Production method of ergothioneine
By pretreating fish bone meal with acid and enzymatic hydrolysis, combined with molasses fermentation, the problems of high raw material costs and low utilization efficiency of complex by-products in microbial fermentation methods have been solved, achieving high-yield and low-cost production of ergothioneine.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- DALIAN POLYTECHNIC UNIVERSITY
- Filing Date
- 2026-03-11
- Publication Date
- 2026-05-12
AI Technical Summary
Existing methods for producing ergothione by microbial fermentation suffer from high raw material costs and the inability of conventional culture media to directly utilize complex industrial and agricultural byproducts, resulting in unstable yields and high costs.
Fish bone meal is pretreated with a specific acid and then enzymatically hydrolyzed to release readily available amino acids and small peptides. These are then fermented with molasses in a specific ratio and fermented using Rhodosporidium toruloides DL-XSY01 yeast, achieving efficient utilization of inexpensive raw materials.
The yield of ergothioneine was increased to 487 mg/L, and the production cost was reduced by more than 5 times, achieving efficient and stable ergothioneine production.
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Figure CN122012639A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for producing ergothioneine, belonging to the field of microbial and food biotechnology. Background Technology
[0002] Ergothioneine (EGT) is a colorless and odorless sulfur-containing histidine derivative, first discovered by Tanret in 1909 from ergot (Egg mites). Claviceps purpurea Ergothioneine is isolated from thiols and exists in two structural states: thiols and thioketones. At physiological pH, it primarily exists in the thioketone form. Its standard redox potential is -60 mV, higher than that of other thiol-containing antioxidants such as glutathione, making it less susceptible to auto-oxidation under physiological conditions. Therefore, ergothioneine is a very stable antioxidant. Ergothioneine plays an important role in scavenging free radicals, maintaining intracellular redox homeostasis, and protecting DNA from oxidative damage by metal ions. Animals and higher plants cannot produce ergothioneine themselves; animals must obtain it from their diet, while higher plants need to obtain it from their surrounding environment. Currently, ergothioneine can be obtained through chemical synthesis, extraction from edible fungi, and microbial fermentation. In recent years, several parallel development paths have emerged for the microbial fermentation preparation technology of ergothioneine. In the field of edible fungi fermentation, patent CN109939027A discloses a strategy of using Hericium erinaceus fermentation combined with precursor addition to achieve an ergothioneine content of over 300 mg / L in the fermentation broth. Patent CN116769850A employs a unique two-stage process of adding hydrogen peroxide and vitamin C to moderate the fermentation of Pleurotus ostreatus, increasing production efficiency by 58.81% compared to the original strain, achieving a yield of 641.76 mg / L. In the field of genetically engineered bacteria, patented technologies have achieved a shake-flask yield of 350 mg / L by constructing recombinant Escherichia coli and controlling fermentation under microaerobic or anaerobic conditions, providing a new approach to reducing production energy consumption. Downstream in industrialization, patent CN115704044A focuses on the extraction process of high-purity products, with the ergothioneine purity reaching over 99% as determined by HPLC. These patent advancements demonstrate that through strain selection, process control, culture medium optimization, and extraction process innovation, ergothioneine fermentation production is continuously developing towards high yield, low cost, and high purity.
[0003] Edible fungi such as enoki mushrooms are the main source of ergothioneine for industrial extraction. However, the extraction rate is limited by the long growth cycle of the raw materials. Furthermore, histidine and histidine methyl ester can be used as substrates for the chemical synthesis of ergothioneine. However, the synthesis process is complex, often using toxic and harmful substances such as methyl iodide and hydrochloric acid, resulting in high production costs and significant environmental pollution from the generated waste gas and waste liquid. In contrast, microbial production of ergothioneine is a cost-effective alternative strategy. Researchers have successfully increased ergothioneine yield by optimizing fermentation conditions and reconstructing metabolic engineering. Utilizing processing byproducts as low-cost substrate substitutes during microbial fermentation will further reduce the production cost of ergothioneine, promote sustainable production, and significantly advance industrial production.
[0004] Sugarcane is a grass that grows in temperate and subtropical regions, widely distributed in Brazil, India, China, and other parts of the world. Molasses is a byproduct of the sugar industry, based on sugarcane, and is abundant and inexpensive. Molasses has a high sugar content (mainly sucrose) and also contains small amounts of vitamins and metal ions. Therefore, compared with other inexpensive substrates, molasses has more nutritional value. Molasses is used as an environmentally friendly and cost-effective carbon source substitute for various bio-based products. In addition to carbon sources, nitrogen sources are also essential nutrients for microbial growth. Fish bone waste is one of the least utilized fishery wastes. Most fish bone waste is either roughly processed or discarded directly as feed or raw material, which not only wastes resources but also causes environmental pollution. Fish bone meal is made from fish bones, heads, or tails and has low edible value. However, fish bone meal has advantages such as high protein content, balanced amino acid composition, and rich minerals and vitamins. It can be used as a nitrogen source substitute for microbial fermentation to produce high-value-added products, further improving its application value and reducing fermentation costs.
