CN104212737B - Streptomyces clavuligerus and application method thereof in production of melanin - Google Patents
Streptomyces clavuligerus and application method thereof in production of melanin Download PDFInfo
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Abstract
The invention discloses a streptomyces clavuligerus and an application method thereof in melanin production, comprising the following steps: the freeze-stored streptomyces clavuligerus needs to be subjected to strain activation and multiplication culture; transferring the seed liquid into a fermentation culture solution for fermentation culture to obtain a fermentation liquid; separating from the fermentation liquor, and drying to obtain the melanin product. The streptomyces clavuligerus is inoculated into a seed culture solution for propagation culture to obtain a seed solution; transferring the seed liquid into a fermentation culture solution for fermentation culture to obtain a fermentation liquid; separating and drying the fermentation liquor to obtain a melanin product; the method is simple, the operation is convenient, the germplasm resources of the melanin-producing bacteria are enriched, the produced melanin has stable performance, the highest yield can reach 0.85g/L, the method with higher efficiency and stable performance is provided for the production of the melanin, and the blank of the existing lack of microbial strains in the production and application of the melanin is made up.
Description
Technical Field
The invention belongs to the technical field of melanin-producing bacteria, and particularly relates to streptomyces clavuligerus and an application method thereof in producing melanin.
Background
Melanin is a substance capable of coloring a mordant and is widely applied to production, life and scientific research. Commonly used pigments can be roughly classified into synthetic pigments and natural pigments. The artificially synthesized pigment is an organic pigment prepared by a chemical synthesis method and mainly prepared from aniline dye separated from coal tar; the natural pigment is generally a pigment prepared by processing a substance existing in nature or a secondary metabolite produced by a culture method.
Compared with synthetic pigments, natural pigments are nontoxic and harmless to human bodies, have high nutritional value and important biological activity, so development and application of the nontoxic and harmless natural pigments become a trend for pigment development. Melanin is one of the most widely used pigments at present, and is a heterogeneous polyphenol polymer with high molecular weight. Generally, melanin is not essential to the growth and development of organisms, but can improve the survival and competitive power of organisms (leaf brightness, age, Verticillium, etc., the stability of melanin of YM421 of the genus Sclerotinia and the antioxidant activity thereof, the journal of bacteriology, 2010, 29(2): 254-; melanin has a variety of biological activities, including ultraviolet absorption, antioxidant, free radical scavenging, etc. (Henson, J.M., Butler, M.J., and Day, A.W. (1999) The dark side of The mycelium: Melanins of phytopathogenic fungi [ J ]. Annu Rev phytopathothocath 37:447 well 471.; Wakamatsu, K., and S.Ito.2002.advanced chemical methods in skin determination. pigment Cell Res.15:174 well 183.), and has a wide application prospect in The medical, food, and cosmetic industries.
Although plant melanin is a main source for extracting natural melanin at present, the main industrial production raw materials of the melanin are black beans and black sesame, the raw material supply is easily influenced by climate and season, and the microbial production of the melanin is not limited by regions and seasons and is easy to industrialize. The microbial melanin has a good competitive advantage in terms of production cost, production efficiency, environmental protection and the like.
Many microorganisms such as fungi, actinomycetes and bacteria can produce melanin, for example, Chinese patent document with publication number CN 102477407A discloses a melanin-producing Escherichia coli engineering bacterium DH5 α 9F2 with preservation number CCTCCNO: M2010244, Chinese patent document with publication number CN102492630A discloses a melanin-producing mutant strain aureobasidium pullulans (Aureobasidi, Pullulan) with preservation number CGMCC No.3337, and document (Corona, screening of high-yield melanin strains and preliminary study of fermentation conditions and melanin structure, institute of academy of sciences (Chengdu Biochemical research institute), 2006) discloses a streptomycete 88, and the yield of melanin of the strain is up to 86mg/100 mL.
Because melanin is widely applied, the research on microbial melanin is more, but most of the research focuses on the aspects of melanin synthesis pathway and bioactivity, and the exploration and discovery of microbial strains for producing melanin are less involved.
Disclosure of Invention
The embodiment of the invention aims to provide an application method of streptomyces clavuligerus in melanin production, and aims to solve the problem that the existing streptomyces clavuligerus lacks microbial strains in melanin production and application.
