CN105506018A

CN105506018A - Strain culture medium for industrially producing L-hydroxyproline through fermentation method and culture method

Info

Publication number: CN105506018A
Application number: CN201511034740.8A
Authority: CN
Inventors: 赵体金; 林俊辉; 彭久合
Original assignee: TIANJIN JINGYE FINE CHEMICALS CO Ltd
Current assignee: TIANJIN JINGYE FINE CHEMICALS CO Ltd
Priority date: 2015-12-31
Filing date: 2015-12-31
Publication date: 2016-04-20

Abstract

Provided are a strain culture medium for industrially producing L-hydroxyproline through a fermentation method and a culture method. The culture medium is composed of yeast powder, peptone, sodium chloride, agar and AMP; the culture method comprises the steps that the pH of the strain culture medium is adjusted to 7.0 through a NaOH solution of 2N, the stain culture medium is subpackaged in 10 ml slant test tubes or 50 ml eggplant-shaped bottles, the slant test tubes or eggplant-shaped bottles are plugged and wrapped with kraft paper, sterilization is conducted, the slant test tubes or eggplant-shaped bottles are taken out to be cooled to 50-60 DEG C, a 10% AMP solution is added into the slant test tubes or eggplant-shaped bottles in a sterile room and shaken to be uniform, the slant test tubes or eggplant-shaped bottles are put in an inclined mode and put in the sterile room for use after the mixture is solidified; a ring of strains are evenly applied on the empty slant faces of the test tubes or eggplant-shaped bottles through an inoculating loop on a clean bench, the test tubes or the eggplant-shaped bottles are put in a culture box with the temperature of 37+/-1.0 DEG C for culture for 24-48 h, and a bacterial lawn grows well and is directly put into a tank or put in a refrigerator at the temperature of 0-5 DEG C for use. According to the strain culture medium for industrially producing the L-hydroxyproline through the fermentation method and the culture method, the strains are selected to be cultured in the prepared solid culture medium, strain culture is good, and the improvement of fermentation level of the L-hydroxyproline, reduction of the production and avoidance of environmental pollution are facilitated.

Description

Fermentation method industrialization skill produces bacterium culture medium and the cultural method of L-oxyproline

Technical field

The present invention relates to bacterium culture medium and the cultural method of fermentable, be specifically related to bacterial classification solid medium and the cultural method of fermentation method suitability for industrialized production L-oxyproline.

Background technology

Oxyproline (Hydroxyproline, Hyp) is imino-acid, is the product after L-PROLINE hydroxylation, and its molecular formula is C ₅h ₉nO ₃.Oxyproline is white flaky crystals or crystalline powder, micro-sweet, and fusing point is 274 DEG C, soluble in water, is slightly soluble in ethanol.Occurring in nature trans-4-oxyproline is comparatively common, trans-and 4-Hydroxyproline is found in animal collagen the earliest.Trans-4-Hydroxyproline is widely used in the aspects such as medicine, chemical industry, animal-feed, nutrition and beauty culture.

At present, the method for the production oxyproline of report mainly contains proteolysis extraction method, chemical synthesis, microbe fermentation method and enzymatic synthesis four kinds both at home and abroad, and wherein proteolysis extraction method is the industrial process generally adopted at present.But, proteolysis extraction method need through strong acid and strong base process, purification step is long, and oxyproline extraction yield is at 4%-7%, extract and raw materials cost high, not only waste large content of starting materials, and waste pollution is serious, along with the increase of current environmental stress and the rising of resource cost of material, traditional proteolysis extraction method is just gradually by market.Microbe fermentation method just develops rapidly with the technique of its environmental protection, raw material and advantage with low cost.

At present, the research of Production by Microorganism Fermentation L-oxyproline is also in laboratory stage, not yet form ripe industrialized producing technology, and the industrialized producing technology of advanced person, especially the solid culture formula of bacterial classification and method have material impact for the fermentation unit of L-oxyproline, cost and three waste discharge.

Summary of the invention

The object of the invention is to: bacterium culture medium and cultural method that a kind of fermentation method suitability for industrialized production L-oxyproline is provided, the bacterial classification adopting this spawn culture formula and technique to obtain is applied to suitability for industrialized production L-oxyproline, is conducive to the fermentation level of raising L-oxyproline, reduces production cost, avoids environmental pollution.

The technical solution used in the present invention:

The bacterium culture medium of fermentation method suitability for industrialized production L-oxyproline is made up of yeast powder, peptone, sodium-chlor, agar, AMP, and its formula is yeast powder 5g/L, peptone 10g/L, sodium-chlor 5g/L, agar 20g/L, AMP50mg/L.

The spawn culture method of fermentation method suitability for industrialized production L-oxyproline, comprise the following steps, first, the NaOH solution of bacterium culture medium 2N adjusts pH to 7.0, is sub-packed in 10ml slant tube or 50ml eggplant bottle, to jump a queue bag kraft paper, 121 DEG C of sterilizings 15 minutes, taking out the AMP solution adding 10% after being cooled to 50 ~ 60 DEG C in sterilisable chamber, is 50ppm to AMP final concentration, shake up rear-inclined 30 ° placement, rearmounted sterilisable chamber to be solidified is for subsequent use; Be uniformly coated on test tube or the blank inclined-plane of eggplant bottle at the standardized ring bacterial classification of Bechtop transfering loop, place in the incubator of 37 ± 1.0 DEG C and cultivate 24 ~ 48 hours, lawn is grown rear directly upper tank or is placed in 0 ~ 5 DEG C of refrigerator for subsequent use.

