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Proliferation method and application of small selective-breeding vitamin C two-step fermentation bacteria

A two-step fermentation and vitamin technology, applied in the direction of bacteria, etc., can solve the problems that the excellent characteristics of the target strain are difficult to be fully expressed, and the difficulty of screening work is increased, so as to achieve the effect of increasing the content and conversion rate of sugar and acid, and reducing the difficulty of breeding

Inactive Publication Date: 2013-10-09
NINGXIA QIYUAN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the single colony of the treated small bacteria is not screened one by one, the unmutated colony and the mutant colony, the positive mutation and the negative mutation colony cannot be reflected separately, so it is difficult to fully express the excellent characteristics of the target strain, which increases the difficulty of the screening work.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Separate the large and small bacteria from the vitamin C two-step bacteria strain 08-1, select a single colony of small bacteria with full colonies and a moderate size, and spread them on the plate one by one by streaking method, and overlap the large bacteria for proliferation and culture, and culture at 28-30°C 2-3 days. Take the proliferating small bacteria and connect them one by one to the slant, and overlap the big bacteria to culture on the slant, and cultivate them at 28-30°C for 2-3 days. Take 2 loops of the lawn on the slant and inoculate it into the seed medium, and culture it with shaking at 200r / min at 28-30°C for 16h. Transfer to the fermentation medium according to 10% inoculum size, and shake at 200r / min at 28-30°C for 3-4 days. The content of gulonic acid and the concentration of residual L-sorbose in the fermentation broth were detected. In this test, a vitamin C two-step bacteria strain S-1 was obtained, the content of gulong acid reached 98.5 m...

Embodiment 2

[0017] Collect a certain number of small bacteria from the vitamin C second-step bacteria starting strain 09-2, irradiate them with a 15w ultraviolet lamp at a distance of 30cm for about 60s, and the mortality rate is 90-95%. , 28-30°C for 2-3 days. After the culture is over, select small single colonies of small bacteria with full colonies and moderate size, apply them to the plate one by one by streaking method, overlap the large bacteria for proliferation and culture, and culture at 28-30°C for 2-3 days. The proliferating small bacteria are connected to the slope one by one, and the large bacteria are overlapped for cultivation on the slope, and cultivated at 28-30°C for 2-3 days. Take 2 loops of the lawn on the slant and inoculate it into the seed medium, and culture it with shaking at 200r / min at 28-30°C for 16h. Transfer to the fermentation medium according to 10% inoculum size, and shake at 200r / min at 28-30°C for 3-4 days. The content of gulonic acid and the concentr...

Embodiment 3

[0019] Collect a certain number of small bacteria from the vitamin C second-step bacteria starting strain 11-45, dilute to a certain concentration, and microscopic examination requires that the colonies are evenly distributed without overlapping. Take 1ml and add it to a sterile plate, spread evenly, and air-dry to form a bacterial film. placed in the target chamber of an ion implantation mutagen machine, and the irradiation dose was 3×10 12 N + / cm 2 After irradiation, the bacterial film is eluted, diluted and spread on the plate, and the large bacteria are overlapped for plate culture, and cultivated at 28-30°C for 2-3 days. After the cultivation, select small single colonies of small bacteria with full colonies, moderate size and neat edges, apply them to the plate one by one by streaking method, overlap the large bacteria for proliferation and culture, and culture at 28-30°C for 2-3 days. After the proliferation, the small bacteria are connected to the slope one by one,...

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Abstract

The invention relates to a proliferation method and an application of small selective-breeding vitamin C two-step fermentation bacteria. The proliferation method comprises the following steps of: diluting small bacteria treated with a physical or chemical method; coating the small bacteria onto a flat plate and culturing for 2 to 3 days; picking single colonies of the small bacteria one by one through inoculating loops; separately coating the single colonies onto the separated flat plate by using a scoring method; picking big bacteria by using the inoculating loops, uniformly inoculating the big bacteria into the flat plate on which the small bacteria are coated and carrying out enrichment culture for 2 to 3 days; inoculating the proliferated small bacteria onto a cant one by one; picking the big bacteria by utilizing inoculating needles, inoculating the big bacterial into the cant on which the small bacteria are coated and culturing the big bacteria; inoculating two loops of cant bacteria mosses which grow well into a seed culture medium and culturing for 16 hours; transferring the cant bacteria mosses into a culture fermentation medium according to 10% of inoculum size and culturing for 3 to 4 days; detecting the content of Keto-L-gulonic acid in a fermentation liquor and the concentration of residual L-sorbose; and selecting target strains according to the statistical analysis of experimental data.

Description

technical field [0001] The invention relates to the technical fields of biology and medicine, in particular to a multiplication method and application in the breeding process of vitamin C two-step fermentation small bacteria. Background technique [0002] Vitamin C (Vitanun C, Vc) is an essential vitamin for human nutrition. It is not only used as an important medical product to treat various diseases, but also widely used in food, feed and cosmetics. With the increase of the application range of vitamin C, the market demand is increasing day by day, so that people continue to study and improve the production technology of VC. [0003] At present, the fermentation production of vitamin C is realized by a two-step method, that is, the mixed fermentation of Bacillus (commonly known as large bacteria, mainly Bacillus megaterium, Bacillus cereus and Bacillus thuringiensis) and Gluconobacter oxidans (commonly known as small bacteria) , the small bacteria are acid-producing bacte...

Claims

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Application Information

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IPC IPC(8): C12N1/20
Inventor 于新燕孙瑞君胡继红陈惠琴
Owner NINGXIA QIYUAN PHARMA
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