[0005] However, existing technologies still have certain limitations. First, carbon and nitrogen sources are expensive, and most reported high-yield strategies still rely on refined raw materials such as glucose, yeast powder, and peptone, with raw material costs accounting for a high proportion of total production costs. Second, the efficiency of raw material utilization needs to be improved. Although industrial and agricultural by-products such as molasses and fish bone meal are abundant and inexpensive, their complex composition and inhibitory substances can easily lead to a decrease in microbial metabolic activity and unstable yields if they are directly used to replace conventional culture media. Third, the process adaptability is insufficient. Research on pretreatment processes, nutrient complementarity mechanisms, and adaptability of complex by-products such as molasses and fish bone meal in ergothioneine fermentation is still inadequate and lacks systematic optimization.
[0006] Therefore, developing a technology that can efficiently utilize low-cost byproducts such as molasses and fish bone meal, and achieve high and stable yields of ergothioneine through appropriate pretreatment and fermentation processes, is of great significance for reducing production costs and promoting the large-scale green manufacturing of ergothioneine. Summary of the Invention
[0007] To address the technical bottlenecks of existing microbial fermentation methods for ergothioneine production, such as high raw material costs and the inability of conventional culture media to directly utilize complex industrial and agricultural byproducts, this invention creatively proposes a method of pretreating fish bone meal with a specific acid followed by enzymatic hydrolysis. This process releases more readily available amino acids and small peptides, which are then fermented in proportion with molasses. This pretreatment method effectively overcomes the inherent problems of high levels of inhibitory components and low nutrient release efficiency in byproducts, achieving highly efficient synergy between two inexpensive raw materials. Consequently, while reducing raw material costs by more than five times, it unexpectedly increases ergothioneine yield to a high level of 487 mg / L.
[0008] The first technical solution provided by the present invention is a method for producing ergothioneine, wherein the method involves microbial fermentation culture using molasses and fish bone meal as raw materials to obtain ergothioneine.
[0009] In some embodiments, the method uses molasses and fish bone meal hydrolysate as carbon and nitrogen sources in the fermentation system, inoculates ergothioneine-producing microbial seed culture, and cultivates it at 20℃~37℃ with aeration. After fermentation is terminated, the bacterial cells are isolated and collected, and ergothioneine is extracted. The yield of ergothioneine produced using the bacterial cells of this invention is 200-400 mg / L.
[0010] In some embodiments, the ergot-producing microorganism is red yeast. Rhodosporidium toruloides DL-XSY01, accession number CGMCC No. 23534.
[0011] In some embodiments, the total sugar content in the fermentation system is 20-60 g / L, and the total protein content is 12-67 g / L.
[0012] In some embodiments, the preparation method of fish bone powder hydrolysate includes the following steps: (1) preparing 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 0-6 mol / L of acid solution; (2) Heat treatment at 70-121℃ for 60 min, cool to a suitable temperature for protease hydrolysis and adjust to a suitable pH; (3) Add protease to the cooled solution and hydrolyze it for 2-8 h at a suitable protease temperature. Heat inactivation of protease terminates the hydrolysis reaction.
[0013] In some embodiments, in step (1), the acid solution includes phosphoric acid, citric acid, hydrochloric acid, or sulfuric acid.
[0014] In some embodiments, in step (2), the protease may be a neutral protease, acidic protease, alkaline protease, papain, trypsin, or pepsin. The enzymatic hydrolysis conditions for the neutral protease are: temperature 40-50℃, pH 6.5-7.5, and dosage 50 U / mg; for the acidic protease, the conditions are: temperature 40-50℃, pH 2.5-5.5, and dosage 50 U / mg; for the alkaline protease, the conditions are: temperature 50-55℃, pH 8.0-11.0, and dosage 15 U / mg; for the papain, the conditions are: temperature 55-65℃, pH 5.7-7.0, and dosage 200 U / mg; for the trypsin, the conditions are: temperature 50-55℃, pH 7.8-8.5, and dosage 130 U / mg; and for the pepsin, the conditions are: temperature 37-40℃, pH 1.5-2.0, and dosage 3000 U / mg. U / mg. The amount of each protease added, based on its final enzyme activity in the reaction system, is 150-200 U / mL.
[0015] In some embodiments, the fermentation system also contains 0–3.0 g / L of nutrients required for the growth of conventional microorganisms, including yeast powder, peptone, malt extract, potassium phosphate, magnesium sulfate, zinc sulfate, manganese sulfate, calcium chloride, ferrous sulfate, etc., and adjusts the pH to 5.5–7.5.
[0016] In some embodiments, ergothionein-producing microorganisms are inoculated into a liquid seed culture medium (20 g / L glucose, 10 g / L yeast extract, 10 g / L peptone, balance water, pH 5.5–6.0) and cultured at 30°C with shaking at 180–200 r / min for 20–36 h to obtain a product containing 10 g / L ergothionein per milliliter. 5 ~10 8 Seed liquid containing live cells.
[0017] In some embodiments, the inoculum of ergothionein-producing microbial seed liquid is 2% to 20% (v / v), and the fermentation is carried out at 20°C to 37°C for 36 h to 240 h with aeration, and the total reducing sugar concentration in the fermentation broth can be reduced to below 1%.
[0018] In some implementations, fermentation is terminated, the cells are collected by solid-liquid separation, and ergothionein is extracted by heat treatment.
[0019] In some implementations, the molasses used may be beet molasses, sugarcane molasses, glucose molasses, or corn molasses.
[0020] In some embodiments, the raw materials for the fish bone meal can be fish heads, fish tails, fish bones, and whole fish with low edible value.
