Screening and fermentation regulation method of 5-hydroxytryptophan functional spore bacteria

Abstract

The invention relates to a screening and fermentation regulation method of 5-hydroxytryptophan functional spore bacteria, which comprises the following steps: collecting abnormal fermentation bacterial liquid or bacterial residue waste, and suspending in a sulfuric acid aqueous solution with the pH value of 1.8-2.5 for 2 days; coating the suspension on a kanamycin resistance screening agar plate, and culturing at 30 DEG C for 48 hours; separating bacterial colonies on the resistant plate, carrying out shake-flask culture in a kanamycin resistant culture medium containing tryptophan, adding IPTG (isopropyl-beta-d-thiogalactoside) for induction in the later stage of index fermentation, detecting that tryptophan in fermentation liquor is converted into 5-hydroxytryptophan, and storing the 5-hydroxytryptophan in a refrigerator at the temperature of 80 DEG C below zero; the method comprises the following steps: resuscitating a strain, culturing and amplifying in a shake flask, transferring into a 5L fermentation tank, fermenting with a culture medium containing 20g / L tryptophan, adding IPTG (isopropyl-beta-d-thiogalactoside) at the later stage of exponential growth to induce transformation, and simultaneously adding cumaric acid and ferulic acid to regulate and control the fermentation rate and the metabolic process. The functional bacterium provided by the invention is high in 5-HTP conversion specificity, only a small amount of isomeride is generated, the isomeride can be easily removed in the purification process, and few polymers are generated in the strain fermentation process.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for screening and fermentation regulation of 5-hydroxytryptophan functional spore bacteria. Background Art

[0002] 5-Hydroxytryptophan (5-HTP) is an amino acid derivative formed by the oxidation of tryptophan. In most European countries, 5-HTP is a commonly prescribed drug for a variety of therapeutic purposes; however, in the North American market, it is sold as an "over-the-counter" dietary supplement. 5-HTP is also considered a potential feed additive for farm animals, with positive effects on animal growth and production performance.

[0003] With the continuous reduction of resources such as the Ghana tree and people's increasing attention to green and environmentally friendly synthesis processes, the use of genetic engineering and metabolic engineering methods to transform engineered bacteria and obtain 5-hydroxytryptophan has gradually attracted people's attention. Summary of the invention

[0004] The present invention aims to provide a method for screening and fermentation regulation of 5-hydroxytryptophan functional spore-forming bacteria.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for screening and fermentation regulation of 5-hydroxytryptophan functional spore-forming bacteria, the method comprising:

[0007] Abnormal fermentation bacterial liquid or bacterial residue waste is collected, and suspended in a sulfuric acid aqueous solution with a pH of 1.8-2.5 for 2 days to allow spores of spore-forming bacteria to form and simultaneously kill bacteriophages in the sample; the suspension is spread on a kanamycin resistance screening agar plate, and cultured at 30° C. for 48 hours; the colonies on the resistant plate are separated, and shake-cultured in a kanamycin resistance medium containing tryptophan, and IPTG is added for induction in the late exponential fermentation period. After testing, tryptophan in the fermentation liquid is converted into 5-hydroxytryptophan, and the screening of 5-hydroxytryptophan functional spore-forming bacteria is completed, and the strain is stored in a -80° C. refrigerator;

[0008] The strains preserved in a -80°C refrigerator were revived, cultured and amplified in a shake flask, and then transferred to a 5L fermenter for fermentation with a culture medium containing 20g / L tryptophan. IPTG was added in the late exponential growth period to induce conversion. Coumaric acid and ferulic acid were added to regulate the fermentation rate and metabolic process to achieve the purpose of optimizing the conversion of tryptophan into 5-hydroxytryptophan. After fermentation regulation, the efficiency of converting tryptophan into 5-HTP was greatly improved to 70-85%. At the same time, the production of metabolic by-products was reduced, providing conditions for a simple purification process of the product.

