Method for Propagating Coenzyme Q10

Abstract

The invention discloses a bacterial strain expansion method for coenzyme Q10. The method comprises the following steps: obtaining a corresponding single colony bacterial strain based on expansion requirements, and uniformly crushing the bacterial strain in a triangular flask containing 9 ml of sterilized physiological saline by using sterilized glass beads to obtain a bacterial suspension; evenly distributing 0.2 ml of the bacterial suspension sucked by using a graduated pipette nozzle on a culture plate; and culturing the culture plate normally in an incubator at 32° C. for 4 hours, and then inverting the culture plate in the incubator at 32° C. for 5-6 days to complete the expansion and improve production practicability.

Description

Technical Field

[0001] The invention relates to the technical field of bacterial strain cultivation, and in particular to a bacterial strain expansion method for coenzyme Q10. Background Art

[0002] The yhD strain is a special strain for the fermentation production of BJ01 (coenzyme Q10) products. The process requires that a single colony grown on a natural selection plate for 5 to 6 days is inoculated into a mother bottle medium shaker culture and then inoculated into a seed tank for production of first-level seed culture. This method requires a huge number of single colonies. The size and color of the single colonies grown by natural selection and coating are not uniform, and individual differences always exist. When the colonies are too small, the operator must ensure that they are not contaminated by human operation when picking a large number of single colonies of about φ2mm in a short time, which tests the operator's carefulness, patience and physical strength. In the process of manual picking, it is inevitable that the operator will be dizzy and cause differences in the selection of single colonies. After being connected to the mother bottle for culture, individual differences may expand, and the uniformity and synchronization of the strains will affect the subsequent fermentation production, resulting in reduced production practicality. Summary of the invention

[0003] The purpose of the present invention is to provide a method for expanding the bacterial strain of coenzyme Q10 to improve production practicability.

[0004] To achieve the above object, the present invention provides a method for expanding the bacterial strain of coenzyme Q10, comprising the following steps:

[0005] Obtain the corresponding single colony strain based on the expansion culture requirements, and use sterilized glass beads to evenly crush it to obtain a bacterial suspension;

[0006] Use a graduated pipette to evenly distribute 0.2 ml of the bacterial suspension on the culture plate;

[0007] The culture plate was inverted and cultured in an incubator at 32° C. for 5-6 days to complete the expansion culture.

[0008] Among them, based on the expansion culture requirements, the corresponding single colony strains are obtained, and sterilized glass beads are used to evenly crush them to obtain a bacterial suspension, including:

[0009] Based on the requirements of expansion cultivation, randomly select a single colony of strains from the three-level strains that have completed the initial cultivation;

[0010] The single bacterial colony was placed in a sterilized triangular flask and evenly crushed using sterilized glass beads to obtain a bacterial suspension.

[0011] The single bacterial colony is placed in a sterilized triangular flask and evenly crushed with sterilized glass beads to obtain a bacterial suspension, including:

[0012] Add the single bacterial colony into a conical flask containing 9 ml of sterilized saline;

[0013] A plurality of sterilized glass beads are added into the conical flask, and the conical flask is shaken at a set driving force and a set time to evenly break the single bacterial colony to obtain a bacterial suspension.

[0014] Wherein, the culture plate is inverted and cultured in an incubator at 32° C. for 5-6 days. Before the expansion culture is completed, the method further comprises:

[0015] The culture plate was normally cultured in an incubator at 32° C. for 4 hours;

[0016] After 4 hours, the culture plate was inverted and cultured in the incubator at 32°C.

[0017] After the 0.2 ml of the bacterial suspension is evenly distributed on the culture plate using a graduated pipette, the method further comprises:

[0018] The culture plate was placed at room temperature until the bacterial suspension was completely absorbed.

