 hours.After cultivation terminates, curdled milk is stirred, namely complete the activation of lactobacterium helveticus, the obtained starter for the preparation of lactobacterium helveticus fermented-milk.

Get the lactobacterium helveticus starter that 10mL prepared and be inoculated into the sterilized 12%(v/v of 500mL) in skimming milk (rate of vaccination is 2v/v%), 37 DEG C of fermentations, after 19 hours, after stirring out curdled milk, are preserved, are obtained lactobacterium helveticus fermented-milk under 4 DEG C of conditions.

2) control fermentation breast

Adopt same method, use lactobacillus bulgaricus (Lactobacillus Bulgaricus, LB340) and thermophilus mattress (Streptococcus Thermophilus, TA40) to make control fermentation breast as fermented bacterium.

Concrete grammar is: aseptically, and respectively picking lactobacillus bulgaricus and thermophilus mattress bacterium colony three ring, add sterilized 12%(w/w respectively by it) skimming milk in, stir.After having inoculated, with aluminium foil sealing, with preventing pollution.Be placed in incubator 37 DEG C and cultivate 19h.After cultivation terminates, curdled milk is stirred, namely complete the activation of lactobacillus bulgaricus and thermophilus mattress, obtained two kinds of starters for the preparation of control fermentation breast.

Get fermentation using lactobacillus bulgaricus agent and the agent of 5mL streptococcus thermophilus fermentation that 5mL prepared, co-inoculation to (rate of vaccination is 2v/v%) in sterilized 12% skimming milk of 500mL, 37 DEG C fermentation 19h after, after stirring out curdled milk, preserve under 4 DEG C of conditions, obtain control fermentation breast.

Two, the confirmation of biologically active polypeptides

1. experimental technique

1) sample preparation

The lactobacterium helveticus fermented-milk respectively prepared by previous step and control fermentation breast, and carry out low-temperature centrifugation in the skimming milk loading centrifuge tube of 12wt%, centrifugal condition is 9000rpm/min, 4 DEG C, 20min.Abandon precipitation after centrifugal, get supernatant liquor.

Supernatant liquor is poured into respectively in ultrafiltration cup, in order to prevent biologically active polypeptides to be oxidized, opening nitrogen pot pressure valve and filling nitrogen, opening magnetic stirring apparatus simultaneously, occurring concentration polarization phenomenon to prevent solution.Make sample be the filter membrane of 10kDa, 3kDa respectively by molecular weight cut-off, collect in time having filtrate to flow out.

In ultra-filtration process, should keep flow speed stability, filtrate is limpid.Flow rate control is at about 1mL/min, and pressure is 0.1 ~ 0.3MPa, collect lactobacterium helveticus fermented-milk, control fermentation breast respectively, and the filtrate of unfermentable 12wt% skimming milk is as experiment sample, control sample and blank, in-4 DEG C of freezen protective.

2) mass spectroscopy

Filtrate (experiment sample) after the lactobacterium helveticus fermented-milk ultrafiltration of collect previous step and the filtrate (blank) after skim milk ultra carry out mass spectroscopy, and Mass Spectrometry Conditions is as follows:

