CN110862928A - A kind of15Preparation method and application of N-labeled mycoprotein - Google Patents

A kind of15Preparation method and application of N-labeled mycoprotein Download PDF

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CN110862928A
CN110862928A CN201911222334.2A CN201911222334A CN110862928A CN 110862928 A CN110862928 A CN 110862928A CN 201911222334 A CN201911222334 A CN 201911222334A CN 110862928 A CN110862928 A CN 110862928A
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fermentation
mycoprotein
labeled
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seed
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夏建业
张薇
刘鹏
陈敏
庄英萍
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Qingdao Institute Of Innovation East China University Of Technology
East China University of Science and Technology
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East China University of Science and Technology
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
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    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry

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Abstract

The invention discloses a15A preparation method and application of N-labeled mycoprotein. The preparation method comprises the following steps: seed culture: inoculating the spore liquid into a seed culture medium, and culturing at 33-35 ℃ at a rotating speed of 120-180 revolutions per minute to obtain a seed liquid; pretreatment: placing the seed liquid in a centrifuge tube, centrifuging, and washing with normal saline to obtain a seed treatment liquid; marking and culturing: adding the seed treatment solution into a fermentation culture medium solution, shaking the flask for fermentation, then placing the mixture into a fermentation tank for continuous fermentation culture, adding the fermentation culture medium solution in a feeding mode in the continuous fermentation process, and obtaining fermentation liquor after terminating the fermentation; protein isolation: filtering the obtained fermentation liquor to obtain thallus, freezing with liquid nitrogen, grinding, and freeze drying to obtain the final product15N-labeled mycoprotein. The preparation method of the invention can efficiently prepare15N is marked into mycoprotein to obtain15And (3) labeling the protein with N.

