CN110819575A - Culture method of bacillus for producing nattokinase - Google Patents

Culture method of bacillus for producing nattokinase Download PDF

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CN110819575A
CN110819575A CN201911265786.9A CN201911265786A CN110819575A CN 110819575 A CN110819575 A CN 110819575A CN 201911265786 A CN201911265786 A CN 201911265786A CN 110819575 A CN110819575 A CN 110819575A
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culture
bacillus natto
culture medium
shake flask
flask reactor
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孙婧
蒋继宏
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Jiangsu Normal University
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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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21062—Subtilisin (3.4.21.62)

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Abstract

A method for culturing Bacillus natto kinase, which comprises: adding a certain amount of carbon source-containing culture medium into a shake flask reactor, and sterilizing at high temperature for later use; inoculating the activated bacillus subtilis seed solution into a shake flask reactor for shake culture, wherein the culture temperature is 28 ℃, the rotation speed of the shake flask reactor is 200-240rpm, the inoculation amount of the seed solution is 1-5%, and the initial pH of a culture medium is 7-7.5. Aiming at the problem of low biomass generally existing in the process of culturing the bacillus natto, the invention improves the carbon source of the culture medium and optimizes the culture condition, and improves the growth condition of the bacillus natto to improve the bacillus natto biomass. The culture method can improve the unit cell density in the fermentation process of the bacillus natto, improve the biomass of the bacillus natto, and simultaneously, the components of the culture medium are simple, thereby being beneficial to subsequent treatment and reducing the production cost.