The embodiment of the invention is realized by the following steps of:
firstly, strain activation is required to be carried out on the streptomyces clavuligerus which is preserved in a freezing way, and the activation conditions are as follows: the temperature is 37 ℃ and the time is 48 h; selecting a single colony on an activation plate to be inoculated in a seed culture solution for multiplication culture;
step two, transferring the seed liquid into a fermentation culture solution for fermentation culture to obtain a fermentation liquid; the conditions of fermentation culture are as follows: the temperature is 25-30 ℃, and the time is 7-8 days; the fermentation culture is shaking culture, and the rotating speed is 180 r/min-220 r/min;
separating and drying the fermentation liquor to obtain a melanin product; centrifuging the fermentation liquor to obtain a supernatant, concentrating the supernatant, adjusting the pH value to 1-2, standing for one day, centrifuging, and taking a precipitate to obtain a melanin crude product; and (3) soaking the melanin crude product in a hydrochloric acid solution of 5-7 mol/L for 4-5 h, washing with absolute ethyl alcohol and acetone in sequence, and drying at normal temperature to constant weight to obtain a melanin pure product.
Further, in the first step, the conditions of the propagation culture are as follows: the temperature is 30-40 ℃, and the time is 12-36 h; more preferably, the conditions of the proliferation culture are: the temperature is 35-37 ℃, and the time is 24-28 h; the rotating speed of the proliferation culture which is shaking culture is preferably 150 r/min-200 r/min.
Further, the inoculation amount of the seed liquid in the second step is preferably 5-15%, and more preferably 10%; the temperature of fermentation culture is preferably 28 ℃, and the time is 7 days; the fermentation culture preferably is carried out at a shaking culture rotation speed of preferably 200 r/min.
Furthermore, the yield of the melanin of the application method of the strain streptomyces clavuligerus in the production of the melanin can reach 0.85g/L at most.
The application method of the streptomyces clavuligerus in the production of melanin provided by the invention comprises the steps of inoculating streptomyces clavuligerus into a seed culture solution for multiplication culture to obtain a seed solution; transferring the seed liquid into a fermentation culture solution for fermentation culture to obtain a fermentation liquid; separating and drying the fermentation liquor to obtain a melanin product; the method is simple, the operation is convenient, the germplasm resources of the melanin-producing bacteria are enriched, the produced melanin has stable performance, the highest yield can reach 0.85g/L, the method with higher efficiency and stable performance is provided for the production of the melanin, and the blank of the existing lack of microbial strains in the production and application of the melanin is made up.
Drawings
FIG. 1 is a flow chart of a method for applying Streptomyces clavuligerus in the production of melanin according to an embodiment of the present invention;
FIG. 2 is a micrograph of Streptomyces clavuligerus provided in an example of the present invention;
FIG. 3 is an electrophoretogram of PCR amplification products of Streptomyces clavuligerus 16SrDNA provided in an embodiment of the present invention;
FIG. 4 is a diagram of an ultraviolet-visible spectrum of melanin produced by Streptomyces clavuligerus according to an embodiment of the present invention;
FIG. 5 is an infrared spectrum of melanin produced by Streptomyces clavuligerus according to an embodiment of the present invention;
FIG. 6 is a graph showing the effect of pH on melanin solution stability provided by an example of the present invention;
FIG. 7 is a graph showing the effect of temperature on melanin solution stability provided by an embodiment of the present invention;
FIG. 8 is a graph showing the effect of UV light on the stability of a melanin solution provided by an embodiment of the present invention;
FIG. 9 is a graph showing the effect of an oxidizing agent on the stability of a melanin solution provided by an embodiment of the present invention;
FIG. 10 is a graph showing the effect of metal ions on melanin solution stability according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The application of the principles of the present invention will be further described with reference to the accompanying drawings and specific embodiments.
As shown in FIG. 1, the application method of the streptomyces clavuligerus strain in the production of melanin comprises the following steps:
s101: the freeze-stored streptomyces clavuligerus needs to be firstly activated under the following conditions: the temperature is 37 ℃ and the time is 48 h; selecting a single colony on an activation plate, inoculating the single colony in a seed culture solution, and performing enrichment culture;
s102: transferring the seed liquid into a fermentation culture solution for fermentation culture to obtain a fermentation liquid; the conditions for the fermentation culture are preferably: the temperature is 25-30 ℃, and the time is 7-8 days; the fermentation culture is preferably shake culture, and the rotating speed is 180-220 r/min;
s103: separating and drying the fermentation liquor to obtain a melanin product; centrifuging the fermentation liquor to obtain a supernatant, concentrating the supernatant, adjusting the pH value to 1-2, standing for one day, centrifuging, and taking a precipitate to obtain a melanin crude product; and (3) soaking the crude melanin pigment product in a hydrochloric acid solution of 5-7 mol/L for 4-5 h, washing with absolute ethyl alcohol and acetone in sequence, and drying at normal temperature to constant weight to obtain a pure melanin product.