Description

A kind of15N-labeled mycoproteinPreparation method and application thereof
Technical Field
The invention relates to the technical field of isotope labeling, in particular to a method for labeling a nuclear magnetic resonance system15A preparation method and application of N-labeled mycoprotein.
Background
Aspergillus niger, a common species of fungi of the genera Aspergillus, Aphyllophorales, Moniliaceae, the class Hyphomycetes, the subdivision Deuteromycotina, is widely distributed in foodstuffs, vegetable products and soils all over the world. Aspergillus niger is an important fermentation industrial strain, can produce amylase, acid protease, cellulase, pectinase, glucose oxidase, citric acid, gluconic acid, gallic acid and the like, and some strains can also convert hydroxyprogesterone into androstene. The growth is proper at 37 ℃ and the minimum relative humidity is 88 percent, which can cause the mildew of grains with higher water content and other industrial equipment. Aspergillus niger can be used for the production of saccharified feeds and biofertilizers. Aspergillus niger can also be used for measuring trace elements such as manganese, copper, molybdenum, zinc and the like and as a mould corrosion test bacterium. Therefore, the application range of the aspergillus niger is very wide, and the aspergillus niger also has very high economic value.
The traditional method for detecting the concentration of mycoprotein is to directly draw a standard curve by using the linear relation between the sample concentration of the UPS2 standard substance and iBAQ, and then to draw the standard curve14iBAQ of N sample proteins absolute concentrations of sample proteins were calculated. However, in the conventional method, enzyme digestion and mass spectrum deviation exist, and errors exist between the calculated protein concentration and the actual protein concentration.
Therefore, it is highly desirable to provide a process for preparing15A method for labeling mycoprotein with N to provide15And the N-labeled high-abundance protein further solves the problems, eliminates the deviation and realizes the absolute quantification of the protein.
Disclosure of Invention
The purpose of the invention is to obtain15N-labeled mycoprotein, to provide a method for efficiently binding15N-labelling to mycoprotein15Method for producing N-labeled mycoprotein, method for producing N-labeled mycoprotein15The abundance of N label is high, then passes15N can realize the pairing of the thallus eggsAbsolute quantification of white.
The invention provides a15The preparation method of the N-labeled mycoprotein comprises the following steps:
seed culture: inoculating the spore liquid into a seed culture medium, and culturing at 33-35 ℃ at a rotating speed of 120-180 revolutions per minute to obtain a seed liquid;
pretreatment: placing a proper amount of the seed liquid into a centrifugal tube, centrifuging for 4-8 minutes at 3500-4500 rpm, and washing with normal saline to obtain a seed treatment liquid;
marking and culturing: adding the seed treatment solution into a fermentation culture medium solution, shaking the flask for fermentation, then placing the mixture into a fermentation tank for continuous fermentation culture, adding the fermentation culture medium solution in a feeding mode in the continuous fermentation process, and obtaining fermentation liquor after terminating the fermentation;
protein isolation: filtering the obtained fermentation liquor to obtain thallus, freezing with liquid nitrogen, grinding, and freeze drying to obtain the final product15N-labeled mycoprotein.
Further, the nitrogen atom of the nitrogen source in the fermentation medium is15And marking by N.
Further, in the fermentation tank, the feeding rate of the fermentation medium solution is 10-16 g/h.
Further, the spore liquid is aspergillus niger CBS513.88 spore liquid.
Further, the pH value of the seed culture medium is 6.2-6.8.
Further, the components of the seed culture medium comprise: anhydrous glucose, corn steep liquor solids, and an antifoaming agent.
Further, the seed culture medium is sterilized before use under the condition of sterilization at 121 ℃ for 30 min.
Further, the components of the fermentation medium include: glucose, CaCl2、MgSO4·7H2O、EDTA、(NH4)2SO4、KH2PO4、NaH2PO4·2H2O、MnSO4·H2O、ZnCl2、CuSO4·5H2O、CoCl2·6H2O、FeSO4·7H2O and an antifoaming agent.
Further, the thalli are frozen by liquid nitrogen and stored at the temperature below minus 80 ℃.
Further, the concentration of the spore liquid is 1 × 106～107/mL。
Further, in the seed culture step, the culture time is 22-26 hours.
Further, in the seed culture step, the culture time is 24 hours.
Further, the time for the shake flask fermentation is 22-26 hours.