Described concentration be 10% AMP solution to be dissolved in distilled water obtained by Ampicillin Trihydrate.

Described bacterial classification comes from Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences, and bacterial strain is a strain EscherichiaColiK12W3110 mutation.

Beneficial effect of the present invention: the present invention selects bacterial classification to cultivate in prepared solid medium, spawn culture is good, is conducive to the fermentation level of raising L-oxyproline, reduces production cost, avoids environmental pollution.

Embodiment

Below in conjunction with specific embodiment, in further detail the present invention is described.Should be understood that these embodiments just in order to demonstrate the invention, but not limit the scope of the invention by any way.

Example 1

The spawn culture method of fermentation method suitability for industrialized production L-oxyproline: the NaOH solution of bacterium culture medium 2N adjusts pH to 7.0, be sub-packed in 10ml slant tube, to jump a queue bag kraft paper, 121 DEG C of sterilizings 15 minutes, in sterilisable chamber, the AMP solution 5ul of 10% is added after taking-up is cooled to 50 DEG C, shake up rear-inclined 30 ° placement, rearmounted sterilisable chamber to be solidified is for subsequent use; Be uniformly coated on test tube slant at the standardized ring bacterial classification of Bechtop transfering loop, place in the incubator of 37 ± 1.0 DEG C and cultivate 24 hours, lawn directly plants female bottle after growing; Wherein bacterial classification derives from Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences, and bacterial strain is a strain EscherichiaColiK12W3110 mutation.Wherein concentration be 10% AMP solution to be dissolved in distilled water obtained by Ampicillin Trihydrate.

Example 2

The spawn culture method of fermentation method suitability for industrialized production L-oxyproline: the NaOH solution of bacterium culture medium 2N adjusts pH to 7.0, be sub-packed in 50ml eggplant bottle, to jump a queue bag kraft paper, 121 DEG C of sterilizings 15 minutes, in sterilisable chamber, the AMP solution 25ul of 10% is added after taking-up is cooled to 55 DEG C, shake up rear-inclined 30 ° placement, rearmounted sterilisable chamber to be solidified is for subsequent use; Be uniformly coated on the blank inclined-plane of eggplant bottle at the standardized ring bacterial classification of Bechtop transfering loop, place in the incubator of 37 ± 1.0 DEG C and cultivate 38 hours, lawn is placed in 0 ~ 5 DEG C of refrigerator for subsequent use after growing; Wherein bacterial classification derives from Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences, and bacterial strain is a strain EscherichiaColiK12W3110 mutation.Wherein concentration be 10% AMP solution to be dissolved in distilled water obtained by Ampicillin Trihydrate.

Example 3

Bacterial classification solid medium is made up of yeast powder, peptone, sodium-chlor, agar, AMP, and its formula is yeast powder 5g/L, peptone 10g/L, sodium-chlor 5g/L, agar 20g/L, AMP50mg/L.

Spawn culture: the NaOH solution of bacterium culture medium 2N adjusts pH to 7.0, be sub-packed in 50ml eggplant bottle, to jump a queue bag kraft paper, 121 DEG C of sterilizings 15 minutes, in sterilisable chamber, the AMP solution 25ul of 10% is added after taking-up is cooled to 60 DEG C, shake up rear-inclined 30 ° placement, rearmounted sterilisable chamber to be solidified is for subsequent use; Be uniformly coated on the blank inclined-plane of eggplant bottle at the standardized ring bacterial classification of Bechtop transfering loop, place in the incubator of 37 ± 1.0 DEG C and cultivate 36 hours, lawn is placed in 0 ~ 5 DEG C of refrigerator for subsequent use after growing; Wherein bacterial classification derives from Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences, and bacterial strain is a strain EscherichiaColiK12W3110 mutation.Wherein concentration be 10% AMP solution to be dissolved in distilled water obtained by Ampicillin Trihydrate.

Claims

1. the bacterium culture medium of fermentation method suitability for industrialized production L-oxyproline, is characterized in that, is made up of yeast powder, peptone, sodium-chlor, agar, AMP, and its formula is yeast powder 5g/L, peptone 10g/L, sodium-chlor 5g/L, agar 20g/L, AMP50mg/L.

2. the spawn culture method of fermentation method suitability for industrialized production L-oxyproline, it is characterized in that, comprise the following steps, first, the NaOH solution of bacterium culture medium 2N adjusts pH to 7.0, be sub-packed in 10ml slant tube or 50ml eggplant bottle, bag kraft paper of jumping a queue, 121 DEG C of sterilizings 15 minutes, in sterilisable chamber, the AMP solution of 10% is added after taking-up is cooled to 50 ~ 60 DEG C, be 50ppm to AMP final concentration, shake up rear-inclined 30 ° placement, rearmounted sterilisable chamber to be solidified is for subsequent use; Be uniformly coated on test tube or the blank inclined-plane of eggplant bottle at the standardized ring bacterial classification of Bechtop transfering loop, place in the incubator of 37 ± 1.0 DEG C and cultivate 24 ~ 48 hours, lawn is grown rear directly upper tank or is placed in 0 ~ 5 DEG C of refrigerator for subsequent use.

3. the spawn culture method of fermentation method suitability for industrialized production L-oxyproline according to claim 2, it is characterized in that, described bacterial classification derives from Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences, and bacterial strain is a strain EscherichiaColiK12W3110 mutation.

4. the spawn culture method of fermentation method suitability for industrialized production L-oxyproline according to claim 2, is characterized in that, the AMP solution of described 10% is dissolved in distilled water obtained by Ampicillin Trihydrate.