Ionic means: ES+

Mass range (m/z): 100-1500

Capillary voltage (Capillary) (kV): 3.0

Sampling spiroid (V): 35.0

Ion source temperature (DEG C)): 100

Desolventizing temperature (DEG C): 350

Desolventizing air-flow (L/hr): 600.0

Collision energy (eV): 6.0

Sweep time (sec): 0.3

The interscan time (sec): 0.02

2. experimental result

The mass spectrum comparing result of the filtrate (experiment sample) after the ultrafiltration of lactobacterium helveticus fermented-milk and the filtrate (blank) after skim milk ultra is shown in Fig. 1 ~ 2.A curve in Fig. 1 is the unleavened skimming milk of 3000Da (blank) crude extract sample, and the B curve in Fig. 1 is 3000Da lactobacterium helveticus fermented-milk crude extract sample (experiment sample).Through relatively finding out, after lactobacterium helveticus fermentation, 3000Da is without in the component of fermented-milk crude extract and 3000Da lactobacterium helveticus fermented skim milk crude extract, great changes will take place, and these components are due to the different retention time of hydrophobicity also difference to some extent.This mass spectrum mass range is 100Da-1500Da, and therefore known skimming milk has the small-molecule substance of absorption relatively less at below 1500Da, 210nm place; And after lactobacterium helveticus fermentation, the material showed increased in this molecular weight ranges, describes in the skimming milk that these polypeptide are not present in without fermentative processing, but just generation after the lactobacterium helveticus fermentation.And we find the prolongation along with fermentation time, the abundance showed increased of polypeptide, further demonstrate that the fermentation by lactobacterium helveticus, in skimming milk, original high molecular weight protein is decomposed, from the many and micromolecule polypeptide of complexity of the single high molecular weight protein amount of becoming.

Fig. 2 is 3000Da without the comparative result of fermented skim milk (blank) crude extract and 3000Da lactobacterium helveticus fermented skim milk (experiment sample) crude extract differing molecular quantity of material abundance difference.Longitudinal axes to show that in skimming milk crude extract contents is verified the molecular weight of answering, and lateral shaft to show in fermented-milk crude extract that contents is verified the molecular weight of answering.By comparing, lactobacterium helveticus fermented-milk can be obtained and without in fermented skim milk, because the molecular weight of the material differed greatly brought that ferments, thus select the parent ion of these mass-to-charge ratioes further to be analyzed by second order ms.

As shown in Figure 1, molecular weight 397.07Da, the retention time material of 6.72 minutes content in skimming milk crude extract higher (peak in Fig. 1-A collection of illustrative plates), and control fermentation Ruzhong does not almost have.And molecular weight is 232.15Da, retention time is that the content of material in fermented-milk and skimming milk of 3.61min is all higher.Therefore, we have selected that content in fermented-milk is higher and content is lower in unleavened skimming milk component has carried out comparison in difference, and comparative result is in table 1.

According to MarkerLynx Marker software analysis, (p>0.05) molecular weight fragment obtaining having significant difference is as shown in table 1, according to abundance and mass-to-charge ratio situation, select 444.24Da, retention time is the sequencing analysis that the polypeptide of 16.20min carries out second order ms.

Table 1:3000Da fermented-milk crude extract and 3000Da skimming milk crude extract comparison in difference

Three, the high-efficient liquid phase technique separating-purifying of biologically active polypeptides

1. laboratory apparatus and reagent

Instrument: kTA protein purification instrument purifier10

Chromatographic column specification: SOURSE5RPC ST4.6/150

Flow velocity: 1mL/min

Temperature: 25 DEG C

Ultraviolet detection wavelength: 215nm

Mobile phase A: the ddH2O containing 2% acetonitrile and 0.05%TFA

Mobile phase B: 100% acetonitrile

Sample size: 1mL

Gradient condition: 0min-7.5min keeps 100%A liquid; 7.5min-42.5minB liquid becomes 50% from 0%; 42.5min-45minB liquid becomes 100% from 50%; 45min-50min keeps 100%B liquid; 50min-72minA liquid becomes 100% from 0%.

2. experimental technique

Sample pre-treatments: sample and control sample and mobile phase A liquid will be tested and half-and-half dilute (volume ratio 1:1 dilutes), as loading sample.Loading sample carries out rp-hplc analysis, and experimental result is shown in Fig. 3.