Further, the time for the shake flask fermentation is 24 hours.
Further, the concentration of the bacteria after the shake bottle fermentation is finished is 1.13-1.20 g of DCW/kg.
Further, the terminating fermentation is: when the carbon dioxide release rate (CER) in the fermentation system decreases.
A use of15Preparation method of N-labeled mycoprotein15And (3) calculating the absolute quantification of the mycoprotein by marking the mycoprotein with the N.
In the invention, the catalyst prepared by the method15The N-labeled mycoprotein is better used for quantifying the mycoprotein. Adding internal standard protein to fit UPS2 standard concentration with iBAQ regression to obtain standard curve, substituting15The iBAQ of the N internal standard protein calculates the absolute concentration of the internal standard protein, and the subsequent sample measurement will be carried out15The N internal standard proteins were mixed with the samples and mass-analyzed according to each14N protein and15the ratio of iBAQ of the N protein was quantified absolutely.
In the present invention, the freeze-drying technique of the mycoprotein is a technique which is conventional in the art.
In an embodiment of the present invention, the seed culture medium comprises: 20g/kg of anhydrous glucose, 20g/kg of corn steep liquor solids and 1mL/L of antifoam.
In one embodiment of the present invention, the hair is a hair brushThe components of the fermentation medium comprise: 50.1g/kg glucose, 0.076g/kg CaCl21g/kg of MgSO 24·7H2O, 0.67g/kg EDTA, 3g/kg (NH)4)2SO43g/kg KH2PO41.69g/kg NaH2PO4·2H2O, 0.04g/kg MnSO4·H2O, 0.02g/kg ZnCl20.015g/kg of CuSO4·5H2O, 0.015g/kg CoCl2·6H2O, 0.3g/kg of FeSO4·7H2O and 1mL/L of an antifoaming agent.
In a specific embodiment of the present invention: in the fermenter, the feed time and the corresponding feed rate of the fermentation medium solution are:
24.22～34.97h：15.75g/h；
34.97～39.52h：21g/h；
39.52～48.5h：10.5g/h。
the invention has the beneficial effects that:
by the preparation method of the invention, the preparation method can well prepare15N-labeling to mycoprotein to obtain15The N-labeled protein is convenient for later-stage various detections. In the preparation method of the present invention, the pretreatment step can remove the nitrogen source in the seed culture medium14Influence of N on thallus and increase in final thallus protein15The abundance of N. The invention adopts a shake flask fermentation method, and can overcome the problem of delay period increase caused by small inoculation amount.
In the method of the invention, the corn steep liquor in the seed liquid is washed away by using normal saline, so that the corn steep liquor in the seed culture medium can be reduced14The influence of N; and a small amount of Aspergillus niger is inoculated for shake flask fermentation, so that the amount of Aspergillus niger in the seed culture medium can be reduced14N, the problem of delay period extension caused by small inoculation amount can be solved, and finally, the marking time of the Aspergillus niger is effectively improved15Abundance of N-tag. By the invention15The mycoprotein prepared by the preparation method of the N-labeled mycoprotein is beneficial to absolute quantification of the protein by adopting an iBAQ quantification method in the later period, not only can eliminate enzyme digestion and mass spectrum deviation, but also can be used as the mycoproteinThe secondary manufacture can be used for many times, and the cost is reduced.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A kind of15The preparation method of the N-labeled mycoprotein comprises the following steps:
seed culture: inoculating the spore liquid into a seed culture medium, and culturing at 33-35 ℃ for 22-26 hours at a rotating speed of 120-180 revolutions per minute to obtain a seed liquid;
pretreatment: placing a proper amount of the seed liquid into a centrifugal tube, centrifuging for 4-8 minutes at 3500-4500 rpm, and washing with normal saline to obtain a seed treatment liquid;
marking and culturing: placing the seed treatment solution in a shake bottle, adding a fermentation culture medium solution, shaking the bottle for fermentation for 24 hours, transferring the bottle to a fermentation tank, performing fermentation culture for 45-50 hours, adding the fermentation culture medium solution in a feeding manner in a fermentation culture engineering, and stopping fermentation when the carbon dioxide release rate (CER) in a fermentation system is reduced to obtain fermentation liquor;
protein isolation: carrying out suction filtration on the obtained fermentation liquor to obtain thalli, freezing the thalli by using liquid nitrogen, grinding, and carrying out freeze drying for 24-26 hours to obtain the bacillus subtilis15N-labeled mycoprotein.
