CN112851753B - Potato protein hydrolysate, preparation and application thereof in adherent cell culture - Google Patents
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Abstract
The invention relates to the technical field of biological pharmacy, in particular to potato protein hydrolysate, preparation and application thereof in adherent cell culture. Comprising a complex active polypeptide and an amino acid; the amino acid content is at least 25wt%. The invention optimizes and improves the preparation process of potato protein hydrolysate, obtains a component smaller than 5kD by a separation and concentration method, and finds 16 peptide fragments by detection and identification when applied to MDCK adherent cell culture, wherein the 16 peptide fragments are main peptide fragments for promoting cell proliferation. Provides a new idea for high-valued utilization of potato protein and provides a reference for developing potato protein hydrolysate to replace part of serum culture medium.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to potato protein hydrolysate, preparation and application thereof in adherent cell culture.
Background
With the rapid development of bioscience research, low-serum culture has become a research hotspot and has a wide application prospect. Through the deep research on mechanisms such as cell nutrition, metabolism, exogenous protein expression mechanism and the like, the development of a serum-free culture medium is further promoted, so that the cell culture technology is widely applied to the fields of biological preparation, vaccine engineering, cells and the like. At present, when cells are cultured, serum with a certain proportion is added into a culture medium to provide nutrition and various growth factors required by cell growth in most cases, potato protein contains rich protein, crude fiber and carbohydrate, has 19 balanced amino acid components, and hydrolysate can provide beneficial nutrient substances for the cells, change the cell growth environment, improve the growth condition and have regulation and control effects on vital activities such as cell metabolism, growth, apoptosis and the like.
Disclosure of Invention
In view of the above technical problem, a first aspect of the present invention provides a potato protein hydrolysate comprising a complex active polypeptide and an amino acid; the amino acid content is at least 25wt%.
As a preferred embodiment of the present invention, the complex active polypeptide comprises at least a polypeptide having the amino acid sequence:
SEQ ID No 1：GQLIVVPQNF
SEQ ID No 2：QLIVVPQNF
SEQ ID No 3：IDPGDSDIIK
SEQ ID No 4：GGGGGYGGGGGYGGGGGGGYGGGR
SEQ ID No 5：VPTVDVSVVDL
SEQ ID No 6：NPEEIPWG
SEQ ID No 7：APGKPPVDY
SEQ ID No 8：APIDVVVPPGNTGLDPS。
as a preferred embodiment of the present invention, the complex active polypeptide further comprises a polypeptide having the amino acid sequence:
SEQ ID No 9：PPVLPPQE
SEQ ID No 10：VPELGPDVNF
SEQ ID No 11：GEGTNTWDRFE。
as a preferred embodiment of the present invention, the amino acids comprise at least a combination of amino acids; the amino acid combination consists of serine, glutamic acid, valine, leucine, tyrosine, aminobutyric acid, histidine, proline and glutamine; the amino acid combination is present in an amount of at least 18wt% based on the mass of the potato protein hydrolysate.
In a second aspect, the present invention provides a method for preparing potato protein hydrolysate as described above, comprising the steps of:
(1) Preparing a substrate: placing potato protein powder into a reaction kettle, adding pure water, and stirring for dissolving to obtain potato protein powder solution;
(2) Enzymolysis: adding protease into the potato protein powder, stirring and mixing uniformly, adding 0.1M/L HCl to adjust the pH to 5.5-7.5, setting the temperature to 40-60 ℃, and carrying out enzymolysis reaction for 3-8 hours;
(3) And (3) inactivation: heating the system after enzymolysis to at least 95 ℃, treating for at least 15min, cooling to room temperature, and collecting crude hydrolysate;
(4) Post-treatment: and centrifuging the crude hydrolysate to collect supernatant, filtering by adopting an ultrafiltration membrane to obtain refined hydrolysate, and drying to obtain the product.
As a preferable technical scheme of the invention, the concentration of the potato protein powder solution is 5-12wt%.
As a preferable technical scheme of the invention, the ratio of the protease to potato protein powder is (5000-35000) m:1g.
As a preferable technical scheme of the invention, the stirring speed in the enzymolysis reaction is 300-900 rpm/min.
In a third aspect, the invention provides the application of potato protein hydrolysate in the MDCK adherence cell culture field.
In a fourth aspect the invention provides a cell culture medium comprising potato protein hydrolysate as described above; the content of the potato protein hydrolysate is 0.3-0.7 g/L.