3. experimental result

As seen from Figure 3, a curve is the wash-out collection of illustrative plates of the RPLC 215nm of control sample, elution time is that 26min place has an obvious absorption peak, all the other peak heights are relatively low, according to the proportional relation of absorption value and peptide bond concentration, can think that its polypeptides matter is less, and kind is single in 12% control fermentation breast 3000Da supernatant liquor (control sample).B curve is the wash-out collection of illustrative plates of lactobacterium helveticus fermented-milk 3000Da supernatant liquor (experiment sample) RPLC 215nm, compared with control fermentation breast, absorption peak showed increased in the anti-phase collection of illustrative plates of lactobacterium helveticus fermented-milk 3000Da supernatant liquor, and be that 21min, 24min and 33min place has three places the most significantly absorption peak in elution time, in experiment, these three peaks are collected, be recorded as fermented-milk isolate B peak, fermented-milk isolate C peak and fermented-milk isolate D peak respectively.

Contrast with the retention time of former fermented-milk isolate B peak, the corresponding Middle Molecular Substance in C peak and D peak according to the polypeptide corresponding to each molecular weight, find that the Material Source of 444.24Da is in the B peak of fermented-milk isolate.

By control fermentation breast and the comparison of lactobacterium helveticus fermented-milk, can find to ferment the fermented-milk obtained through lactobacterium helveticus, its contain than to kefir milk approved for distribution more horn of plenty, molecular weight is less than the peptide material of 3000Da.These polypeptides matters are because the intracellular enzyme of the large protein in former skimming milk secreted by lactobacterium helveticus and extracellular enzyme decompose, and discharge that some polypeptide fragments and free amino acid formed.Extracellular enzyme secreted by milk-acid bacteria has non-specific or specific cutting to beta-casein fragment in dairy products.Usually these polypeptides matters obtained by fermentable, very likely have certain biological activity.If use lactobacillus bulgaricus and thermophilus streptococcus combinations produce common sour milk, because the output of polypeptide is few, kind is single, and biological activity is relatively low.

According to RPLC principle, the poor material of hydrophobicity, due to more weak with separator column solid phase bonding force, first elutes from separator column, and the good material of hydrophobicity and the effect of separator column solid phase bound are comparatively greatly, after be eluted from separator column.Can obtain thus, three its hydrophobicitys of isolate arrange in the following order: >D peak, >C peak, lactobacterium helveticus fermented-milk isolate B peak.Through collecting operation, obtaining the sample of B peak value, adopting Vacuum Freezing & Drying Technology to carry out lyophilize ,-4 DEG C of freezings, as the experiment material of follow-up mass spectroscopy.

Four, the mass spectroscopy purifying of biologically active polypeptides and determined amino acid sequence

1. experimental technique

(1) chromatographic condition:

Instrument: Waters ACQUITY UPLC ultra-high efficiency liquid phase-electron spray(ES)-level Four bar-flight time matter instrument

Chromatographic column specification: BEH C18 chromatographic column

Flow velocity: 0.4mL/min

Temperature: 45 DEG C

Ultraviolet detection wavelength: 210nm

Sample size: 7 μ L

Gradient condition: 0min-3min keeps 99%A liquid, 1%B liquid; 3min-9minB liquid becomes 5%, A liquid from 1% and becomes 95% from 99%; 9min-15minB liquid becomes 10%, A liquid from 5% and becomes 90% from 95%; 15min-21min%B liquid becomes 25%, A liquid from 10% and becomes 75% from 90%; 21min-24min, B liquid becomes 40%, A from 25% and also becomes 60% from 75%; 24min-27min, B liquid becomes 80%, A liquid from 40% and becomes 20% from 60%; 27min-27.5min keep 80%B liquid, 20%A liquid; 27.5min-28min, B liquid becomes 5%, A liquid from 80% and becomes 95% from 20%; 28min-28.5min, B liquid becomes 1%, A liquid from 5% and becomes 99% from 95%; 28.5min-30min, keep 99%A liquid, 1%B liquid.A liquid: the ddH2O containing 2% acetonitrile and 0.05%TFA; B liquid: 100% acetonitrile

(2) Mass Spectrometry Conditions:

