Rhizopus oryzae for producing ergothioneine
A technology for ergothioneine and Rhizopus oryzae, applied in the field of microorganisms, can solve the problems of long fermentation period, slow growth of mushroom mycelium, and high production cost, and achieves fast growth rate, simple post-processing procedure, and no product inhibition. Effect
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Embodiment 1
[0027] Embodiment 1: Screening of filamentous fungi resistant to ergothioneine
[0028] Collect food waste samples from 30 areas in Huzhou City, Zhejiang Province, resuspend them with sterile water, filter them with sterile absorbent cotton, and coat the suspension with solids containing penicillin, kanamycin, chloramphenicol and ergothioneine Medium (glucose 20g / L, peptone 10g / L, yeast extract 5g / L, penicillin (100μg / ml), kanamycin (50μg / ml), chloramphenicol (25μg / ml), ergothioneine 1g / L, agar 20g / L, pH7.0), cultivated at 28-30°C for 4-5 days. Pick vigorously growing filamentous microorganisms and transfer them to new solid plates for pure culture. After more than two months, a total of more than 600 strains of microorganisms with different shapes were obtained, most of which were molds, which were used for the next step of shake flask fermentation screening.
Embodiment 2
[0029] Embodiment 2: the microbial screening of producing ergothioneine
[0030] More than 600 strains of microorganisms obtained in Example 1 were inoculated into test tubes of liquid medium (glucose 20g / L, peptone 10g / L, yeast extract 5g / L, pH7.0) respectively, and each bacterial strain was inoculated with 3 filling volumes for 4ml test tubes. 30°C, 230rpm shaker culture for 72h.
[0031] After the cultivation, the three test tube culture fluids of each bacterial strain were combined, centrifuged to get the supernatant, and the content of ergothioneine was detected by HPLC. The production of ergothioneine could be detected in the fermentation broth of 19 strains of microorganisms. The ergothioneine content in two of the microorganisms was relatively high, and they were named SH444 and SH445 respectively.
Embodiment 3
[0032] Embodiment 3: bacterial strain identification
[0033] The bacterial strains SH444 and SH445 obtained in Example 2 were sent to the Institute of Microbiology, Chinese Academy of Sciences for strain identification, and according to data such as cultivation, i.e. cell micromorphological characteristics, physiological and biochemical characteristics and rRNA gene sequence, it was determined that SH444 was Rhizopus oryzae; Determine SH445 as Trichoderma reesei. Specifically:
[0034] 3.1 Strain SH444
[0035] The colonies are sparse and fluffy, the hyphae are white, the rhizomes are slightly dark brown, the stolons crawl, the rhizoids are well developed, chemo-heterotrophic, aerobic. The bacterial strain was observed under a microscope, and the shape was strip-shaped, colorless and transparent. Such as figure 1 and figure 2 shown.
[0036] Its rRNA gene sequence is:
[0037] GAAGGATCATTAATTATGTTAAAGCGCCTTACCTCTTAGGGTTTCCTCTGGGGTAAGTGATTGCTTCTACACTGTGAAAATTTGGCTGAGAG...
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