Search

CN-122012639-A - Ergothioneine production method

CN122012639ACN 122012639 ACN122012639 ACN 122012639ACN-122012639-A

Abstract

The invention discloses a production method of ergothioneine, belonging to the technical field of microorganisms and food biology. The invention adopts specific acid to pretreat the fishbone powder, combines with enzymolysis technology, releases more easily-utilized amino acid and small peptide, and carries out proportional fermentation with molasses. The pretreatment method effectively solves the inherent problems of multiple inhibition components and low nutrition release efficiency in byproducts, and realizes the efficient synergy of two cheap raw materials, thereby unexpectedly increasing the yield of ergothioneine to a high level of 487 mg/L while reducing the cost of the raw materials by more than 5 times. The ergothioneine prepared by the invention is a very stable antioxidant and plays an important role in removing free radicals in vivo, maintaining redox steady state in cells, protecting DNA from oxidative damage of metal ions and the like. Can be used as raw material for food, cosmetic or pharmaceutical processing.

Inventors

  • ZHANG SUFANG
  • LI ZHIJIE
  • Dong Naihui
  • JIA YISONG
  • DAI YIWEI
  • JI CHAOFAN
  • CHEN YINGXI
  • QIN NINGBO
  • LIN XINPING

Assignees

  • 大连工业大学

Dates

Publication Date
20260512
Application Date
20260311

Claims (10)

  1. 1. A production method of ergothioneine is characterized in that molasses and fish bone powder hydrolysate are used as carbon sources and nitrogen sources in a fermentation system, ergothioneine-producing microorganism seed liquid is inoculated, ventilation culture is carried out at 20-37 ℃, and after fermentation is ended, thalli are separated and collected, and ergothioneine is obtained through extraction.
  2. 2. The method according to claim 1, wherein the ergothioneine-producing microorganism is rhodotorula Rhodosporidium toruloides DL-XSY01, accession number CGMCC No. 23534.
  3. 3. The method according to claim 1, wherein the total sugar content in the fermentation system is 20-60 g/L and the total protein content is 12-67 g/L.
  4. 4. The method according to claim 1, wherein the method for preparing the fish bone powder hydrolysate comprises the steps of (1) preparing the fish bone powder into an aqueous solution with a total concentration of 20 g/L-600 g/L and a protein content of 1.2-70 g/L, and adding an acid solution with a concentration of 0-6 mol/L; (2) Heating at 70-121 deg.C to 60 min, cooling to suitable protease hydrolysis temperature and adjusting to suitable pH; (3) Adding protease into the cooled solution, hydrolyzing 2-8 h at proper temperature, and heat inactivating protease to stop hydrolysis reaction.
  5. 5. The method of claim 4, wherein in step (1), the acid solution comprises phosphoric acid, citric acid, hydrochloric acid, or sulfuric acid.
  6. 6. The method according to claim 4, wherein in the step (2), the protease is neutral protease, acidic protease, alkaline protease, papain, trypsin or pepsin, the enzymolysis condition of the neutral protease is that the temperature is 40-50 ℃, the pH is 6.5-7.5, the addition amount is 50U/mg, the enzymolysis condition of the acidic protease is that the temperature is 40-50 ℃, the pH is 2.5-5.5, the addition amount is 50U/mg, the enzymolysis condition of the alkaline protease is that the temperature is 50-55 ℃, the pH is 8.0-11.0, the addition amount is 15U/mg, the enzymolysis condition of the papain is that the temperature is 55-65 ℃, the addition amount is 200U/mg, the enzymolysis condition of the trypsin is that the temperature is 50-55 ℃, the pH is 7.8-8.5, the addition amount is 130U/mg, the enzymolysis condition of the pepsin is that the temperature is 37-40 ℃, the pH is 1-3000.5, and the addition amount is 3000U.0.
  7. 7. The method according to claim 1, wherein the fermentation system further comprises 0-3.0 g/L of nutrient components required by conventional microorganism growth, including yeast powder, peptone, malt extract, potassium phosphate, magnesium sulfate, zinc sulfate, manganese sulfate, calcium chloride, ferrous sulfate, etc., and the pH is adjusted to 5.5-7.5.
  8. 8. The process according to claim 1, wherein the molasses used is beet molasses, cane molasses, grape molasses or corn molasses.
  9. 9. The method according to claim 1, wherein the seed solution of ergothioneine-producing microorganism has an inoculum size of 2% -20% (v/v), and is aerated and cultured at 20 ℃ -37 ℃ for 36-240 hours, and the total reducing sugar concentration in the fermentation broth can be reduced to below 1%.
  10. 10. Use of the method according to any one of claims 1 to 9 for increasing the fermentation yield of ergothioneine.