[0021] The second technical solution provided by the present invention is the application of the method described in the first technical solution in increasing the fermentation yield of ergothionein.
[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Ergothioneine has a high yield. When fermented in a 5L bioreactor, the yield of ergothioneine can reach 487mg / L.
[0023] (2) This invention utilizes inexpensive processing by-products (molasses and fish bone meal) as raw materials for microbial fermentation, which reduces the production cost by more than 5 times compared with conventional culture medium fermentation. It is a new technology for high-value utilization of resources.
[0024] (3) Wide range of applications. The ergothioneine prepared by this invention is a very stable antioxidant that plays an important role in scavenging free radicals in the body, maintaining intracellular redox homeostasis, and protecting DNA from oxidative damage by metal ions. It can be used as a raw material in food, cosmetics, or pharmaceutical processing. Attached Figure Description
[0025] Figure 1 To utilize molasses and fish bone meal enzymatic hydrolysates as substrates Rhodosporidium toruloides A simplified process flow diagram for the production of ergothioneine using DL-XSY01. Detailed Implementation
[0026] refer to Figure 1 The preferred embodiments of the present invention will be described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.
[0027] Test method: Ergothionein determination using high-performance liquid chromatography (HPLC): An Agilent 1260 HPLC system (California, USA) was used for the quantitative determination of ergothionein in the extract. The conditions were as follows: Agilent ZORBAX-SB-C18 column (4.6 × 250 mm, 5 μm; Agilent, CA, USA); mobile phase A (water):B (methanol) = 99:1, isocratic elution, flow rate 0.7 mL / min; temperature: 30 ℃; injection volume: 5 μL; UV detection wavelength set to 257 nm. Peak areas were integrated, and a standard curve was plotted based on results obtained from commercially available ergothionein standards. The ergothionein content in the sample was calculated based on the peak area and the slope of the standard curve.
[0028] Raw materials used in the examples: (1) The red yeast used in this invention Rhodosporidium toruloidesDL-XSY01 is deposited by the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 23534 and has been published in patent CN115287203A.
[0029] (2) YPD liquid culture medium: 20.0g peptone, 10.0g yeast powder, 20.0g glucose, distilled water to 1L, pH adjusted to 7.0, autoclave for 20min.
[0030] (3) YPD solid culture medium: 20.0g peptone, 10.0g yeast powder, 20.0g glucose, 15g agar, distilled water to 1L, pH adjusted to 7.0, autoclaved for 20min, and then poured into plates.
[0031] (4) Fish bone meal: TDAS Company, Peru, with a protein content of 10.15% (w / w).
[0032] (5) Sugarcane molasses: Guangxi Guimi Biotechnology Co., Ltd., with a total sugar content of 50.02% (w / w).
[0033] (6) Beet molasses: Botian Sugar Industry (Zhangbei) Co., Ltd., with a total sugar content of 49.78% (w / w).
[0034] (7) Corn molasses: Heilongjiang Haotian Corn Development Co., Ltd., its total sugar content is 51.78% (w / w).
[0035] (8) Neutral protease: Shanghai Maclean Biochemical Technology Co., Ltd., Product No.: D832687 (50 U / mg).
[0036] (9) Acidic protease: Shanghai Maclean Biochemical Technology Co., Ltd., Product No.: P885915 (50 U / mg).
[0037] (10) Alkaline protease: Shanghai Maclean Biochemical Technology Co., Ltd., Product No.: P750258 (15 U / mg).
[0038] (11) Papain: Shanghai Maclean Biochemical Technology Co., Ltd., Product No.: P6321 (200 U / mg).
[0039] (12) Pepsin: Shanghai Maclean Biochemical Technology Co., Ltd., Product No.: P6322 (3000 U / mg).
[0040] Example 1: Raw material processing, culture medium preparation, and fermentation The fish bone meal was heat-treated at 121℃ for 60 min using 0.5 mol / L phosphate solution and 200 g / L fish bone meal. The pH was adjusted to the optimal pH for neutral protease (pH 7). After cooling, the fish bone meal was enzymatically hydrolyzed with neutral protease (hydrolysis temperature 45℃, hydrolysis time 6 h; neutral protease was added to the hydrolysis system to achieve an enzyme activity of 160 U / mL, with a specific activity of 50 U / mg). The concentrations of beet molasses and fish bone meal hydrolysates in the culture medium were 20 g / L (corresponding to a total sugar content of 10 g / L) and 180 g / L (corresponding to a total protein content of 27 g / L), respectively. The pH was adjusted to 5.5. After sterilization at 121℃ for 15 min, red yeast was inoculated at a 2% (v / v) inoculum. R. toruloides DL-XSY01 was cultured at 30℃ with aeration for 36 hours. Fermentation was then stopped, and the fermentation broth was centrifuged at 8000 r / min for 10 min to collect the cells.
[0041] Add 1 mL of sterile water to every 100 mg of wet bacterial cells, vortex, resuspend the cells, then treat in a 90℃ water bath for 30 min, cool, centrifuge at 8000 r / min for 10 min, collect the supernatant, filter through a 0.22 µm aqueous filter membrane, and determine the ergothioneine yield using high performance liquid chromatography (HPLC). The yield was 253 mg / L. Compared with fermentation on YPD medium under the same conditions, the production cost was reduced by 300%.