Ionic means: ES+

Mass range (m/z): 100-1500

Capillary voltage (Capillary) (kV): 3.0

Sampling spiroid (V): 35.0

Ion source temperature (DEG C)): 100

Desolventizing temperature (DEG C): 350

Desolventizing air-flow (L/hr): 600.0

Collision energy (eV): 6.0

Sweep time (sec): 0.3

The interscan time (sec): 0.02

Second order ms parent ion quality (m/z): 444.24

According to above-mentioned experiment condition, utilize ultra-high efficiency liquid phase-electron spray(ES)-level Four bar-flight time mass spectrum, obtain first mass spectrometric figure, second order ms figure that lactobacterium helveticus fermented-milk isolate B peak middle-molecular-weihydroxyethyl is the polypeptide of 444.24Da, and by Masslynx computed in software aminoacid sequence, the results are shown in Figure 4-6.

2. experimental result

Calculate through Masslynx software analysis, to obtain molecular weight be the aminoacid sequence of the active polypeptide fragment of 444.25Da is Ser-Leu-Pro-Gln(SLPQ), be designated as SEQ ID NO:1.This fragment derives from lactobacterium helveticus fermented-milk isolate B peak, corresponding with the residue sequence of 84 ~ No. 87 of beta-casein, and the genbank sequence number of beta-casein aminoacid sequence is AAA30431.1, and sequence is shown in SEQ ID NO:3.

The promotion immunity of organisms activity experiment of embodiment 2 biologically active peptides

One, mtt assay measures the vitro lymphocyte proliferation capacity experimental of biologically active polypeptides SLPQ

1) experiment material and instrument:

Reagent and material: laboratory animal balb/c mouse (male 6-8 age in week, Shanghai Communications University's agricultural and biological institute animal experimental center); After lactobacterium helveticus fermentation, mass spectroscopy is separated the milk-derived biologically active polypeptides SLPQ obtained; Mouse lymphocyte extracting solution (purchased from Suo Laibao company); RPMI1640 substratum (purchased from GIBCO company); 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide bromine salt (MTT, purchased from Amresco company); ConA (ConA, available from Sigma); Bovine serum albumin (BSA, purchased from Genebase company); Stomach en-(available from Sigma); Pancreatin (CorolasePP, purchased from AB company).

Instrument: LRH-250F biochemical cultivation case, the permanent Science and Technology Ltd. in Shanghai; GL-22M high speed freezing centrifuge, Shanghai Lu Xiang instrument whizzer Instrument Ltd.; Hera cell150CO ₂incubator, Heraeus company; Dragon Wellscan MK3 microplate reader, Labsystems company; ALPHA1-2-LD vacuum freeze drier, Christ company; Ultra Performance Liquid Chromatography-quadrupole time-of-flight mass spectrometer, waters company.

2) experimental technique:

Get mouse spleen under aseptic condition, extract mouse lymphocyte with lymphocyte extracting solution, carry out first culture.With complete RPMI1640 nutrient solution, cell density is adjusted to 2.5 × 10 ⁶individual/mL.Add successively in 96 porocyte culture plates: 100 μ L mouse lymphocyte suspensions, 100 μ L RPMI1640 complete culture solutions, 20 μ L ConAs, 100 μ L samples.In addition, arrange blank group (pH7.2 ~ 7.4, the PBS of 3mol/L) and negative control group (500 μ g/mL BSA), research shows that it does not affect for vitro lymphocyte proliferation.Often organize 3 parallel laboratory test samples.At 5%CO ₂after cultivating 68h in 37 DEG C of incubators, under aseptic condition, every hole adds 20 μ L MTT, continues to cultivate 4h, careful abandoning supernatant, and every hole adds 100 μ L dimethyl sulfoxide (DMSO), and 37 DEG C of biochemical cultivation case hatching 10min, shake up, measure light absorption value by microplate reader at 570nm place.

Vitro lymphocyte proliferation ability stimulation index represents, method of calculation are as follows:

In formula: A1 is the light absorption value of blank under 570nm place; A2 is the light absorption value of negative control group under 570nm place, and A3 is the light absorption value of experimental group under 570nm place.

