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Tetragenococcus halophilus strain high effectively using arginine and preventing accumulation of citrulline

A technology of Tetracoccus halophilus and arginine, applied in the field of microorganisms, can solve the problems of not meeting the requirements of industrial applications and having a large impact, and achieve the effect of improving the efficiency of the strain and improving the safety.

Active Publication Date: 2017-10-03
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the previous research, the halophilic tetradococcus R23CCTCC NO:M2013480 screened by the inventor has a complete ADI pathway, can utilize arginine without accumulating citrulline, and is currently the most capable of utilizing arginine and citrulline halophilic tetrad, but its ability to utilize arginine and citrulline is greatly affected by arginine concentration, glucose concentration and environmental factors
R23 exhibits the accumulation of citrulline in the environment of more than 3g / L arginine, and can no longer consume citrulline in the system, but the average concentration of arginine in the actual moromi fermentation environment is 5g / L, R23 has Does not meet industrial application requirements

Method used

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  • Tetragenococcus halophilus strain high effectively using arginine and preventing accumulation of citrulline
  • Tetragenococcus halophilus strain high effectively using arginine and preventing accumulation of citrulline
  • Tetragenococcus halophilus strain high effectively using arginine and preventing accumulation of citrulline

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Mutation breeding of halophilic tetrad

[0034] Inoculate the halophilic Tetradendococcus R23 into the MRS liquid medium containing 100g / L NaCl, culture it statically at 30°C until the logarithmic growth phase, discard the supernatant after centrifugation, and wash the bacteria with PBS buffer (pH7.0) 2 ~3 times later, add 5mL PBS buffer solution to the centrifuge tube to suspend the bacteria, and then carry out ultraviolet mutagenesis and plasma mutagenesis respectively.

[0035] (1) UV mutagenesis of Tetradneococcus halophilus R23

[0036] ① Take 5mL of the bacterial suspension and drop it on the sterilized and cooled Petri dish, and carry out ultraviolet mutagenesis under ultraviolet light, and the mutagenesis time is respectively . Placed at 30cm under a 15w UV lamp, irradiated for 60s, 90s, 120s respectively, each treatment was repeated 3 times, and stood in a dark incubator for 30min after each UV irradiation.

[0037] ② Dilute the mutated bacterial su...

Embodiment 2

[0048] Example 2 Analysis of the Ability of Utilization of Arginine and Citrulline by Halotetradine Bacteria

[0049] Pick a single colony of tetrad halophilus L3H9 activated on the plate and inoculate it in the MRS liquid medium containing 100g / LNaCl, culture it statically at 30°C for 4 days, centrifuge at 8000rpm for 10min to get the bacteria, add 1mL of phosphate buffer ( PBS, pH7.0) to suspend the bacteria.

[0050] Add 3 mL of amino acid utilization medium containing 0.5 g / L citrulline, 3 g / L arginine, 4 g / L arginine, and 5 g / L arginine to a 5 mL centrifuge tube, inoculate 300 μL of bacterial solution, and incubate at 30 °C After 5 days of static culture, the content of arginine, citrulline and ornithine in the medium was detected by HPLC. The utilization rate is greater than the citrulline utilization rate, therefore, under high salt and high arginine conditions, the halophilic Tetradendococcus efficiently utilizes arginine and does not accumulate citrulline. The speci...

Embodiment 3

[0055] Example 3 Analysis of arginine utilization ability of halophilic tetrad at different temperatures

[0056] In the actual process of soy sauce fermentation, there will be many temperature changes, so the ability of Tetragenococcus halophilus L3H9 to utilize arginine at different temperatures was investigated.

[0057] Pick a single colony of tetrad halophilus L3H9 activated on the plate and inoculate it in a liquid medium of 100g / L NaCl, culture it statically at 30°C for 4 days, centrifuge at 8000rpm for 10min to get the bacteria, add 1mL of phosphate buffer saline (PBS, pH7.0) suspended bacteria.

[0058] Add 3mL of amino acid utilization medium containing 5g / L arginine and 0.5g / L citrulline to a 5mL centrifuge tube, inoculate 300μL of bacterial solution, and place them at 20°C, 25°C, and 30°C for static culture for 5 days. The content of arginine, citrulline and ornithine in the culture medium was detected by the method of HPLC, and the specific results were as follow...

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Abstract

The invention discloses a Tetragenococcus halophilus strain high effectively using arginine and preventing accumulation of citrulline and belongs to the field of microorganisms. The Tetragenococcus halophilus L3H9 is preserved in the China center for type culture collection (CCTCC) in the Wuhan University of China and has a preservation number CCTCC NO: M 2017250. The Tetragenococcus halophilus strain can high effectively use arginine and prevent accumulation of citrulline under conditions of high salt and high arginine, has the potential to reduce the ethyl carbamate content in the environment and has potential application values in improvement of Tetragenococcus halophilus industrial application performances, development of a soy sauce industry, control or reduction of ethyl carbamate content of the fermentation system and improvement of the safety of fermented foods.

Description

technical field [0001] The invention relates to a strain of halophilic tetradococcus which efficiently utilizes arginine and does not accumulate citrulline, and belongs to the field of microorganisms. Background technique [0002] Soy sauce is a traditional fermented condiment in my country, and my country is a big country in the production and export of soy sauce. Soy sauce is a condiment with unique flavor formed by long-term fermentation using soybeans, wheat, bran, etc. as raw materials, through the joint action of various microorganisms. Soy sauce contains a variety of soluble proteins, polypeptides, amino acids, polysaccharides, alcohols, esters, organic acids, rich vitamins, inorganic salts, etc., and its nutritional value is very rich. [0003] Ethyl carbamate (EC) is a naturally occurring by-product in the production of fermented food and alcoholic beverages. It is carcinogenic and genotoxic, and can cause lung tumors, lymphoma, liver cancer, skin cancer, etc. On ...

Claims

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Application Information

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IPC IPC(8): C12N1/20A23L27/24A23L29/00C12R1/01
CPCA23L27/24A23L29/065C12N1/20C12N1/205C12R2001/01
Inventor 雷庆子方芳王博陈坚堵国成
Owner JIANGNAN UNIV
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