[0042] Example 2: Raw material processing, culture medium preparation, and fermentation Fish bone meal was heat-treated at 121℃ for 60 min using phosphoric acid solution. The pH was adjusted to the optimal level for acidic protease at 3.5. After cooling, the fish bone meal was enzymatically hydrolyzed with acidic protease (hydrolysis temperature: 45℃; hydrolysis time: 6 h; acidic protease was added to the hydrolysis system to achieve an enzyme activity of 160 U / mL, with a specific activity of 50 U / mg). The concentrations of beet molasses and fish bone meal hydrolysates in the culture medium were 20 g / L (corresponding to a total sugar content of 10 g / L) and 180 g / L (corresponding to a total protein content of 27 g / L), respectively. The pH was adjusted to 6.0. After sterilization at 121℃ for 15 min, red yeast was inoculated at a 4% (v / v) inoculum. R. toruloides DL-XSY01 was cultured at 30℃ with aeration for 96 h, and then post-treated according to the method in Example 1. The yield of ergothioneine was 298 mg / L. Compared with fermentation on YPD medium under the same conditions, the production cost was reduced by 389%.
[0043] Example 3: Raw material processing, culture medium preparation, and fermentation Using phosphoric acid solution and fish bone meal, the mixture was heat-treated at 121℃ for 60 min to adjust the pH to the optimal level for alkaline protease (pH 9). After cooling, the fish bone meal was enzymatically hydrolyzed with alkaline protease (hydrolysis temperature: 50℃; hydrolysis time: 6 h; alkaline protease was added to the hydrolysis system to achieve an enzyme activity of 160 U / mL, with a specific activity of 15 U / mg). The contents of beet molasses and fish bone meal hydrolysates in the culture medium were 20 g / L (corresponding to a total sugar content of 10 g / L) and 180 g / L (corresponding to a total protein content of 27 g / L), respectively. The pH was adjusted to 6.0. After sterilization at 121℃ for 15 min, Rhodopsinia toruloides DL-XSY01 was inoculated at a 6% (v / v) inoculum and cultured at 30℃ with aeration for 96 h. Post-treatment was performed according to the method in Example 1, yielding ergothioneine at a yield of 243 mg / L. Compared with conventional culture medium fermentation under the same conditions, the production cost was reduced by 319%.
[0044] Example 4: Raw material processing, culture medium preparation and fermentation Fish bone meal was heat-treated at 121℃ for 60 min using phosphoric acid solution to adjust to the optimal pH for papain. After cooling, the fish bone meal was enzymatically hydrolyzed with papain (hydrolysis temperature: 55℃; hydrolysis time: 6 h; papain was added to the hydrolysis system to achieve an enzyme activity of 160 U / mL, with a specific activity of 200 U / mg). The concentrations of sugarcane molasses and fish bone meal hydrolysates in the culture medium were 30 g / L (corresponding to a total sugar content of 15 g / L) and 270 g / L (corresponding to a total protein content of 40 g / L), respectively, and the pH was adjusted to 6.5. After sterilization at 121℃ for 15 min, red yeast was inoculated at an 8% (v / v) inoculum. R. toruloides DL-XSY01 was cultured at 30℃ with aeration for 108 h, and then post-treated according to the method in Example 1. The yield of ergothioneine was 364 mg / L, which reduced the production cost by 461% compared with conventional culture medium fermentation under the same conditions.
[0045] Example 5: Raw material processing, culture medium preparation and fermentation Fish bone meal was heat-treated at 121℃ for 60 min using phosphoric acid solution to adjust to the optimal pH for pepsin. After cooling, the fish bone meal was enzymatically hydrolyzed with pepsin (hydrolysis temperature: 37℃; hydrolysis time: 6 h; pepsin was added to the hydrolysis system to achieve an enzyme activity of 160 U / mL, with a specific activity of 3000 U / mg). The concentrations of sugarcane molasses and fish bone meal hydrolysates in the culture medium were 60 g / L (corresponding to a total sugar content of 30 g / L) and 300 g / L (corresponding to a total protein content of 45 g / L), respectively, and the pH was adjusted to 7.5. After sterilization at 121℃ for 15 min, red yeast was inoculated at a 10% (v / v) inoculum. R. toruloidesDL-XSY01 was cultured at 30℃ with aeration for 144 h, and then post-treated according to the method in Example 1. The yield of ergothioneine was 253 mg / L, which reduced the production cost by 331% compared with conventional culture medium fermentation under the same conditions.
[0046] Example 6: Raw material processing, culture medium preparation and fermentation Fish bone meal was heat-treated at 121℃ for 60 min using hydrochloric acid solution to adjust to the optimal pH for neutral protease. After cooling, the fish bone meal was enzymatically hydrolyzed with neutral protease (hydrolysis temperature: 45℃; hydrolysis time: 6 h; neutral protease was added to the hydrolysis system to achieve an enzyme activity of 160 U / mL, with a specific activity of 50 U / mg). The concentrations of sugarcane molasses and fish bone meal hydrolysates in the culture medium were 20 g / L (corresponding to a total sugar content of 10 g / L) and 270 g / L (corresponding to a total protein content of 40 g / L), respectively. The pH was adjusted to 7.0. After sterilization at 121℃ for 15 min, red yeast was inoculated at a 12% (v / v) inoculum. R. toruloides DL-XSY01 was cultured at 30℃ with aeration for 120 h, and then post-treated according to the method in Example 1. The yield of ergothioneine was 224 mg / L, which reduced the production cost by 298% compared with conventional culture medium fermentation under the same conditions.