The beneficial effects are that: the preparation process of potato protein hydrolysate is optimized, and simultaneously, a component smaller than 5kD is obtained through a separation and concentration method, and is applied to MDCK adherent cell culture, 16 peptide fragments are found through detection and identification, and the 16 peptide fragments are main peptide fragments for promoting cell proliferation. Provides a new idea for high-valued utilization of potato protein and provides a reference for developing potato protein hydrolysate to replace part of serum culture medium.
Specifically, the invention obtains optimal preparation process parameters of potato protein hydrolysate, namely the temperature is 50 ℃, the pH is 6.2, and E/S=24000: 1m/g, and the reaction time is 6 hours, and the hydrolysis reaches 42.52 percent. The hydrolysate with the cell size smaller than 5kD obtained by ultrafiltration concentration is continuously cultured for more than 5 generations, and the cell viability of each group is greater than 94% after each generation of cell digestion count, so that stable continuous passage can be realized. By adding potatoes with different concentrations less than 5kD into low serum basal medium DMEMProtein hydrolysates 5g/L+5% NBS, 4g/L+5% NBS, 3g/L+5% NBS, 2g/L+5% NBS, 1g/L+5% NBS, 0.5g/L+5% NBS. The MDCK adherent cells are continuously cultured, and the results of 6 concentration gradient experiments show that the optimal addition concentration of potato protein hydrolysate is 0.5g/L+5% NBS. According to the cell growth curve, the cell growth curve shows that the maximum cell proliferation concentration is reached in each group at 120 hours, and the viable cell densities of 10% NBS and 5% NBS of the control group are 6.99X10 respectively 5 cell•mL-1、6.17×10 5 cell.mL-1, the living cell density of the experimental group reaches 7.23×10 5 cell.mL-1, substantially identical to the 10% NBS group, 17.18% higher than the 5% NBS group; the cell doubling time of the experimental group is 17.67h without significant difference (P > 0.05) from 17.42h of the 10% NBS group, and compared with 19.22h of the 5% NBS group, the cell doubling time is obviously shortened, and the difference is significant (P < 0.05); cell specific growth rate of experimental group is 0.0154+/-0.00022 h -1 And 0.0153+ -0.00038 h of 10% NBS group -1 There was no significant difference (P > 0.05), but significantly higher than 0.0149± 0.00022h for the 5% nbs group -1 (P < 0.05), the comparative analysis found that 1g/L potato protein hydrolysate can almost replace 5% of serum, and has 16 peptide fragments with promotion effect on cell growth: GQLIVVPQNF, QLIVVPQNF, GQLIVVPQN, LIVVPQNF, IDPGDSDII, DPGDSDIIK, GGGGGYGGGGGYGGGGGGGYGGGR, VPTVDVSVVDL, NPEEIPWG, NPEEIPW, APGKPPVDY, APIDVVVPPGNTGLDPS, PPVLPPQE, PVLPPQEP, VPELGPDVNF, GEGTNTWDRFE respectively.
Drawings
FIG. 1 is an electron microscopy image of cell morphology of serial subculture with potato protein hydrolysates added at different concentrations; wherein the first row is from left to right 10% NBS, 5% NBS, 0.5g/LPPH+5% NBS, 1g/LPPH+5% NBS, the second row is from left to right 2g/LPPH+5% NBS, 3g/LPPH+5% NBS, 4g/LPPH+5% NBS, 5g/LPPH+5% NBS.
FIG. 2 is a graph of cell growth.
FIG. 3 shows the result of SDS-PAGE gel electrophoresis, wherein M is a protein molecular weight standard; a is potato protein as such; b is potato protein hydrolysate.
FIG. 4 is a graph showing the effect of hydrolysis time on the degree of hydrolysis.
FIG. 5 is a graph showing the effect of hydrolysis temperature on the degree of hydrolysis.
FIG. 6 is the effect of enzyme/substrate ratio on the degree of hydrolysis.
FIG. 7 is a graph showing the effect of pH on the degree of hydrolysis.
Detailed Description
The technical features of the technical solution provided in the present invention will be further clearly and completely described in connection with the detailed description below, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The words "preferred," "more preferred," and the like in the present disclosure refer to embodiments of the present disclosure that may provide certain benefits in some instances. However, other embodiments may be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
It should be understood that all numbers expressing, for example, amounts of ingredients used in the specification and claims, except in any operating example or otherwise indicated, are to be understood as being modified in all instances by the term "about".
In a first aspect the invention provides a potato protein hydrolysate comprising a complex active polypeptide and an amino acid; the amino acid content is at least 25wt%.