[0009] Compared with the prior art, the present invention has the following beneficial effects: the functional bacteria provided by the present invention have high specificity in converting 5-HTP, only produce a small amount of isomers which can be easily removed during the purification process, and rarely produce polymers during the bacterial fermentation process, thereby greatly improving the industrial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is the infrared detection spectrum of the 5-HTP product produced in the embodiment of the present invention. DETAILED DESCRIPTION

[0011] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0012] The embodiment of the present invention provides a method for screening and fermentation regulation of 5-hydroxytryptophan functional spore-forming bacteria, the method comprising:

[0013] Under certain conditions, the structure of the spores changes, and after the forespore stage, a complete spore is formed. The spores have strong resistance to heat, radiation and chemicals. The spores are resistant to stress, such as being able to withstand high temperatures of 60°C for a long time, being able to survive at 120°C for 20 minutes, and being resistant to acid and alkali.

[0014] Abnormal fermentation bacterial liquid or bacterial residue waste from a biochemical company in Baoding was collected and suspended in a sulfuric acid aqueous solution with a pH of 1.8-2.5 for 2 days to allow spores to form and kill bacteriophages in the sample; the suspension was spread on a kanamycin resistance screening agar plate and cultured at 30°C for 48 hours; the colonies on the resistant plate were separated and cultured in a shake flask in a kanamycin resistance medium containing tryptophan, IPTG was added for induction in the late exponential fermentation period, and the tryptophan in the fermentation liquid was detected to be converted into 5-hydroxytryptophan, completing the screening of 5-hydroxytryptophan functional spore bacteria and storing the strain in a -80°C refrigerator;

[0015] The strains preserved in a -80°C refrigerator were revived, cultured and amplified in a shake flask, and then transferred to a 5L fermenter for fermentation with a culture medium containing 20g / L tryptophan. IPTG was added in the late exponential growth period to induce conversion. Coumaric acid and ferulic acid were added to regulate the fermentation rate and metabolic process to achieve the purpose of optimizing the conversion of tryptophan into 5-hydroxytryptophan. After fermentation regulation, the efficiency of converting tryptophan into 5-HTP was greatly improved to 70-85%. At the same time, the production of metabolic by-products was reduced, providing conditions for a simple purification process of the product.

[0016] Figure 1This is the infrared detection spectrum of the 5-HTP product produced by the functional bacteria in the embodiment of the present invention, wherein the black line is the 5-HTP standard (USP) and the red line is the 5-HTP product.

[0017] It should be noted that, in this article, the term "comprises" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0018] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for screening and fermentation regulation of 5-hydroxytryptophan functional spore-forming bacteria, characterized in that: The method comprises: Abnormal fermentation bacterial liquid or bacterial residue waste is collected, and suspended in a sulfuric acid aqueous solution with a pH of 1.8-2.5 for 2 days to allow spores of spore-forming bacteria to form and kill bacteriophages in the sample; the suspension is spread on a kanamycin resistance screening agar plate and cultured at 30°C for 48 hours; the colonies on the resistant plate are separated and cultured in a shake flask in a kanamycin (25-50ug / mL) resistant culture medium containing tryptophan (the culture medium formula is 2% sucrose, 2% yeast extract, 1.5% beef extract, 0.06% MgSO4·7H2O, 0.0009% FeSO4·7H2O), IPTG is added for induction in the late exponential fermentation period, and it is detected that tryptophan in the fermentation liquid is converted into 5-hydroxytryptophan, and the 5-hydroxytryptophan functional spore-forming bacteria screening is completed and the strain is stored in a -80°C refrigerator; The strain was stored in a -80°C refrigerator and revived. After culture and expansion in a shake flask, it was transferred to a 5L fermenter and fermented with a medium containing 20g / L tryptophan. IPTG (concentration 0.1-1mM) was added in the late exponential growth period (OD600 was 0.6-0.8) to induce conversion. Coumaric acid and ferulic acid (0.01-0.2mg / L) were added to regulate the fermentation rate and metabolic process. After fermentation regulation, the conversion of tryptophan into 5-HTP reached 70-85%.

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