[0047] Example 7: Raw material processing, culture medium preparation and fermentation Fish bone meal was heat-treated at 121℃ for 60 min using hydrochloric acid solution to adjust to the optimal pH for acidic protease. After cooling, the fish bone meal was enzymatically hydrolyzed with acidic protease (hydrolysis temperature: 40℃; hydrolysis time: 6 h; acidic protease was added to the hydrolysis system to achieve an enzyme activity of 160 U / mL, with a specific activity of 50 U / mg). The glucose and fish bone meal hydrolysate concentrations in the culture medium were 50 g / L (corresponding to a total sugar content of 50 g / L) and 450 g / L (corresponding to a total protein content of 67 g / L), respectively, and the pH was adjusted to 5.5. After sterilization at 121℃ for 15 min, red yeast was inoculated at a 14% (v / v) inoculum. R. toruloides DL-XSY01 was cultured at 30℃ with aeration for 168 h, and then post-treated according to the method in Example 1. The yield of ergothioneine was 278 mg / L, which reduced the production cost by 355% compared with conventional culture medium fermentation under the same conditions.
[0048] Example 8: Raw material processing, culture medium preparation and fermentation Fish bone meal was heat-treated at 121°C for 60 min using hydrochloric acid solution to adjust to the optimal pH for alkaline protease. After cooling, the fish bone meal was enzymatically hydrolyzed with alkaline protease (hydrolysis temperature: 50°C; hydrolysis time: 6 h; alkaline protease was added to the hydrolysis system to achieve an enzyme activity of 160 U / mL, with a specific activity of 15 U / mg). The glucose and fish bone meal hydrolysate concentrations in the culture medium were 20 g / L (corresponding to a total sugar content of 20 g / L) and 80 g / L (corresponding to a total protein content of 12 g / L), respectively, and the pH was adjusted to 6.5. After sterilization at 121°C for 15 min, red yeast was inoculated at a 16% (v / v) inoculum. R. toruloides DL-XSY01 was cultured at 30℃ with aeration for 216 h, and then post-treated according to the method in Example 1. The yield of ergothioneine was 198 mg / L, which reduced the production cost by 290% compared with conventional culture medium fermentation under the same conditions.
[0049] Example 9: Raw material processing, culture medium preparation and fermentation Fish bone meal was heat-treated at 121°C for 60 min using hydrochloric acid solution to adjust to the optimal pH for papain. After cooling, the fish bone meal was enzymatically hydrolyzed with papain (hydrolysis temperature: 55°C; hydrolysis time: 6 h; papain was added to the hydrolysis system to achieve an enzyme activity of 160 U / mL, with a specific activity of 200 U / mg). The concentrations of corn molasses and fish bone meal hydrolysates in the culture medium were 10 g / L (corresponding to a total sugar content of 5.2 g / L) and 90 g / L (corresponding to a total protein content of 13.5 g / L), respectively, and the pH was adjusted to 7.0. After sterilization at 121°C for 15 min, Rhodopsinia tortuloides DL-XSY01 was inoculated at a 20% (v / v) inoculum and cultured at 30°C with aeration for 240 h. Following the post-treatment method of Example 1, the yield of ergothioneine was 291 mg / L, representing a 398% reduction in production cost compared to conventional culture medium fermentation under the same conditions.
[0050] Example 10: Raw material processing, culture medium preparation and fermentation Fish bone meal was heat-treated at 80°C for 60 min using phosphoric acid solution. The pH was adjusted to the optimal level for neutral protease. After cooling, the fish bone meal was enzymatically hydrolyzed with neutral protease (hydrolysis temperature: 45°C; hydrolysis time: 6 h; neutral protease was added to the hydrolysis system to achieve an enzyme activity of 160 U / mL, with a specific activity of 50 U / mg). The culture medium contained 20 g / L corn molasses and 180 g / L fish bone meal hydrolysates (corresponding to a total sugar content of 10.4 g / L) and 180 g / L fish bone meal hydrolysates (corresponding to a total protein content of 27 g / L), respectively, and the pH was adjusted to 7.5. After sterilization at 121°C for 15 min, red yeast was inoculated at an inoculum size of 18% (v / v). R. toruloidesDL-XSY01 was cultured at 30℃ with aeration for 216 h, and then post-treated according to the method in Example 1. The yield of ergothioneine was 201 mg / L, which reduced the production cost by 306% compared with conventional culture medium fermentation under the same conditions.
[0051] Example 11: Raw material processing, culture medium preparation and fermentation Fish bone meal was heat-treated at 80°C for 60 min using phosphoric acid solution. The pH was adjusted to the optimal level for acidic protease. After cooling, the fish bone meal was enzymatically hydrolyzed with acidic protease (hydrolysis temperature: 40°C; hydrolysis time: 6 h; acidic protease was added to the hydrolysis system to achieve an enzyme activity of 160 U / mL, with a specific activity of 50 U / mg). The concentrations of corn molasses and fish bone meal hydrolysates in the culture medium were 30 g / L (corresponding to a total sugar content of 15.5 g / L) and 270 g / L (corresponding to a total protein content of 40 g / L), respectively. The pH was adjusted to 5.5. After sterilization at 121°C for 15 min, red yeast was inoculated at a 16% (v / v) inoculum. R. toruloides DL-XSY01 was cultured at 30℃ for 192 h with aeration, and then post-treated according to the method in Example 1. The yield of ergothioneine was 328 mg / L, which reduced the production cost by 419% compared with conventional culture medium fermentation under the same conditions.