Applicants used a fully automated amino acid analyzer on the potato protein hydrolysates described above to detect free amino acids in the hydrolysates. Specifically, the potato protein hydrolysate and 5% of 5-sulfosalicylic acid were dissolved in pure water, centrifuged at 1200rpm/min for 15min, the proteins were removed, and the supernatant was diluted in a double ratio. The chromatographic conditions are as follows: sample analysis system is biological body fluid system, exchange column: 4.6X10 mm, 3nm sulfonic acid type cationic resin as filler, separating column temperature: 50 ℃, reaction column temperature: the sample volume was 20. Mu.L at 135 ℃.
The potato protein hydrolysate in the present application comprises at least 25wt% and the above amino acids as tested by the above experiment; further preferably, the potato protein hydrolysate comprises at least 30wt% and the above amino acids; further, the potato protein hydrolysate comprises at least 35wt% of amino acids and the above; further, the potato protein hydrolysate comprises at least 37.1wt% amino acids.
In some embodiments, the amino acids comprise at least a combination of amino acids; the amino acid combination consists of serine, glutamic acid, valine, leucine, tyrosine, aminobutyric acid, histidine, proline and glutamine; the amino acid combination is present in an amount of at least 18wt% based on the mass of the potato protein hydrolysate; further, the amino acid combination is present in an amount of at least 20% by weight and more based on the mass of the potato protein hydrolysate.
In some preferred embodiments, the amino acid further comprises phosphoserine, aspartic acid, threonine, glycine, alanine, citrulline, cystine, methionine, phenylalanine, ornithine, lysine, arginine, asparagine.
Applicants analyzed the potato protein hydrolysate polypeptide information described herein using nanoliter liquid chromatography-quadrupole orbitrap mass spectrometry. The method comprises the following steps:
1. desalting and quantifying peptide: 50mg of potato protein hydrolysate dry powder described in this application was weighed, dissolved in 100 μl of 0.1% TFA (trifluoroacetic acid), centrifuged to obtain the supernatant, dried with the appropriate amount of peptide fragments, desalted using C18StageTip, and dried in vacuo. After drying the peptide was reconstituted with 0.1% TFA and OD280 was used to determine the concentration of the peptide for analysis.
2. Mass spectrometry: nanoliter reversed phase chromatographic mobile phase A is 98 percent of water, 0.1 percent of formic acid and 2 percent of acetonitrile, and nanoliter reversed phase chromatographic mobile phase B is 2 percent of water, 0.1 percent of formic acid and 98 percent of acetonitrile. Sample is introduced into TrapColumn (100 [ mu ] m, 20, mm, 5 [ mu ] m, C18, dr. Maisch GmbH) and then subjected to gradient separation through a chromatographic column (75 [ mu ] m, 150, mm, 3 [ mu ] m, C18, dr. Maisch GmbH) at a flow rate of 300nl/min. The liquid phase separation gradient is as follows: 0-3min, linear gradient of liquid B from 2% to 7%;3-48min, and the linear gradient of the liquid B is from 7% to 35%;48-53min, linear gradient of liquid B from 35% to 90%;53-60min, and the solution B is maintained at 90%. After peptide separation, DDA (data dependent acquisition) mass spectrometry was performed with a Q-exact plus mass spectrometer for a period of 60min.
Detection mode: positive ion, parent ion scan range: 350-1800 m/z, primary mass spectrum resolution: 70,000@m/z 200, agctarget:3e6, first order MaximumIT:50ms. Peptide fragment secondary mass spectrometry was collected as follows: secondary mass spectrum (MS 2 scan) of 20 highest intensity parent ions was triggered after each full scan (Fullscan), secondary mass spectrum resolution: 17,500@ m/z 200, AGC target: 1e5, second order Maximum IT:45ms, MS 2 Activation Type: HCD，Isolation window：2.0m/z，Normalizedcollision energy：28。
3. Database retrieval: the mass spectrum database retrieval software adopted is MaxQuant1.6.1.0; the following protein databases were used: from UniprotProtein Database, the species is patto (potato), a total of 75051 protein sequences.
The potato protein hydrolysate described in the present application was found to comprise at least the polypeptide of 8 as determined by the above-described experimental analysis.