In step S101, the conditions of the growth culture are preferably: the temperature is 30-40 ℃, and the time is 12-36 h; more preferably, the conditions of the proliferation culture are: the temperature is 35-37 ℃, and the time is 24-28 h; more preferably, the propagation culture is shake culture, and the rotating speed is 150-200 r/min.
In the step S102, the inoculation amount of the seed liquid is preferably 5-15%, and more preferably 10%; the conditions for the fermentation culture are preferably: the temperature is 28 ℃, and the time is 7 days; the fermentation culture is preferably carried out at a shaking table culture rotating speed of 200 r/min;
under the set fermentation conditions, the yield of melanin of the streptomyces clavuligerus can reach 0.85g/L at most;
the invention will be further described with reference to specific embodiments thereof:
example 1, strain isolation and characterization:
(1) strain separation:
collecting soil samples from different places of the Jiaxing park, selecting soil with the depth of 5-20 cm below the soil surface during sampling, and taking the soil back to a laboratory by using sterile paper bags; after air drying and grinding, 1g of soil sample is added with 99mL of deionized water to prepare soil suspension, and the soil suspension is sequentially diluted to 10-5、10-6(ii) a Get 10-5、10-60.1mL of diluent is respectively coated on a screening culture medium, cultured at 37 ℃, selected to produce colonies with early melanin production time and dark color, and then subjected to streaking separation and purification for multiple times to obtain single colonies;
the formula of the screening culture medium is as follows: 3g of beef extract, 10g of peptone, 5g of NaCl, 15-20g of agar, 7.0-7.2 of pHs and 1000mL of water;
(2) morphological identification:
inoculating the single colony producing melanin into a Gao-Shi synthetic first culture medium for 3 days, and observing the characteristics of the colony: the bacterial colony is basically characterized in that the bacterial colony is compact in texture, compact in velvet-like surface, firm, dry and wrinkled; the strain was observed under a microscope, and the results are shown in FIG. 2;
(3) and (3) molecular identification:
the genomic DNA of this strain was extracted according to the experimental procedure of the literature ((Orsini M, Romano-Spica V.A microwave-based method for nucleic acid isolation from environmental samples. Let Appl Microbiol,2001,33:17-20.) and its 16sDNA sequence was amplified using primers with the genomic DNA as template, the sequences of which were as follows:
an upstream primer: 5'-AGAGTTTGATCCTGGCTCAGAACGAAC-3', respectively;
a downstream primer: 5'-TACGGTTACCTTGTTACGACTTCACCCC-3' the flow of the air in the air conditioner,
the amplified product was subjected to agarose gel electrophoresis, and the results are shown in FIG. 3, as can be seen from FIG. 3, the length of the PCR product was about 1500bp, which is consistent with the expected length when the primers were designed in advance, and the sequencing was performed by Shanghai Biotechnology services, Inc., and the base sequence is shown as SEQ ID No.3 (providing the PCR product sequence, listed in the "sequence table part" at the end of the text), and the sequencing results were registered in the ncbi website GenBank under the accession number: JN 088281.1. The gene comparison result shows that the similarity of the strain screened by the invention and the 16SrRNA of the streptomyces clavuligerus which produces melanin and is reported in the literature is higher and reaches 98 percent, the strain is sent to the common microorganism center of China general microbiological culture Collection center at 18 months 3 and 2014, the preservation number is CGMCC No.8934, the depositor is Liuxiao of Jiaxing institute, and the address is No.3 of Xilu 1 of the morning area in Beijing city; institute of microbiology, national academy of sciences; the referenced biological materials: jx-01; suggested classification nomenclature: streptomyces clavuligerus, Streptomyces clavuligerus; the biological material is received by the China microbial culture collection center at 18 days 03 and 2014, is registered in a book and is stored for 30 years, the viability of the biological material is detected by the China microbial culture collection center at 18 days 03 and 2014, and the result is survival;
example 2, fermentation of strains to produce melanin:
(1) selecting bacterial colony from test tube for activating bacterial strain, and culturing at 37 deg.C for 48 hr; the formula of the activation medium is as follows:
(2) selecting a single colony on an activation plate, transferring the single colony to a seed culture solution for enrichment culture, and culturing at 37 ℃ for 48h at the rotating speed of 200 r/min; the formula of the seed culture solution is (g/L): soluble starch 2g, KNO30.1g，K2HP040.05g，NaCl0.05g，MgSO4·7H2O0.05g，FeS04·7H2O0.01g， pH7.2-7.4。
(3) Inoculating the seed liquid into a fermentation culture solution for fermentation culture, and performing shake culture at 28 ℃ for 7 days at a rotation speed of 200r/min until the fermentation liquid is black;
(4) centrifuging black fermentation liquor at 10000r/min, collecting supernatant, performing rotary evaporation concentration, adjusting the pH of the concentrated solution to 2 with 6mol/L HCL, standing for 24h, centrifuging at 10000r/min, and precipitating to obtain a melanin crude product; soaking the precipitate in 6mol/LHCl solution for 4h, washing with anhydrous ethanol and acetone for 2 times, and drying at room temperature to constant weight to obtain melanin pure product.