[0019] The invention discloses a bacterial strain expansion method for coenzyme Q10. The method comprises the following steps: obtaining a corresponding single colony bacterial strain based on the expansion requirement, and evenly crushing the bacterial strain in a triangular flask containing 9 ml of sterilized physiological saline by using sterilized glass beads to obtain a bacterial suspension; evenly distributing 0.2 ml of the bacterial suspension on a culture plate by using a graduated pipette nozzle; and culturing the culture plate normally in an incubator at 32° C. for 4 hours, and then inverting the culture plate in an incubator at 32° C. for 5-6 days to complete the expansion, thereby improving production practicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The present invention provides a schematic diagram of the steps of a method for bacterial expansion of coenzyme Q10.

[0022] Figure 2 It is a schematic diagram of the single colony strain distribution provided by the present invention. DETAILED DESCRIPTION

[0023] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0024] In the description of the present invention, “plurality” means two or more than two, unless otherwise clearly and specifically defined.

[0025] See also Figure 1 The present invention provides a method for expanding the bacterial strain of coenzyme Q10, comprising the following steps:

[0026] S101. Obtain corresponding single colony strains based on the expansion culture requirements, and use sterilized glass beads to evenly crush them to obtain a bacterial suspension.

[0027] Specifically, based on the expansion culture requirements, a single colony of bacteria is randomly selected from the tertiary bacteria that have completed the initial cultivation. The expansion culture requirements are uniform color, roundness and fullness, and meeting the requirements of the tertiary bacteria. Then, the single colony of bacteria is placed in a triangular flask containing 9 ml of sterilized physiological saline, and a plurality of sterilized glass beads are added to the triangular flask. Under a set driving force and a set time, the triangular flask is shaken to evenly break the single colony of bacteria to obtain a bacterial suspension. The number of glass beads used is limited according to the volume of the triangular flask, and the surface of the glass beads must be smooth and free of cracks, etc., to avoid damage to the structure of the single colony of bacteria or uneven breakage. The driving force used for shaking is manually controlled by hand to ensure that the collision of the glass beads with the triangular flask will not break the triangular flask during the shaking process. The shaking time is generally set to about 3 minutes. Too much shaking will cause the structure of the single colony of bacteria to be destroyed, and too little shaking will cause uneven breakage.

[0028] S102, using a graduated pipette nozzle to evenly distribute 0.2 ml of the bacterial suspension on the culture plate.

[0029] Specifically, 0.2 ml of the bacterial suspension is sucked with a complete 1 ml graduated pipette, and the 0.2 ml of the bacterial suspension is evenly dotted on a culture plate with a diameter of 150 mm using a graduated pipette nozzle. Specifically, 0.2 ml of the bacterial suspension is sucked with a graduated pipette, and 10-12 single colonies are added per block, the size of each single colony is 6-8 mm, and the distance between two adjacent single colonies is about 30 mm. Figure 2As shown, the culture plate after the spotting is placed at room temperature until the bacterial suspension is completely absorbed, wherein the culture plate can be coated with some sterilizing agent in advance for sterilization to ensure the sterility of the culture process, and the culture plate has all the nutrient solution required for expansion culture.

[0030] S103, inverting the culture plate in a 32° C. incubator and culturing for 5-6 days to complete the expansion culture.

[0031] Specifically, first, the culture plate is normally cultured in an incubator at 32° C. for 4 hours. Normal culture means that it is placed normally for culture. After 4 hours, the culture plate is inverted and continued to be cultured in the incubator at 32° C. for 5-6 days to complete the expansion culture.

[0032] The method further comprises:

[0033] Obtain the cultured bacterial species, cultivate the cultured bacterial species to the third-level bacterial species according to the cultivation process, and sterilize all utensils.

[0034] Specifically, first, obtain the special strain for fermentation production of BJ01 (coenzyme Q10) products: yhD strain, and then culture the cultured strain to a third-level strain according to the conventional strain culture process. This process takes about 5-6 days, and during the culture process, sterilization is required to avoid affecting the subsequent expansion process. In order to improve the efficiency of expansion, the triangular flask, the physiological saline, the graduated pipette, the graduated pipette nozzle, the glass beads and the culture plate that will be used are sterilized in advance to ensure the purity of the expansion process and the stability of the results.