In some embodiments, the complex active polypeptide comprises at least a polypeptide having the amino acid sequence:
SEQ ID No 1：GQLIVVPQNF
SEQ ID No 2：QLIVVPQNF
SEQ ID No 3：IDPGDSDIIK
SEQ ID No 4：GGGGGYGGGGGYGGGGGGGYGGGR
SEQ ID No 5：VPTVDVSVVDL
SEQ ID No 6：NPEEIPWG
SEQ ID No 7：APGKPPVDY
SEQ ID No 8：APIDVVVPPGNTGLDPS。
further preferred, the complex active polypeptide further comprises a polypeptide having the amino acid sequence:
SEQ ID No 9：PPVLPPQE
SEQ ID No 10：VPELGPDVNF
SEQ ID No 11：GEGTNTWDRFE。
further preferred, the complex active polypeptide further comprises a polypeptide having the amino acid sequence:
SEQ ID No 12：GQLIVVPQN
SEQ ID No 13：LIVVPQNF
SEQ ID No 14：DPGDSDIIK
SEQ ID No 15：NPEEIPW
SEQ ID No 16：PVLPPQEP。
in a second aspect, the present invention provides a method for preparing potato protein hydrolysate as described above, comprising the steps of:
(1) Preparing a substrate: placing potato protein powder into a reaction kettle, adding pure water, and stirring for dissolving to obtain potato protein powder solution;
(2) Enzymolysis: adding protease into the potato protein powder, stirring and mixing uniformly, adding 0.1M/L HCl to adjust the pH to 5.5-7.5, setting the temperature to 40-60 ℃, and carrying out enzymolysis reaction for 3-8 hours;
(3) And (3) inactivation: heating the system after enzymolysis to at least 95 ℃, treating for at least 15min, cooling to room temperature, and collecting crude hydrolysate;
(4) Post-treatment: and centrifuging the crude hydrolysate to collect supernatant, filtering by adopting an ultrafiltration membrane to obtain refined hydrolysate, and drying to obtain the product.
In some embodiments, the concentration of the potato protein powder solution is 5-12 wt%; further preferably, the concentration of the potato protein powder solution is 8wt%.
The applicant carried out a one-factor hydrolysis experiment using the Degree of Hydrolysis (DH) as an evaluation index. The influence of different enzymolysis time, temperature, pH and enzyme-to-substrate ratio (E/S) on the hydrolysis effect is studied, and the optimal hydrolysis conditions of all hydrolysis factors are determined. According to the single factor experimental result, the response surface experimental design is carried out by taking the temperature (A), the pH (B) and the E/S (C) as independent variables and DH as response values. Box-Behnken (BBD) experiments were designed as shown in Table 1 using Design-Expert8.0.6, three replicates per set. The effect of each hydrolysis factor on the degree of hydrolysis was compared by analysis of variance.
TABLE 1Box-Behnken experiment design factor level coding table
| Encoding | Temperature (. Degree. C.) A | pH B | E/S(U/g) C |
| -1 | 40 | 5.5 | 5000 |
| 0 | 50 | 6.5 | 20000 |
| 1 | 60 | 7.5 | 35000 |
Statistical analysis is carried out on experimental data by using Design-expert8.06 software to obtain a quadratic polynomial regression equation: degree of Hydrolysis (DH) = 42.446-1.57375 a-3.275 b+4.01625 c-1.0725 ab+1.715 ac+3.3475 bc-6.918 b 2-3.8705 c 2-5.873 c 2.
Analysis of variance of regression equation fitting model of degree of hydrolysis as shown in Table 2, pLess than 0.001 is very significant, which indicates that model fitting is true; the mismatch term p= 0.2067 is not significant, which indicates that the model is stable and has good fitting degree. Factor B, C, A 2 、B 2 、C 2 The influence of BC on the degree of hydrolysis is extremely remarkable, the influence of A on the degree of hydrolysis is remarkable, and only the factors AB and AC are not remarkable. The influence on the degree of hydrolysis can be known according to the absolute value of the coefficient estimated value, and the influence is as follows: the process conditions have the following influence on the degree of hydrolysis: c (C)>B>A, i.e. E/S>pH>Temperature. Wherein, the determination coefficient R of the model 2 adj= 0.9501, and is related to the prediction coefficient PredR 2 And the experimental model and the experimental value are good in fitting degree, and the method can be used for predicting and optimizing the hydrolysis degree extraction process. Therefore, the quadratic regression equation can objectively predict the law of the hydrolysis degree after enzymolysis along with the change of each factor.
TABLE 2 analysis of variance
Wherein p <0.05 represents significant differences; * Representing the difference being very significant, p <0.01.