Description

Culture method of bacillus for producing nattokinase
Technical Field
The invention relates to the technical field of functional food and biological energy; in particular to a culture process for improving the biomass of the bacillus natto in the process of culturing the bacillus natto.
Background
Bacillus subtilis has many attractive properties, and in recent years, the research on Bacillus subtilis has been advanced, and the physiological properties and genetic background as well as biochemical mechanism of Bacillus subtilis have been intensively studied. As a gram-positive bacterium, Bacillus subtilis is an industrially used expression host for the production of heterologous proteins. Bacillus subtilis has many advantages, such as being approved by the FDA in the united states, and having biosafety; has the characteristic of strong protein secretion; mature fermentation process and the like.
Natto is a traditional soybean food in Japan, and bacillus natto is a microorganism separated from the fermentation process of natto, belongs to bacillus subtilis, and has the functions of decomposing macromolecular substances, decomposing biological macromolecules into micromolecular active substances (such as oligosaccharide substances) and enabling a human body to digest and absorb more easily. The bacillus natto can also synthesize nattokinase, which is an orally-taken protein with fibrinolytic activity, has the pharmacological effects of improving blood circulation, reducing blood viscosity, improving blood circulation and reducing thrombosis risk, and is widely applied to the fields of food, medicine, health-care products and the like. Nattokinase is also an effective enzyme for the vitrolysis of drugs, acting mainly to induce posterior detachment of the vitreous ring. Because of the nature of bacillus natto, it is often used to produce nattokinase. Meanwhile, the nattokinase produced by the bacillus natto can be naturally secreted to the outside of cells, thereby being very beneficial to subsequent separation and purification.
Because of the excellent characteristics of the bacillus natto, the bacillus natto has wide application in the aspects of biological pharmacy, biological fresh-keeping, animal breeding, food addition, microecological preparation and the like, and has excellent application prospect and production potential in industry for improving the biomass of the bacillus natto.
Disclosure of Invention
The invention aims to provide a culture method of bacillus for producing nattokinase, which improves the biomass of the bacillus natto by optimizing the culture condition of the bacillus natto.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a method for culturing Bacillus natto kinase comprises the following steps:
s1: adding a certain amount of carbon source-containing culture medium into a shake flask reactor, and sterilizing at high temperature for later use;
s2: inoculating the activated bacillus subtilis seed solution into a shake flask reactor for shake culture, wherein the culture temperature is 28 ℃, the rotation speed of the shake flask reactor is 200-240rpm, the inoculation amount of the seed solution is 1-5%, and the initial pH of a culture medium is 7-7.5.
Further, the components of the culture medium comprise the following components in parts by weight:
0.1% KH2PO40.2% of Na2HPO42% tryptone, 0.02% CaCl20.05% of MgSO 42% of carbon source and the balance of water.
Further, the carbon source is sorbose, maltose, trehalose, xylitol or sucrose.
Further, the carbon source is maltose.
Further, the step S2 specifically includes: inoculating the activated bacillus subtilis seed solution into a shake flask reactor for shake culture, wherein the culture temperature is 28 ℃, the rotation speed of the shake flask reactor is 240rpm, the inoculation amount of the seed solution is 2%, and the initial pH of a culture medium is 7-7.5.
Compared with the prior art, the invention has the beneficial effects that:
aiming at the problem of low biomass generally existing in the process of culturing the bacillus natto, the invention improves the carbon source of the culture medium and further optimizes the culture condition, and improves the growth condition of the bacillus natto to improve the bacillus natto biomass. The culture method can improve the unit cell density in the fermentation process of the bacillus natto, improve the biomass of the bacillus natto, and simultaneously, the components of the culture medium are simple, thereby being beneficial to subsequent treatment and reducing the production cost.
Drawings
FIG. 1 shows the growth of Bacillus natto in shake flask reactor of example 1 in five different carbon sources (sorbose, maltose, trehalose, xylitol, sucrose) liquid culture medium at 180rpm under 1% inoculum size;
FIG. 2 shows the growth of Bacillus natto in a shake flask reactor in a maltose liquid medium under the conditions of 180rpm, 2%/3%/4%/5% inoculum size in a culture system in example 2 of the present invention;
FIG. 3 shows the growth of Bacillus natto in shake flask reactor in example 3 of the present invention in a culture system in maltose liquid medium at 200/220/240rpm under 2% inoculum size.
The specific implementation mode is as follows:
the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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
In the culture process for improving the biomass of the bacillus natto and thus improving the yield of the nattokinase, a shake flask reactor is used, five different carbon sources (sorbose, maltose, trehalose, xylitol and sucrose) liquid culture media are selected, and the bacillus natto is inoculated into a culture system under the culture conditions of 180rpm and 1% inoculation amount.
The specific method comprises the steps of adding a single culture medium into a 250mL shake flask reactor, inoculating 1% activated bacillus subtilis seed solution (the initial OD value is 0.2-0.6) after high-temperature sterilization, and carrying out shaking culture at the rotation speed of 180rpm and the temperature of 28 ℃. The OD value was measured.
Wherein the culture medium contains 0.1 wt% of KH2PO40.2% by weight of Na2HPO4 Tryptone 2% by weight, CaCl 0.02% by weight20.05% by weight of MgSO4Carbon source (sorbose, maltose, trehalose, xylitol, sucrose) at 2% by weight and pH 7-7.5.
In the embodiment, the bacillus subtilis natto is preserved by China center for culture Collection of industrial microorganisms, and is numbered CICC 10262.
FIG. 1 shows the variation trend of Bacillus natto biomass characterized by OD value in different carbon source culture medium systems. As can be seen from FIG. 1, the biomass of Bacillus natto was significantly increased throughout the entire cultivation period using the maltose medium, the OD value reached 44.3 by culturing Bacillus natto using the maltose medium, and 48 hours was determined to be a suitable cultivation period.
Example 2
In the culture process for improving the biomass of the bacillus natto, a shake flask reactor is utilized, four different inoculation amounts (2%/3%/4%/5%) are selected, and the bacillus natto is inoculated into a culture system by a maltose liquid culture medium under the culture condition of 180 rpm.
The specific method comprises the steps of adding a maltose culture medium into a 250mL shake flask reactor, sterilizing at high temperature, activating the bacillus subtilis seed solution (the initial OD value is 0.2-0.6), and carrying out shaking culture under the conditions of different inoculation amounts (2%/3%/4%/5%), the temperature is 28 ℃, and the rotation speed is 180 rpm. The OD value was measured.
The maltose medium was the same as in example 1.
The bacillus subtilis natto is the same as the specific example 1.
FIG. 2 shows the variation trend of Bacillus natto biomass characterized by OD values in different rotating speed culture systems. As shown in FIG. 2, when 2% of the inoculum size was used, the OD value of Bacillus natto cultured was 46.2. The biomass can be increased by 4% compared to example 1.
Example 3
In the culture process for improving the biomass of the bacillus natto, a shake flask reactor is utilized, three different rotating speeds (200/220/240rpm) are selected, and the bacillus natto is inoculated into a culture system under the culture conditions of a maltose liquid culture medium and 2% inoculation amount.
The specific method comprises adding maltose culture medium into 250mL shake flask reactor, sterilizing at high temperature, inoculating 2% activated Bacillus subtilis seed solution (initial OD value of 0.2-0.6), and performing shake culture at 28 deg.C and different rotation speeds (200/220/240 rpm). The OD value was measured.
The maltose medium was the same as in example 1.
The bacillus subtilis natto is the same as the specific example 1.
FIG. 3 shows the variation trend of Bacillus natto biomass characterized by OD values in different rotating speed culture systems. As shown in FIG. 3, when the rotation speed was 240rpm, the OD value of Bacillus natto was 62.4 by culturing Bacillus natto. The biomass can be increased by 23% compared to example 1.