[0035] The present invention adopts a simple method of adding physiological saline glass beads to a single bacterial colony to crush and evenly prepare a bacterial suspension, and then using the bacterial suspension for spot plate culture to obtain a spot plate single bacterial colony with relatively uniform size and shape, uniform color, synchronous growth and absolutely guaranteed uniform seed source. After inoculating a mother bottle with the spot plate single bacterial colony, the effect of fermentation after inoculating a mother bottle with a natural breeding single bacterial colony can also be achieved, and it is relatively stable. The most critical thing is that the operability of inoculating a mother bottle with a spot plate bacterial colony is greatly improved, and the possibility of pollution caused by our personnel operation is more efficiently reduced. The operation time of inoculating the same number of production mother bottles with spot plate single bacterial colonies by the two methods is shortened by 2 / 3 or even more, and the labor intensity is also reduced. At the same time, the culture medium and culture cost of the spot plate bacterial colony are not increased. Therefore, this method has stronger production practicality than the natural breeding single bacterial colony required by the process, and the usage is in the decreasing experimental stage. It is very likely to achieve that 10 spot plate single bacterial colonies can achieve the same effect as 650 breeding single bacterial colonies.

[0036] The experimental data of two methods for culturing single colonies are as follows:

[0037]

[0038] The experimental data of two methods for culturing single colonies are as follows:

[0039]

[0040] The invention discloses a bacterial strain expansion method for coenzyme Q10. The method comprises the following steps: obtaining a corresponding single colony bacterial strain based on the expansion requirement, and evenly crushing the bacterial strain in a triangular flask containing 9 ml of sterilized physiological saline by using sterilized glass beads to obtain a bacterial suspension; evenly distributing 0.2 ml of the bacterial suspension on a culture plate by using a graduated pipette nozzle; and culturing the culture plate normally in an incubator at 32° C. for 4 hours, and then inverting the culture plate in an incubator at 32° C. for 5-6 days to complete the expansion, thereby improving production practicability.

[0041] What is disclosed above is only a preferred embodiment of the present invention, and it certainly cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A method for the bacterial expansion of coenzyme Q10, characterized in that: The following steps are involved: Obtain the corresponding single colony strain based on the expansion culture requirements, and use sterilized glass beads to evenly crush it to obtain a bacterial suspension; Use a graduated pipette to evenly distribute 0.2 ml of the bacterial suspension on the culture plate; Incubate the culture plate upside down in a 32° C. incubator for 5-6 days to complete the expansion culture; Obtain the corresponding single colony strains based on the expansion requirements, and use sterilized glass beads to evenly crush them to obtain a bacterial suspension, including: Based on the requirements of expansion cultivation, randomly select a single colony of strains from the three-level strains that have completed the initial cultivation; The single bacterial colony is placed in a sterilized triangular flask and evenly crushed using sterilized glass beads to obtain a bacterial suspension; The single bacterial colony is placed in a sterilized triangular flask and evenly crushed with sterilized glass beads to obtain a bacterial suspension, including: Add the single bacterial colony into a conical flask containing 9 ml of sterilized saline; Adding a plurality of sterilized glass beads into the triangular flask, shaking the triangular flask under a set driving force and a set time to evenly break the single bacterial colony to obtain a bacterial suspension; Using a graduated pipette to evenly distribute 0.2 ml of the bacterial suspension on a culture plate, including: using a graduated pipette to distribute 0.2 ml of the bacterial suspension, according to 10-12 single colonies / block, the size of each single colony is 6-8 mm, and the distance between two adjacent single colonies is 30 mm; The culture plate is inverted and cultured in an incubator at 32° C. for 5-6 days. Before the expansion culture is completed, the method further comprises: The culture plate was normally cultured in an incubator at 32° C. for 4 hours; After 4 hours, the culture plate was inverted and cultured in the incubator at 32°C.

2. The method for growing coenzyme Q10 according to claim 1, characterized in that: After using a graduated pipette nozzle to evenly distribute 0.2 ml of the bacterial suspension on the culture plate, the method further comprises: The culture plate was placed at room temperature until the bacterial suspension was completely absorbed.

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