In some embodiments, the ratio of protease to potato protein powder is (5000-35000) m:1g; further, the ratio of protease to potato protein powder is 24000m:1g.
Further preferably, the pH of the system in the step (2) is 6.2.
Further preferably, the temperature of the system in the enzymolysis in the step (2) is 50 ℃.
In some embodiments, the stirring speed in the enzymolysis reaction is 300-900 rpm/min.
Further preferably, the stirring speed in the enzymolysis reaction is 600rpm/min.
As can be seen from the hydrolysis experimental results in figures 4-7, the hydrolysis process parameters of potato protein are further optimized and improved to obtain the optimal hydrolysis parameters, namely 50 ℃ and pH6.2, E/S24000U/g, and the hydrolysis degree reaches 43.491% in the reaction for 6 hours.
In some embodiments, the post-treatment method in step (4) comprises centrifugation: the crude hydrolysate was centrifuged at 4200rpm for 20min and the supernatant was collected to remove incompletely hydrolyzed proteins, inactivated enzymes and insoluble impurities.
Further, the post-treatment method in the step (4) further comprises ultrafiltration concentration: ultrafiltering the supernatant with 5kD ultrafilter membrane to obtain hydrolysate with molecular weight less than 5 kD.
Further, the post-treatment method in step (4) further comprises vacuum freeze drying: and the refined hydrolysate is freeze-dried by adopting a vacuum freeze-drying (-45-25 ℃ linear cooling and heating) method, so that the activity of the hydrolysate is ensured and the hydrolysate is easy to store.
In a third aspect, the invention provides the application of potato protein hydrolysate in the MDCK adherence cell culture field.
The applicant has carried out cell culture experiments on potato protein hydrolysates in the present application, in particular:
1. cell resuscitation: taking 2 MDCK adherent cells frozen in liquid nitrogen, rapidly thawing in 37 ℃ water, sterilizing, inoculating into a cell bottle, culturing in DMEM, placing culture solution with volume of 10mL in 5% CO 2 Culturing in a 37 ℃ incubator for 48 hours, sampling, counting and passaging. And (5) carrying out subsequent experiments when the cell activity rate reaches more than 97%.
2.1, cell serial subculture: the potato protein hydrolysate is used as a blank control group, the components of the potato protein hydrolysate PPH prepared in a laboratory, which is smaller than 5kD, are used as experimental groups, the potato protein hydrolysate is added into a culture medium containing 5% of serum at different concentrations, and the influence of different addition amounts of the hydrolysate on the passage stability and the growth characteristics of cells is compared through continuous cell subculture.
2.2, screening of the addition amount of potato protein hydrolysate: respectively weighing 1g, 2g, 3g, 4g and 5g of potato protein hydrolysate freeze-dried powder, uniformly mixing and dissolving in 1L of DMEM culture medium containing 5% serum, and filtering and sterilizing for later use at the final concentration of 1g/L, 2g/L, 3g/L, 4g/L and 5g/L respectively and at the final concentration of 0.22 mu m. Dogs were treatedKidney cells (MDCK) at 1×10 5 cell concentration of-1 cells was inoculated in T25 cell flasks, placed in an incubator, passaged and counted, and cell growth curves were drawn.
2.3, analysis of cell growth characteristics: the growth promoting effect of the cells is studied through continuous culture of the cells, cell growth curves, maximum proliferation concentration, multiplication time, specific growth rate and the like.
The cell morphology of continuous subculture with potato protein hydrolysate added at different concentrations as shown in FIG. 1, wherein the control group, 10% NBS and 5% NBS, and the prepared potato protein hydrolysate added at a concentration of 0.5g/LPPH+5% NBS, were able to stably maintain the normal morphology of MDCK adherent cells.
The results of the cell growth characteristics analysis are shown in FIG. 2. The maximum proliferation density, doubling time, specific growth rate of the cells are shown in Table 3.
TABLE 3 Table 3
| Maximum proliferation concentration (105 cells mL-1) | Doubling time (h) | Specific growth Rate (h-1) | |
| 5%NBS | 4.91 | 18.92 | 0.0141±0.0002 |
| 10%NBS | 4.93 | 19.26 | 0.0140±0.0003 |
| 1g/L+5%NBS | 7.88 | 16.10 | 0.0157±0.0003 |
| 0.5g/L+5%NBS | 5.97 | 16.59 | 0.0147±0.0003 |
The maximum proliferation concentration of each group of experimental cells is reached at 120h, the multiplication time of 0.5g/L+5% NBS and 1g/L+5% NBS120h of the experimental group added with protein hydrolysates at different concentrations is 16.10h and 16.59h respectively, which are smaller than 18.92h and 19.26h of the control group, and the specific growth rate of 120h of the experimental group is 0.0157+/-0.0003, 0.0147+/-0.0003 respectively, which is significantly higher than that of the control group, namely 0.0141+/-0.0002 and 0.0140+/-0.0003 (P < 0.05).