Claims (5)

1. A method for culturing Bacillus natto kinase, which is characterized by comprising the following steps:
s1: adding a certain amount of carbon source-containing culture medium into a shake flask reactor, and sterilizing at high temperature for later use;
s2: inoculating the activated bacillus subtilis seed solution into a shake flask reactor for shake culture, wherein the culture temperature is 28 ℃, the rotation speed of the shake flask reactor is 200-240rpm, the inoculation amount of the seed solution is 1-5%, and the initial pH of a culture medium is 7-7.5.
2. The method for culturing Bacillus natto kinase producing strains according to claim 1, wherein the culture medium comprises the following components in parts by weight:
0.1% KH2PO40.2% of Na2HPO42% tryptone, 0.02% CaCl20.05% of MgSO42% of carbon source and the balance of water.
3. The method for culturing Bacillus natto kinase producing bacteria according to claim 2, wherein the carbon source is sorbose, maltose, trehalose, xylitol or sucrose.
4. The method for culturing Bacillus natto kinase producing bacteria according to claim 3, wherein the carbon source is maltose.
5. The method for culturing Bacillus natto kinase according to claim 1, wherein the step S2 specifically comprises: inoculating the activated bacillus subtilis seed solution into a shake flask reactor for shake culture, wherein the culture temperature is 28 ℃, the rotation speed of the shake flask reactor is 240rpm, the inoculation amount of the seed solution is 2%, and the initial pH of a culture medium is 7-7.5.
CN201911265786.9A 2019-12-11 2019-12-11 Culture method of bacillus for producing nattokinase Pending CN110819575A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113215073A (en) * 2021-06-15 2021-08-06 中国科学院合肥物质科学研究院 Preparation method and genetic transformation method of bacillus natto competent cells

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107099487A (en) * 2017-06-27 2017-08-29 南京工业大学 Bacillus subtilis with high nattokinase secretion and application thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107099487A (en) * 2017-06-27 2017-08-29 南京工业大学 Bacillus subtilis with high nattokinase secretion and application thereof

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吴婷婷等: "纳豆芽孢杆菌最适发酵条件的研究进展", 《农产品加工》 *
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113215073A (en) * 2021-06-15 2021-08-06 中国科学院合肥物质科学研究院 Preparation method and genetic transformation method of bacillus natto competent cells

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Application publication date: 20200221