[0052] Example 12: Raw material processing, culture medium preparation and fermentation Fish bone meal was heat-treated at 80°C for 60 min using phosphoric acid solution. The pH was adjusted to the optimal level for alkaline protease. After cooling, the fish bone meal was enzymatically hydrolyzed with alkaline protease (hydrolysis temperature: 50°C; hydrolysis time: 6 h; alkaline protease was added to the hydrolysis system to achieve an enzyme activity of 160 U / mL, with a specific activity of 15 U / mg). The concentrations of beet molasses and fish bone meal hydrolysates in the culture medium were 40 g / L (corresponding to a total sugar content of 20 g / L) and 360 g / L (corresponding to a total protein content of 54 g / L), respectively. The pH was adjusted to 6.0. After sterilization at 121°C for 15 min, red yeast was inoculated at a 14% (v / v) inoculum. R. toruloides DL-XSY01 was cultured at 30℃ with aeration for 168 h, and then post-treated according to the method in Example 1. The yield of ergothioneine was 308 mg / L, which reduced the production cost by 388% compared with conventional culture medium fermentation under the same conditions.
[0053] Example 13: Raw material processing, culture medium preparation and fermentation Fish bone meal was heat-treated at 80°C for 60 min using phosphoric acid solution to adjust to the optimal pH for papain. After cooling, the fish bone meal was enzymatically hydrolyzed with papain (hydrolysis temperature: 55°C; hydrolysis time: 6 h; papain was added to the hydrolysis system to achieve an enzyme activity of 160 U / mL, with a specific activity of 200 U / mg). The concentrations of beet molasses and fish bone meal hydrolysates in the culture medium were 50 g / L (corresponding to a total sugar content of 25 g / L) and 450 g / L (corresponding to a total protein content of 67.5 g / L), respectively, and the pH was adjusted to 6.5. After sterilization at 121°C for 15 min, red yeast was inoculated at a 12% (v / v) inoculum. R. toruloides DL-XSY01 was cultured at 30℃ with aeration for 144 h, and then post-treated according to the method in Example 1. The yield of ergothioneine was 338 mg / L, which reduced the production cost by 357% compared with conventional culture medium fermentation under the same conditions.
[0054] Example 14: Raw material processing, culture medium preparation and fermentation Fish bone meal was heat-treated at 80℃ for 60 min using hydrochloric acid solution. The pH was adjusted to the optimal level for neutral protease. After cooling, the fish bone meal was enzymatically hydrolyzed with neutral protease (hydrolysis temperature: 45℃; hydrolysis time: 6 h; neutral protease was added to the hydrolysis system to achieve an enzyme activity of 160 U / mL, with a specific activity of 50 U / mg). The concentrations of beet molasses and fish bone meal hydrolysates in the culture medium were 100 g / L (corresponding to a total sugar content of 50 g / L) and 500 g / L (corresponding to a total protein content of 75 g / L), respectively. The pH was adjusted to 7.0. After sterilization at 121℃ for 15 min, red yeast was inoculated at a 10% (v / v) inoculum. R. toruloides DL-XSY01 was cultured at 30℃ with aeration for 120 h, and then post-treated according to the method in Example 1. The yield of ergothioneine was 245 mg / L, which reduced the production cost by 354% compared with conventional culture medium fermentation under the same conditions.
[0055] Example 15: Raw material processing, culture medium preparation and fermentation Fish bone meal was heat-treated at 80℃ for 60 min using hydrochloric acid solution to adjust to the optimal pH for papain. After cooling, the fish bone meal was enzymatically hydrolyzed with papain (hydrolysis temperature: 40℃; hydrolysis time: 6 h; papain was added to the hydrolysis system to achieve an enzyme activity of 160 U / mL, with a specific activity of 200 U / mg). The contents of sugarcane molasses and fish bone meal hydrolysates in the culture medium were 20 g / L (corresponding to a total sugar content of 10 g / L) and 380 g / L (corresponding to a total protein content of 57 g / L), respectively, and the pH was adjusted to 6.5. After sterilization at 121℃ for 15 min, red yeast was inoculated at a 10% (v / v) inoculum. R. toruloides DL-XSY01 was cultured at 30℃ with aeration for 168 h, and then post-treated according to the method in Example 1. The yield of ergothioneine was 487 mg / L, which reduced the production cost by 540% compared with conventional culture medium fermentation under the same conditions.
[0056] Example 16: Raw material processing, culture medium preparation and fermentation Fish bone meal was heat-treated at 80℃ for 60 min using hydrochloric acid solution. The pH was adjusted to the optimal level for alkaline protease. After cooling, the fish bone meal was enzymatically hydrolyzed with alkaline protease (hydrolysis temperature: 50℃; hydrolysis time: 6 h; alkaline protease was added to the hydrolysis system to achieve an enzyme activity of 160 U / mL, with a specific activity of 15 U / mg). The culture medium contained 60 g / L of sugarcane molasses and 440 g / L of fish bone meal hydrolysate (corresponding to a total sugar content of 30 g / L) and a total protein content of 66 g / L, respectively, and the pH was adjusted to 7.0. After sterilization at 121℃ for 15 min, red yeast was inoculated at an 8% (v / v) inoculum. R. toruloides DL-XSY01 was cultured at 30℃ with aeration for 144 h, and then post-treated according to the method in Example 1. The yield of ergothioneine was 215 mg / L, which reduced the production cost by 310% compared with conventional culture medium fermentation under the same conditions.