The applicant carried out SDS-PAGE gel electrophoresis on the potato protein hydrolysate, and specifically analyzed the molecular weight of the potato protein hydrolysate by using the SDS-PAGE gel electrophoresis method. Preparing 12% separating gel, uniformly mixing and adding into a glass plate; the sample concentration is 1mg/mL, the sample is boiled and denatured at 100 ℃ for 10min, and the loading amount is 20 mu L. Electrophoresis was performed at 80V for 2 hours at low temperature, and the color was removed after staining with Coomassie brilliant blue. The results are shown in FIG. 3. As can be seen from FIG. 3, before potato protein is hydrolyzed, the main molecular weight bands of the protein are distributed near 72kD and 34kD and above 17kD, potato protein is hydrolyzed into a plurality of small molecular substances after full enzymolysis, the molecular weight is more less than 17kD, and only insignificant light bands above 17kD show that potato protein in potato protein hydrolysate has been fully hydrolyzed and the molecular weight is less than 17kD, possibly some amino acids, polypeptides, salts and the like. Gel electrophoresis results show that the efficiency of the flavourzyme for enzymolysis of potato protein is higher.
In a fourth aspect the invention provides a cell culture medium comprising potato protein hydrolysate as described above; the content of the potato protein hydrolysate is 0.3-0.7 g/L.
Further preferably, the potato protein hydrolysate is present in an amount of 0.5g/L.
The present invention will be specifically described below by way of examples. It is noted herein that the following examples are given solely for the purpose of further illustration and are not to be construed as limitations on the scope of the invention, as will be apparent to those skilled in the art in light of the foregoing disclosure.
Examples: the embodiment provides a potato protein hydrolysate, which is prepared by the following steps:
(1) Preparing a substrate: according to the mass fraction of 8%, weighing 800g of potato protein powder, placing the potato protein powder in a 10L reaction kettle, injecting 10L of pure water, and fully stirring and dissolving.
(2) Enzymolysis: adding a corresponding amount of flavourzyme according to E:S=24000:1 (u:g), fully stirring and uniformly mixing, regulating the pH to about 6.2 by 0.1M/L HCl, setting the temperature to 50 ℃, and carrying out enzymolysis at the rotating speed of 600rpm/min for 6 hours.
(3) Inactivation of flavourzyme: and heating the enzymolysis liquid to above 95 ℃ for 15min to completely inactivate the flavourzyme. Cooling to room temperature by condensate water circulation and collecting crude hydrolysate.
(4) And (3) centrifuging: the crude hydrolysate was centrifuged at 4200rpm for 20min and the supernatant was collected to remove incompletely hydrolyzed proteins, inactivated enzymes and insoluble impurities.
(5) Ultrafiltration concentration: ultrafiltering the supernatant with 5kD ultrafilter membrane to obtain hydrolysate with molecular weight less than 5 kD.
(6) Vacuum freeze drying: and the refined hydrolysate is freeze-dried by adopting a vacuum freeze-drying (linear temperature reduction and temperature increase at-45-25 ℃) method, so that the activity of the hydrolysate is ensured and the hydrolysate is easy to store. Potato protein hydrolysate with the hydrolysis degree reaching 43.491% is obtained.