In the fermentation tank, the feeding rate of the fermentation medium solution is 10-16 g/h. The spore liquid is Aspergillus niger CBS513.88 spore liquid. The pH value of the seed culture medium is 6.2-6.8. The seed culture medium is sterilized before use under the condition of sterilization at 121 ℃ for 30 min. The nitrogen atom of the nitrogen source in the fermentation medium is15And marking by N. The thalli are frozen and stored at the temperature below minus 80 ℃. The components of the seed culture medium comprise: without waterGlucose, corn steep liquor solids, and antifoam. The fermentation medium comprises the following components: glucose, CaCl2、MgSO4·7H2O、EDTA、(NH4)2SO4、KH2PO4、NaH2PO4·2H2O、MnSO4·H2O、ZnCl2、CuSO4·5H2O、CoCl2·6H2O、FeSO4·7H2O and an antifoaming agent.
Example 2
A kind of15The preparation method of the N-labeled mycoprotein comprises the following steps:
seed culture: taking 1.4mL of spore with the number of 106Inoculating the spore solution into a seed bottle containing a seed culture medium, and culturing at a rotation speed of 150rpm at 34 ℃ for 24 hours to obtain a seed solution;
pretreatment: placing 4mL (0.033g) of the seed solution into a 10mL centrifuge tube, centrifuging for 5 minutes at 4000rpm, washing with physiological saline three times, removing corn steep liquor to obtain a seed treatment solution, and avoiding the phenomenon of the seed treatment solution in a seed culture medium14The influence of N;
marking and culturing: placing the seed treatment solution in a baffle shake flask containing a fermentation medium, carrying out shake flask fermentation for 24 hours, transferring to a 1L fermentation tank, adding the fermentation medium solution in a material supplementing manner in the fermentation process, and carrying out fermentation culture for 48 hours to obtain a fermentation broth, wherein the volume of the fermentation broth is 587 mL;
protein isolation: filtering the obtained fermentation liquor to obtain thallus, freezing with liquid nitrogen, grinding, and freeze-drying for 24 hr to obtain15N-labeled mycoprotein.
In the fermentation tank, the feeding rate of the fermentation medium solution is 10-16 g/h. The spore liquid is Aspergillus niger CBS513.88 spore liquid. The pH value of the seed culture medium is 6.2-6.8. The seed culture medium is sterilized before use under the condition of sterilization at 121 ℃ for 30 min. The nitrogen atom of the nitrogen source in the fermentation medium is15And marking by N.
In this example, the components and amounts of the seed culture medium are shown in table 1:
TABLE 1
Composition (I) The actual amount of the active ingredient
Anhydrous glucose 4g/200g
Corn steep liquor solid (CSS) 4g/200g
Defoaming agent 100μL/100mL
In this example, the pH of the seed medium was adjusted to 6.5 using 4M sodium hydroxide.
In this example, in the shake flask fermentation step, the mixture is separately filled into 2 500mL baffle shake flasks, each flask containing 100 mL. The culture conditions of the shake flask fermentation are as follows: the temperature was 34 (+ -0.5) deg.C; the rotating speed is 150 rpm; the period is 24 h.
In this example, the components and amounts of the fermentation medium are shown in table 2:
TABLE 2
Composition (I) Actual dosage (g/600g)
Glucose 30.06
CaCl2 0.0456
MgSO4·7H2O 0.6
EDTA 0.402
(NH4)2SO4 1.8
KH2PO4 1.8
NaH2PO4·2H2O 1.014
MnSO4·H2O 0.024
ZnCl2 0.012
CuSO4·5H2O 0.009
CoCl2·6H2O 0.009
FeSO4·7H2O 0.18
Defoaming agent 0.6mL
In the fermentation medium, 30.06g of the glucose is added into 100g of deionized water to a constant volume (separately sterilized, and the glucose solution is sterilized at 121 ℃ for 60min) to obtain a glucose solution; the other ingredients were brought to 440g with deionized water.
In this embodiment, in the fermentation tank, the conditions of the fermentation culture are as follows: the ventilation volume is 3L/min; the temperature was 34 (+ -0.5) deg.C; the fermentation period is 144h (6 days); the rotating speed is 400 rpm; the pH was 4.5 (+ -0.1) and was automatically adjusted with NaOH.
In this example, the feed rate of the fermentation medium in the fermentor (1L fermentor) was:
24.22～34.97h：15.75g/h；
34.97～39.52h：21g/h；
39.52～48.5h：10.5g/h。
in the embodiment, sampling is carried out when the shake flask is fermented for 24 hours, and the concentration of bacteria in the reaction system is measured to be 1.170 gDCW/kg; sampling when fermenting for 24h after transferring into a fermentation tank, and measuring the bacterial concentration in the reaction system to be 2.4377 gDCW/kg; sampling is carried out when fermentation is carried out for 48 hours after the fermentation tank is transferred, and the bacterial concentration in the reaction system is measured to be 4.7488 gDCW/kg.