[0057] Example 17: Raw material processing, culture medium preparation and fermentation Fish bone meal was heat-treated at 80℃ for 45 min using hydrochloric acid solution to adjust to the optimal pH for papain. After cooling, the fish bone meal was enzymatically hydrolyzed with papain (hydrolysis temperature: 55℃; hydrolysis time: 6 h; papain was added to the hydrolysis system to achieve an enzyme activity of 160 U / mL, with a specific activity of 200 U / mg). The contents of sugarcane molasses and fish bone meal hydrolysates in the culture medium were 10 g / L (corresponding to a total sugar content of 5 g / L) and 140 g / L (corresponding to a total protein content of 21 g / L), respectively, and the pH was adjusted to 7.0. After sterilization at 121℃ for 15 min, red yeast was inoculated at a 6% (v / v) inoculum. R. toruloides DL-XSY01 was cultured at 30℃ with aeration for 120 h, and then post-treated according to the method in Example 1. The yield of ergothioneine was 269 mg / L, which reduced the production cost by 373% compared with conventional culture medium fermentation under the same conditions.
[0058] Example 18: Raw material processing, culture medium preparation and fermentation Fish bone meal was heat-treated at 121°C for 15 min using hydrochloric acid solution. The pH was adjusted to the optimal level for neutral protease. After cooling, the fish bone meal was enzymatically hydrolyzed with neutral protease (hydrolysis temperature: 45°C; hydrolysis time: 6 h; neutral protease was added to the hydrolysis system to achieve an enzyme activity of 160 U / mL, with a specific activity of 50 U / mg). The concentrations of glucose molasses and fish bone meal hydrolysate in the culture medium were 20 g / L (corresponding to a total sugar content of 10 g / L) and 280 g / L (corresponding to a total protein content of 31.5 g / L), respectively, and the pH was adjusted to 7.5. After sterilization at 121°C for 15 min, red yeast was inoculated at a 4% (v / v) inoculum. R. toruloides DL-XSY01 was cultured at 30℃ with aeration for 96 h, and then post-treated according to the method in Example 1. The yield of ergothioneine was 220 mg / L, which reduced the production cost by 279% compared with conventional culture medium fermentation under the same conditions.
[0059] Example 19: Raw material processing, culture medium preparation and fermentation Fish bone meal was heat-treated at 80℃ for 45 min using hydrochloric acid solution. The pH was adjusted to the optimal level for acidic protease. After cooling, the fish bone meal was enzymatically hydrolyzed with acidic protease (hydrolysis temperature: 40℃; hydrolysis time: 6 h; acidic protease was added to the hydrolysis system to achieve an enzyme activity of 160 U / mL, with a specific activity of 50 U / mg). The concentrations of glucose molasses and fish bone meal hydrolysate in the culture medium were 30 g / L (corresponding to a total sugar content of 15 g / L) and 270 g / L (corresponding to a total protein content of 40 g / L), respectively. The pH was adjusted to 5.5. After sterilization at 121℃ for 15 min, red yeast was inoculated at a 2% (v / v) inoculum. R. toruloides DL-XSY01 was cultured at 30℃ with aeration for 72 h, and then post-treated according to the method in Example 1. The yield of ergothioneine was 225 mg / L, which reduced the production cost by 319% compared with conventional culture medium fermentation under the same conditions.
[0060] Table 1. Ergothioneine yields in Examples 1-19
[0061] As shown in Table 1, based on the experimental data, the following conclusions can be drawn: Among all examples, Example 15 yielded the highest ergothioneine production, reaching 487 mg / L. The optimal process combination was: treatment with hydrochloric acid solution, using sugarcane molasses as the carbon source, adding papain, and controlling the initial pH at 6.5 and the inoculum size at 10% during fermentation. These results demonstrate that efficient ergothioneine production can be achieved through the synergistic effect of specific process parameters.
[0062] Comparative Example 1 The fish bone powder hydrolysate from Example 1 is omitted, while other conditions and parameters are the same as in Example 1.
[0063] Comparative Example 2 The molasses in Example 1 is omitted, and other conditions and parameters are the same as in Example 1.
[0064] Comparative Example 3 Replace the molasses in Example 1 with crude glycerol, and keep all other conditions and parameters the same as in Example 1.
[0065] Comparative Example 4 The fish bone meal hydrolysate in Example 1 was replaced with soybean meal, and other conditions and parameters were the same as in Example 1.
[0066] Comparative Example 5 The fish bone powder enzymatic hydrolysate in Example 6 is omitted, while other conditions and parameters are the same as in Example 6.
[0067] Comparative Example 6 The molasses in Example 6 is omitted, and other conditions and parameters are the same as in Example 6.
[0068] Comparative Example 7 Replace the molasses in Example 6 with crude glycerol, and keep all other conditions and parameters the same as in Example 6.