The potato protein hydrolysates described above were tested for free amino acid content and the results are shown in table 4 below:
TABLE 4 Table 4
| Sequence number No. | Amino acid (Amino acid) | （wt%） |
| 1 | Phosphoserine P-Ser | 0.309 |
| 2 | Asp of aspartic acid | 1.233 |
| 3 | Threonine Thr | 1.673 |
| 4 | Serine Ser | 2.339 |
| 5 | Glutamic acid Glu | 2.393 |
| 6 | Glycine Gly | 1.063 |
| 7 | Alanine Ala | 1.857 |
| 8 | Citrulline Cit | 0.117 |
| 9 | Valvaline Val | 2.345 |
| 10 | Cystine Cys | 0.269 |
| 11 | Methionine Met | 0.628 |
| 12 | Leucine Leu | 2.281 |
| 13 | Tyrosine Tyr | 3.909 |
| 14 | Phe-phenylalanine | 1.644 |
| 15 | Aminobutyric acid g-ABA | 2.628 |
| 16 | Orn ornithine | 0.060 |
| 17 | Lysine Lys | 0.031 |
| 18 | Histidine His | 2.404 |
| 19 | Arginine Arg | 0.993 |
| 20 | Proline Pro | 2.972 |
| 21 | Asparagine AspNH2 | 0.168 |
| 22 | GluNH2 glutamine | 2.511 |
The potato protein hydrolysate was subjected to nanoliter liquid chromatography-quadrupole orbitrap mass spectrometry analysis, and the results are shown in table 5 below:
TABLE 5
| Sequence number | Sequence | Mass | m/ | Length | |
| 1 | GQLIVVPQNF | 1113.6182 | 557.817 | 10 | |
| 2 | QLIVVPQNF | 1056.5968 | 529.3063 | 9 | |
| 3 | GQLIVVPQN | 966.5498 | 484.2831 | 9 | |
| 4 | LIVVPQNF | 1242.6608 | 622.3385 | 11 | |
| 5 | IDPGDSDIIK | 1071.5448 | 536.7811 | 10 | |
| 6 | DPGDSDIIK | 958.4607 | 480.2375 | 9 | |
| 7 | GGGGGYGGGGGYGGGGGGGYGGGR | 1803.7308 | 902.8743 | 24 | |
| 8 | VPTVDVSVVDL | 1141.623 | 571.8196 | 11 | |
| 9 | NPEEIPWG | 940.429 | 471.222 | 8 | |
| 10 | NPEEIPW | 883.4075 | 442.711 | 7 | |
| 11 | APGKPPVDY | 942.481 | 472.2483 | 9 | |
| 12 | APIDVVVPPGNTGLDPS | 1646.8516 | 824.4367 | 17 | |
| 13 | PPVLPPQE | 875.4752 | 438.7458 | 8 | |
| 14 | PVLPPQEP | 875.4752 | 438.7458 | 8 | |
| 15 | VPELGPDVNF | 1085.5393 | 543.7784 | 10 | |
| 16 | GEGTNTWDRFE | 1654.6682 | 828.3419 | 14 |
The preparation process parameters of potato protein hydrolysate are optimized and obtained in the invention: the temperature is 50 ℃, the pH is 6.2, the E/S is 24000U/g, and the hydrolysis degree reaches 43.491 percent when the reaction is carried out for 6 hours. The component of the prepared potato protein hydrolysate PPH smaller than 5kD has obvious effect of promoting MDCK adherent cell proliferation, and the optimal addition concentration of PPH is 0.5g/L.
The less than 5kD fraction, which has a promoting effect on cell growth, contains 16 peptide fragments: GQLIVVPQNF, QLIVVPQNF, GQLIVVPQN, LIVVPQNF, IDPGDSDII, DPGDSDIIK, GGGGGYGGGGGYGGGGGGGYGGGR, VPTVDVSVVDL, NPEEIPWG, NPEEIPW, APGKPPVDY, APIDVVVPPGNTGLDPS, PPVLPPQE, PVLPPQEP, VPELGPDVNF, GEGTNTWDRFE, respectively, the 16 peptides are presumed to be those peptides in potato protein hydrolysate which act to promote growth of animal cells.
The research result can improve the comprehensive utilization rate of potatoes and the added value of byproducts, and provides reference for developing culture media or medicines containing potato protein hydrolysates.
The foregoing examples are illustrative only and serve to explain some features of the method of the invention. The appended claims are intended to claim the broadest possible scope and the embodiments presented herein are merely illustrative of selected implementations based on combinations of all possible embodiments. It is, therefore, not the intention of the applicant that the appended claims be limited by the choice of examples illustrating the features of the invention. Some numerical ranges used in the claims also include sub-ranges within which variations in these ranges should also be construed as being covered by the appended claims where possible.