3) experimental result and analysis

Experimental result is in table 2.As shown in Table 2, be that under the condition of 500 μ g/mL, the stimulation index of milk-derived biologically active peptides SLPQ is greater than BSA in the mass concentration of biologically active peptides SLPQ, illustrate that SLPQ can stimulate the propagation of external mouse lymphocyte to a certain extent.And the stimulation index of SLPQ reaches 1.298, and negative control group has significant difference (P<0.05).Therefore, can assert that this lactobacterium helveticus fermented-milk is separated to active polypeptide SLPQ there is the ability significantly promoting mouse lymphocyte propagation, can eat as a kind of healthcare products or additive, the specific immunity ability of animal and human's body can be improved.

Table 2 biologically active polypeptides SLPQ is on the impact of vitro lymphocyte proliferation

Note: * labelled notation, for compare with negative control, has significant difference (P < 0.05).

Two, mtt assay measures the macrophages in vitro multiplication capacity experiment of biologically active polypeptides SLPQ

1) experiment reagent and instrument

Reagent: laboratory animal balb/c mouse (male 6-8 all ages) Shanghai Communications University's agricultural and biological institute animal experimental center; Lactobacterium helveticus fermentation mass spectroscopy is separated the milk-derived biologically active polypeptides SLPQ obtained; 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide bromine salt (MTT) Amresco company; LPS(lipopolysaccharides) Sigma company; Bovine serum albumin (Bovine Serum Albumin, BSA) Genebase company; Three lysates, containing the aqueous solution of 10%SDS, 5% isopropylcarbinol and 0.012mol/L HCl.

Plant and instrument: the permanent Science and Technology Ltd. in LRH-250F biochemical cultivation case Shanghai; GL-22M high speed freezing centrifuge Shanghai Lu Xiang instrument whizzer Instrument Ltd.; Hera cell150CO ₂incubator Heraeus company; Dragon WellscanMK3 microplate reader Labsystems company.

2) test method:

The 2%(w/w of balb/c mouse peritoneal injection 2mL) sterilizing starch solution, continuously injection three days, inject disconnected neck after 24 hours for the last time and put to death.Peel off skin of abdomen, draw 4 DEG C of phosphate buffered saline buffers (PBS) with syringe and repeatedly rinse abdominal cavity, after centrifuge tube collects washing fluid, centrifugal (1000rpm, 4 DEG C) abandon supernatant after 10 minutes, wash twice with 4 DEG C of RPMI1640 complete culture solutions (containing 10%FBS), cell viability examination is done in 0.2% trypan blue solution dyeing, confirms that the vigor scavenger cell that has collected accounts for more than 95%.After cell counting count board reading, adjustment cell concn to 2 × 10 ⁵individual/mL.

96 porocyte culture plates are added with 100 μ L/mL, 37 DEG C, 5%CO by blowing and beating to the cell suspension suspended completely ₂cultivate under environment after 4 hours, inhale and abandon liquid in hole, carefully clean at the bottom of cell culture plate well with 37 DEG C of RPMI1640 complete culture solutions, wash away not adherent cell and cell debris, obtain the adherent peritoneal macrophage after purifying.Every hole adds 0.2mLRPMI1640 perfect medium, tests after being dissolved in substratum in advance with little peptide sample and LPS and adds, and starts cell cultures.

Adding number of cells is 2 × 10 ⁵the cell suspension 100 μ L/ hole of/mL, adds RPMI1640 complete culture solution (10%FBS) the 200 μ L/ hole containing biologically active polypeptides SLPQ (100,500 μ g/mL) after adherent purifying; Arranging negative control group, is the RPMI1640 complete culture solution (10%FBS) of 500 μ g/mL BSA; And blank group cultured continuously 48 hours, inflammation group at the 24 little LPS that add constantly to final concentration 100ng/mL.44 littlely add 5%MTT20 μ L/ hole constantly, three lysates in 100 μ L/ holes are added to stop cultivating after reaching 48 hours, after dissolving overnight, survey the absorbance (OD570) in each hole under wavelength 570nm by microplate reader, the calculation formula of growth index (Growth Indices) is as follows:

Wherein, blank nutrient solution is the RPMI1640 complete culture solution containing 10%FBS.