Test example 1
The abundance of the mycoprotein obtained in example 2 of the invention was determined and the experimental data recorded as follows:
analyzing by an isotope ratio mass spectrometer to obtain the mycoprotein15The abundance of N is as high as 97.83%. It can be seen that the preparation method of the present invention can effectively reduce15N is labeled into mycoprotein, and15the marked abundance of N is high, and the effect is obvious.15The N high-abundance marked mycoprotein is beneficial to absolute quantification of mycoprotein in the later period.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A kind of15The preparation method of the N-labeled mycoprotein is characterized by comprising the following steps:
seed culture: inoculating the spore liquid into a seed culture medium, and culturing at 33-35 ℃ at a rotating speed of 120-180 revolutions per minute to obtain a seed liquid;
pretreatment: placing the seed liquid in a centrifugal tube, centrifuging for 4-8 minutes at 3500-4500 rpm, and washing with normal saline to obtain a seed treatment liquid;
marking and culturing: adding the seed treatment solution into a fermentation culture medium solution, shaking the bottle for fermentation, transferring the bottle to a fermentation tank for fermentation culture, adding the fermentation culture medium solution in a feeding manner in the fermentation culture process, and obtaining fermentation broth after terminating the fermentation;
protein isolation: filtering the obtained fermentation liquor to obtain thallus, freezing with liquid nitrogen, grinding, and freeze drying to obtain the final product15N-labeled mycoprotein.
2. The method of claim 115The preparation method of the N-labeled mycoprotein is characterized in that the nitrogen atom of the nitrogen source in the fermentation medium is15And marking by N.
3. The method of claim 1 or 215The preparation method of the N-labeled mycoprotein is characterized in that in the fermentation tank, the feeding rate of the fermentation medium solution is 10-16 g/h.
4. The method of claim 115The preparation method of the N-labeled mycoprotein is characterized in that the spore liquid is Aspergillus niger CBS513.88 spore liquid.
5. According to claim 1Described in15The preparation method of the N-labeled mycoprotein is characterized in that the pH value of the seed culture medium is 6.2-6.8.
6. The method of claim 515The preparation method of the N-labeled mycoprotein is characterized in that the seed culture medium comprises the following components: anhydrous glucose, corn steep liquor solids, and an antifoaming agent.
7. A composition according to any one of claims 1 to 315The preparation method of the N-labeled mycoprotein is characterized in that the fermentation medium comprises the following components: glucose, CaCl2、MgSO4·7H2O、EDTA、(NH4)2SO4、KH2PO4、NaH2PO4·2H2O、MnSO4·H2O、ZnCl2、CuSO4·5H2O、CoCl2·6H2O、FeSO4·7H2O and an antifoaming agent.
8. The method of claim 115The preparation method of the N-labeled mycoprotein is characterized in that the fermentation termination is as follows: when the carbon dioxide release rate (CER) in the fermentation system decreases.
9. The method of claim 115A process for producing an N-labeled mycoprotein, characterized in that the concentration of the spore liquid is 1X 106～107/mL。
10. Prepared by the preparation method of any one of claims 1-915And (3) calculating the absolute quantification of the mycoprotein by marking the mycoprotein with the N.
CN201911222334.2A 2019-12-03 2019-12-03 A kind of15Preparation method and application of N-labeled mycoprotein Pending CN110862928A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101235402A (en) * 2007-02-01 2008-08-06 上海化工研究院 Fermentation technique for producing stability isotope 15N marking L-leucine
CN106417194A (en) * 2016-12-09 2017-02-22 广州微因生物科技有限公司 N15 stable isotope labeling method for insect protein quantification and tracing

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101235402A (en) * 2007-02-01 2008-08-06 上海化工研究院 Fermentation technique for producing stability isotope 15N marking L-leucine
CN106417194A (en) * 2016-12-09 2017-02-22 广州微因生物科技有限公司 N15 stable isotope labeling method for insect protein quantification and tracing

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HONGZHONG LU等: "Integrated isotope-assisted metabolomics and 13C metabolic flux analysis reveals metabolic flux redistribution for high glucoamylase production by Aspergillus niger", 《MICROB CELL FACT》 *
周长林: "《微生物学》", 31 August 2015, 中国医药科技出版社 *

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Application publication date: 20200306