Sequence listing
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I D P G D S D I I K
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1 5 10 15 20
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N P E E I P W G
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N P E E I P W
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1 5
Claims (7)
1. Potato protein hydrolysate, characterized in that it comprises a complex active polypeptide and an amino acid; the amino acid content is at least 25wt%;
the preparation method of the potato protein hydrolysate comprises the following steps:
(1) Preparing a substrate: placing potato protein powder into a reaction kettle, adding pure water, and stirring for dissolving to obtain potato protein powder solution;
(2) Enzymolysis: adding flavourzyme into the potato protein powder, stirring and uniformly mixing, adding 0.1M/L HCl to adjust the pH to 5.5-7.5, setting the temperature to 40-60 ℃, and carrying out enzymolysis reaction for 3-8 hours;
(3) And (3) inactivation: heating the system after enzymolysis to at least 95 ℃, treating for at least 15min, cooling to room temperature, and collecting crude hydrolysate;
(4) Post-treatment: centrifuging the crude hydrolysate to collect supernatant, ultrafiltering with 5kD molecular weight cut-off ultrafiltration membrane, collecting hydrolysate with molecular weight smaller than 5kD to obtain refined hydrolysate, and drying to obtain the final product;
the ratio of the protease to the potato protein powder is (5000-35000) U:1g;
the stirring speed in the enzymolysis reaction is 300-900 rpm/min.
2. Potato protein hydrolysate according to claim 1, wherein the complex active polypeptide comprises at least a polypeptide of the amino acid sequence:
SEQ ID No 1：GQLIVVPQNF
SEQ ID No 2：QLIVVPQNF
SEQ ID No 3：IDPGDSDIIK
SEQ ID No 4：GGGGGYGGGGGYGGGGGGGYGGGR
SEQ ID No 5：VPTVDVSVVDL
SEQ ID No 6：NPEEIPWG
SEQ ID No 7：APGKPPVDY
SEQ ID No 8：APIDVVVPPGNTGLDPS。
3. potato protein hydrolysate according to claim 2, wherein the complex active polypeptide further comprises a polypeptide of the amino acid sequence:
SEQ ID No 9：PPVLPPQE
SEQ ID No 10：VPELGPDVNF
SEQ ID No 11：GEGTNTWDRFE。
4. potato protein hydrolysate according to claim 1, characterized in that the amino acids comprise at least a combination of amino acids; the amino acid combination consists of serine, glutamic acid, valine, leucine, tyrosine, aminobutyric acid, histidine, proline and glutamine; the amino acid combination is present in an amount of at least 18wt% based on the mass of the potato protein hydrolysate.
5. The method for preparing potato protein hydrolysate according to claim 1, wherein the concentration of the potato protein powder solution is 5-12wt%.
6. The use of potato protein hydrolysate according to any one of claims 1 to 4 for MDCK adherent cell culture.
7. A cell culture medium, characterized in that it comprises a potato protein hydrolysate according to any one of claims 1-4; the content of the potato protein hydrolysate is 0.3-0.7 g/L.
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| CN102037134A (en) * | 2008-05-20 | 2011-04-27 | 泉标食品有限公司 | Method for producing corn gluten hydrolysate and corn gluten hydrolysate using the same |
| CN109247447A (en) * | 2017-07-12 | 2019-01-22 | 安琪酵母股份有限公司 | Substitute the yeast hydrolyate and preparation method thereof of plasma protein powder production creep feed |
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| GB752886A (en) * | 1954-01-11 | 1956-07-18 | Wise Potato Chip Company | Method of producing protein hydrolyzate from potatoes and potato waste |
| US20120077268A1 (en) * | 2003-03-03 | 2012-03-29 | Glaxosmithkline Biologicals, S.A. | Animal-free cell culture method |
| JP4924971B2 (en) * | 2007-07-31 | 2012-04-25 | コスモ食品株式会社 | Protein composition containing protein derived from potato and its partially decomposed product and method for extracting them |
| CN102115729B (en) * | 2009-12-31 | 2013-01-23 | 北京清大天一科技有限公司 | Method for culturing baby hamster kidney (BHK) 21 cell in serum-free way, and vaccine preparation method |
| KR102148298B1 (en) * | 2017-04-24 | 2020-08-26 | ㈜로제타엑소좀 | Method for preparing nano particle-free cell culture media |
| CN111172311B (en) * | 2020-01-08 | 2023-06-30 | 西北民族大学 | Dual-fluorescence quantitative PCR detection kit for soybean walnut allergen genes |
| CN111875671A (en) * | 2020-07-23 | 2020-11-03 | 西北民族大学 | Soybean protein hydrolysis additive for serum-free culture medium and preparation method thereof |
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| CN102037134A (en) * | 2008-05-20 | 2011-04-27 | 泉标食品有限公司 | Method for producing corn gluten hydrolysate and corn gluten hydrolysate using the same |
| CN109247447A (en) * | 2017-07-12 | 2019-01-22 | 安琪酵母股份有限公司 | Substitute the yeast hydrolyate and preparation method thereof of plasma protein powder production creep feed |
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