3) experimental result

Experimental result is in table 3, and in experimental group, the interpolation concentration of biologically active polypeptides SLPQ is respectively 500,100 μ g/mL.In normal group, compare with negative control group, when concentration is 500 μ g/mL, there is pole significant difference (P<0.01).Illustrate that biologically active polypeptides SLPQ has the ability promoting macrophage proliferation, effectively can improve the non-specific immunity of body.

The impact that table 3 biologically active polypeptides SLPQ breeds macrophages in vitro

Note: * * labelled notation, for compare with negative control, has significant difference (P < 0.01).

Three, short scavenger cell Secretion of Nitric Oxide amount experiment (Griess method) of biologically active polypeptides SLPQ

1. experiment reagent and equipment

1) reagent: laboratory animal balb/c mouse (male 6-8 all ages) Shanghai Communications University's agricultural and biological institute animal experimental center; Synthesize little peptide SLPQ(to be synthesized by Shanghai Qiang Yao Bioisystech Co., Ltd); LPS, Sigma company; Nitrogen protoxide detection kit, green skies biotechnology research produced.

2) plant and instrument

The permanent Science and Technology Ltd. in LRH-250F biochemical cultivation case Shanghai; GL-22M high speed freezing centrifuge Shanghai Lu Xiang instrument whizzer Instrument Ltd.; Hera cell150CO2 incubator Heraeus company; Dragon Wellscan MK3 microplate reader Labsystems company.

2. test method

Adding number of cells is 2 × 10 ⁶the cell suspension 100 μ L/ hole of/mL, adds RPMI1640 complete culture solution (containing 10%FBS) the 200 μ L/ hole containing peptide after adherent purifying, arrange the cell blank group not adding little peptide.Inflammation group littlely adds LPS to final concentration 1 μ g/mL constantly 24, cultured continuously is after 48 hours, collect nutrient solution supernatant 50 μ L/ hole, Griess reagent 1 and each 50 μ L/ holes of Griess reagent 2 are added successively in nutrient solution supernatant, room temperature reaction, after 10 minutes, measures absorbance (OD540) under 540nm wavelength.

3. experimental result

Experimental result is in table 4, compared with cell blank, normal group has significant difference (P < 0.05) when SLPQ concentration is 500 μ g/mL, illustrate that SLPQ has the ability promoting the secretion of scavenger cell induced nitrous oxide, effectively can promote the immunological competence of scavenger cell.

In addition, nitrogen protoxide is as one of the index of severity of inflammation, in inflammation group, the SLPQ that maximum concentration arrives 500 μ g/mL does not cause the significance of Secretion of Nitric Oxide amount to increase yet, under the inflammatory conditions caused at LPS is described, SLPQ can not the degree of exacerbate inflammation further, has the security of use.

Table 4 biologically active polypeptides SLPQ is on the impact of the scavenger cell nitrogen protoxide amount of inducing

Embodiment 3 biologically active polypeptides SLPQ pipe intestinal digesting stability

(1) in-vitro simulated pipe intestinal digesting

Gl tract digestion experiment is mainly divided into two steps to carry out.First, compound concentration is 500 μ g/mL biologically active polypeptides SLPQ solution, and be add stomach en-in 500 μ g/mL SLPQ solution in concentration, ratio is that every gram of SLPQ 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, adds pancreatin, ratio is that every gram of SLPQ 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 digestive ferment inactivation, reaction solution freeze concentration is dry, make dry powder, under being stored in-20 DEG C of conditions, for subsequent use.

(2) quality of digestion product and determined amino acid sequence

Get the postdigestive sample powder 0.2mg of in-vitro simulated intestines and stomach, add 50 μ L water and 450 μ L dehydrated alcohols, put into-20 DEG C of refrigerator 20min after abundant concussion, centrifugal 30min under 15000rpm speed conditions, get supernatant liquor 400 μ L and carry out UPLC-Q-TOF-MS analysis.