Description

Ergothioneine production method Technical Field The invention relates to a production method of ergothioneine, belonging to the technical field of microorganisms and food biology. Background Ergothioneine (Ergothioneine, EGT) is a colorless and odorless sulphur-containing histidine derivative, which was originally isolated from ergot (CLAVICEPS PURPUREA) from Tanret in 1909, has two structural states, thiol and thione, and exists mainly in the form of thione at physiological pH. The standard oxidation-reduction potential of the ergothioneine is-60 mV which is higher than that of other thiol-containing antioxidant substances such as glutathione and the like, so that the ergothioneine is not easy to undergo autoxidation under physiological conditions, and therefore, the ergothioneine is a very stable antioxidant. Ergothioneine plays an important role in scavenging free radicals in vivo, maintaining redox homeostasis in cells, protecting DNA from oxidative damage by metal ions, and the like. Animals and higher plants are not themselves capable of producing ergothioneine, and animals must be from dietary sources, whereas higher plants need to obtain ergothioneine from their surroundings. Currently, ergothioneine can be obtained by chemical synthesis, edible fungus extraction, and microbial fermentation production. In recent years, microbial fermentation preparation technology of ergothioneine has formed a plurality of paths for parallel development. In the aspect of edible fungus fermentation, patent CN109939027A discloses a strategy of utilizing hericium erinaceus fermentation and combining precursor addition, so that the content of ergothioneine in fermentation broth reaches more than 300 mg/L, while patent CN116769850A adopts a unique process of adding hydrogen peroxide and vitamin C in a double-stage manner to alleviate, stimulates the fermentation of pleurotus citrinopileatus, improves the production efficiency by 58.81 percent compared with the original strain, and achieves the yield of 641.76 mg/L. In the field of genetic engineering bacteria, the technology has the advantages that the shake flask yield is 350 mg/L by constructing recombinant escherichia coli and controlling fermentation to be carried out under a micro-aerobic or anaerobic condition, and a new idea is provided for reducing production energy consumption. Downstream of the industrialization, patent CN115704044a focuses on the extraction process of high purity products, the purity of the ergothioneine prepared by this process can reach more than 99% by HPLC detection. The progress of the patents shows that the fermentation production of ergothioneine is continuously developed towards high yield, low cost and high purity through the innovation of strain breeding, process control, culture medium optimization and extraction processes. Edible fungi such as flammulina velutipes are a main source for industrially extracting ergothioneine. But is limited by long raw material growth period and low extraction rate. In addition, histidine and histidine methyl esters can be used as substrates for the chemical synthesis of ergothioneine. However, the synthesis steps are complex, various toxic and harmful substances such as methyl iodide, hydrochloric acid and the like are often used in the synthesis process, the production cost is high, and the pollution of the generated waste gas and waste liquid to the environment is large. In contrast, the use of microorganisms to produce ergothioneine is a cost-effective alternative strategy. Researchers have successfully improved the yield of ergothioneine by optimizing fermentation conditions and rebuilding metabolic engineering. The processing byproducts are used as low-cost substrate substitutes in the microbial fermentation process, so that the production cost of ergothioneine is further reduced, sustainable production is facilitated, and the method has an important promotion effect on industrial production. Sugarcane is a gramineae plant grown in temperate and subtropical regions and is widely distributed throughout the world of Brazil, india, china, etc. Molasses is a byproduct of sugar industry based on sugar cane, and has abundant reserves and low price. Molasses has a high sugar content (mainly sucrose) and also contains small amounts of vitamins and metal ions. Molasses therefore has more nutrients than other inexpensive substrates. Molasses is used as an environmentally friendly and cost effective carbon source substitute for various bio-based products. In addition to the carbon source, the nitrogen source is also an indispensable nutrient element in the microbial growth process. Fishbone waste is one of the fishery waste with lower utilization rate. Most of fishbone wastes are roughly processed or directly discarded as feed and raw materials, so that resources are wasted and environmental pollution is caused. The fishbone powder is processed by fishbone, fish head or fish tail, and has low edible value