[0069] Comparative Example 8 The fish bone meal hydrolysate in Example 6 was replaced with soybean meal, and other conditions and parameters were the same as in Example 6.
[0070] Comparative Example 9 The fish bone powder enzymatic hydrolysate in Example 6 is omitted, and other conditions and parameters are the same as in Example 9.
[0071] Comparative Example 10 The molasses in Example 6 is omitted, and other conditions and parameters are the same as in Example 9.
[0072] Comparative Example 11 The molasses in Example 6 was replaced with crude glycerol, and other conditions and parameters were the same as in Example 9.
[0073] Comparative Example 12 The fish bone meal hydrolysate in Example 6 was replaced with soybean meal, and other conditions and parameters were the same as in Example 9.
[0074] Comparative Example 13 The amount of molasses added was increased by 50%, while other conditions and parameters remained the same as in Example 15.
[0075] Comparative Example 14 The amount of fish bone powder added was increased by 50%, while other conditions and parameters remained the same as in Example 15.
[0076] Comparative Example 15 The amount of molasses added was increased by 100%, and other conditions and parameters were the same as in Example 15.
[0077] Comparative Example 16 The amount of fish bone powder added was increased by 100%, while other conditions and parameters remained the same as in Example 15.
[0078] Table 2 Experimental Results
[0079] As shown in Table 2, the comparative examples (such as using crude glycerol as a carbon source, not adding a nitrogen source, using soybean meal instead of fish bone meal, or not undergoing enzymatic hydrolysis) generally had low yields, with the highest being only 105 mg / L, far lower than the examples. In contrast, the examples, through the combination of "hydrochloric acid pretreatment + papain hydrolysis + molasses carbon source + fish bone meal nitrogen source", significantly improved the yield. Among them, Example 15 (10 g / L sugarcane molasses, 57 g / L fish bone meal, and acidic protease treatment) achieved a yield of 487 mg / L, which was the best among all experimental groups, proving that this process condition is the key to the efficient production of ergothioneine.
[0080] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A method for producing ergothioneine, characterized in that, The method involves using molasses and fish bone meal hydrolysate as carbon and nitrogen sources in a fermentation system, inoculating it with ergothionein-producing microbial seed liquid, culturing it at 20℃~37℃ with aeration, and separating and collecting the bacterial cells after fermentation is terminated to extract ergothionein.
2. The method according to claim 1, characterized in that, The ergot-producing microorganism is red yeast. Rhodosporidium toruloides DL-XSY01, accession number CGMCC No. 23534.
3. The method according to claim 1, characterized in that, The total sugar content in the fermentation system is 20-60 g / L, and the total protein content is 12-67 g / L.
4. The method according to claim 1, characterized in that, The preparation method of fish bone powder hydrolysate includes the following steps: (1) Prepare an aqueous solution of fish bone powder with a total concentration of 20 g / L-600 g / L and a protein content of 1.2-70 g / L, and add 0-6 mol / L acid solution; (2) Heat treatment at 70-121℃ for 60 min, cool to a suitable temperature for protease hydrolysis and adjust to a suitable pH; (3) Add protease to the cooled solution and hydrolyze it for 2-8 h at a suitable protease temperature. Heat inactivation of protease terminates the hydrolysis reaction.
5. The method according to claim 4, characterized in that, In step (1), the acid solution includes phosphoric acid, citric acid, hydrochloric acid or sulfuric acid.
6. The method according to claim 4, characterized in that, In step (2), the protease can be a neutral protease, acidic protease, alkaline protease, papain, trypsin, or pepsin. The enzymatic hydrolysis conditions for the neutral protease are: temperature 40-50℃, pH 6.5-7.5, and dosage of 50 U / mg; the enzymatic hydrolysis conditions for the acidic protease are: temperature 40-50℃, pH 2.5-5.5, and dosage of 50 U / mg; the enzymatic hydrolysis conditions for the alkaline protease are: temperature 50-55℃, pH 8.0-11.0, and dosage of 15 U / mg; the enzymatic hydrolysis conditions for the papain are: temperature 55-65℃, pH 5.7-7.0, and dosage of 200 U / mg; and the enzymatic hydrolysis conditions for the trypsin are: temperature 50-55℃, pH 7.8-8.5, and dosage of 130 U / mg. U / mg; the enzymatic hydrolysis conditions of the pepsin are: temperature 37-40℃, pH 1.5-2.0, and addition amount of 3000 U / mg.
7. The method according to claim 1, characterized in that, The fermentation system also contains 0–3.0 g / L of nutrients required for the growth of conventional microorganisms, including yeast powder, peptone, malt extract, potassium phosphate, magnesium sulfate, zinc sulfate, manganese sulfate, calcium chloride, ferrous sulfate, etc., and adjusts the pH to 5.5–7.
5.
8. The method according to claim 1, characterized in that, The molasses used can be beet molasses, sugarcane molasses, glucose molasses, or corn molasses.
9. The method according to claim 1, characterized in that, The inoculation amount of ergothionein-producing microbial seed liquid is 2% to 20% (v / v). After aeration culture at 20℃ to 37℃ for 36 h to 240 h, the total reducing sugar concentration in the fermentation broth can be reduced to below 1%.
10. The application of the method according to any one of claims 1 to 9 in increasing ergothioneine fermentation yield.