UPLC condition: Hypersil GOLD C18 chromatographic column (100mm*2.1mm, 1.9 μm, 190 ) (ThermoScientific Co.); Mobile phase A: 0.1% aqueous formic acid, Mobile phase B: containing 0.1% formic acid acetonitrile; Adopt the gradient elution program from the A phase of 99% to 50%A phase; Flow velocity 0.4mL/min; Column temperature: 45 DEG C; Sample size: 5 μ L.

Q-TOF-MS condition: time-of-flight mass spectrometer, mass spectrum adopts electron spray ionisation source (ESI), positive ion mode.And carry out real-time accurate mass correction with the leucine enkephalin of 200ng/mL.Mass scan range is m/z80-1000, and sweep time is 0.3s.Capillary voltage 3kV; Taper hole voltage 35V; First mass spectrometric collision energy is respectively 4; Ion source temperature 100 DEG C; Desolventizing temperature degree and flow are respectively 300 DEG C, 500L/h.

According to above-mentioned experiment condition, biologically active polypeptides SLPQ industry UPLC-Q-TOF-MS before and after digestive ferment process analyzes, in obtained total ion current figure (Fig. 7), in blank, pepsin group, stomach en--pancreatin treatment group, all only occur that 2 molecular weight are the main peak of 444.24Da, molecular weight is identical with SLPQ, and each process retention time and peak area close.2 main peaks of stomach en--pancreatin treatment group are extracted, adopt Q-TOF-MS to analyze and obtain corresponding mass spectrum (Fig. 8 and Fig. 9), the first mass spectrometric figure at two peaks is identical, the nuclear-cytoplasmic ratio of essential substance is 444.23, prove that 2 peak components are identical, be same molecular weight second main peak due to first time wash-out incomplete formed.In total ion current figure, the molecular weight of main peak is 444.24Da, and explanation is substrate SLPQ.Above-mentioned the results show biologically active polypeptides SLPQ can not be degraded under Gl tract digestion condition, after digestive tube, still can play had biological activity.

Claims

1. aminoacid sequence is a preparation method for the biologically active polypeptides of Ser-Leu-Pro-Gln, and step is as follows:

1) ferment: added in skimming milk by lactobacterium helveticus CICC6024 and carry out anaerobically fermenting, obtain lactobacterium helveticus fermented-milk, the condition of described anaerobically fermenting is: leavening temperature 36 ~ 38 DEG C, fermentation time 15 ~ 20h;

2) slightly the carrying of polypeptide: to step 1) lactobacterium helveticus fermented-milk carry out low-temperature centrifugation separation, get supernatant liquor, the condition of described low-temperature centrifugation is: 4 DEG C, 8000 ~ 10000rpm, centrifugal 15 ~ 30min;

3) purifying of polypeptide:

A. to step 2) supernatant liquor carry out uf processing, collect filtrate;

The filtrate of b. collecting adopts reverse chromatography column SOURSE 5RPC ST to carry out RPLC separation, and collecting elution time is the polypeptide mixture of 21min;

C. be separated with ultra-high efficiency liquid phase-electron spray(ES)-level Four bar-flight time matter instrument and obtain polypeptide SLPQ;

Step 3) molecular weight cut-off of filter membrane that adopts of ultrafiltration process described in a is respectively 10kDa and 3kDa; In described ultra-filtration process, pressure range is 0.1 ~ 0.3MPa, and filtrate flow velocity is 0.8 ~ 1.2mL/min.

2. aminoacid sequence is the purposes in the food of biologically active polypeptides in preparation enhancing body immunizing power of Ser-Leu-Pro-Gln, healthcare products and medicine, and described purposes specifically refers to that described biologically active polypeptides promotes lymphopoiesis in preparation, promotes macrophage proliferation, purposes in the food of the short scavenger cell nitrogen protoxide amount of inducing, healthcare products and medicine.