Example 3, melanin basis data were obtained:
a series of tests were carried out on the melanin samples obtained in the preparation of example 2, in order to obtain the basic data of the melanin:
(1) structural characterization:
ultraviolet-visible full-waveband scanning and infrared spectrum analysis are carried out on the melanin, and the results are shown in fig. 4 and fig. 5;
as can be seen from FIG. 3, the melanin has a strong absorption peak at about 240nm, no characteristic peak in the visible light region, and the absorbance decreases with the increase of the wavelength, which is similar to the melanin produced by streptomycin G-HD-4 in the literature (Zhengchenna, Fangbaishan, Rou Ju Xiang, etc.. the extraction and physicochemical properties of the melanin produced by Streptomyces G-HD-4. university of Huaqiao (Nature science edition), 2009,30(3): 292-;
as can be seen from FIG. 5, the infrared spectrum of the melanin is due to-OH and-NH2Will be at 3000nm (3333 cm)-1) An absorption peak is near the surface, and the carbonyl group aggregation can make it at 6000nm (1667 cm)-1) There is an absorption peak near the pigment, and the two marker peaks of the standard melanin of Sigma are respectively positioned at 3405cm according to the record of the literature (Zhang Jianping, Cai Jun, Deng music, etc.. wild Bc strain melanin production research, microbiological report, 2006, (1):25-31.)-1 and 1625cm-1Here, the two marker peaks of melanin of the sample of the present invention are located at 3419.69cm, respectively-1And 1652.04cm-1The infrared spectrogram of the melanin is proved to be very similar to that of standard melanin;
(2) stability:
1) influence of pH on melanin stability:
the influence of pH value on natural pigment is large, 0.001g pure melanin is dissolved in 10mL deionized water with different pH values, the change of solution absorption value is examined, and as shown in FIG. 6, the solubility of the melanin is increased along with the increase of the pH value of the deionized water as shown in FIG. 6: when the pH value is less than or equal to 4, the melanin is basically insoluble, when the pH value is between 4 and 7, the absorbance value of the melanin is rapidly increased, the absorbance is in a stable rising trend after the pH value is more than 7, and the absorbance reaches the maximum when the pH value is 13, which is consistent with the property of acid precipitation and alkali dissolution of the melanin;
2) effect of temperature on melanin stability:
preparing melanin solution with concentration of 0.1g/L, pH of 9 from the pure melanin, respectively placing in water bath at 20 deg.C, 40 deg.C, 60 deg.C, 80 deg.C and 100 deg.C for 2 hr, and measuring OD400As shown in FIG. 7, it can be seen from FIG. 7 that the melanin is stable at a temperature of 20 ℃ to 60 ℃; the melanin is slightly decomposed at the temperature of more than 80 ℃, but the residual rate is still more than 95 percent, which shows that the melanin has better stability at the temperature of between 20 and 100 ℃;
3) effect of uv on melanin stability:
preparing melanin solution with concentration of 0.1g/L, pH of 9 from the pure melanin, irradiating under 265nm ultraviolet lamp for 20min, 40min, 60min, 80min, and 100min, respectively, and measuring OD400As shown in fig. 8, it can be seen from fig. 8 that the residual rate of absorbance of the melanin solution after 80min irradiation was 97.23%, indicating that melanin has good stability to ultraviolet rays;
4) effect of oxidizing agent on melanin stability:
preparing melanin solution with concentration of 0.1g/L, pH of 9 by using the pure melanin product, and adding H with different final concentrations into the melanin solution2O2Standing at room temperature for 1h, and determining OD of each solution400The results are shown in FIG. 9, as can be seen from FIG. 9, with H2O2The concentration is increased, the absorbance value of the melanin solution is in a descending trend, when H is2O2At a concentration of 3%, the residual rate of melanin is 75%, and it can be seen that the stability of melanin to strong oxidizing agents is not high, so that contact with strong oxidizing agents is to be avoided during application;
5) effect of metal ions on melanin stability:
the pure melanin product was used to prepare a melanin solution with a concentration of 9 of 0.1g/L, pH, and the effect of different amounts of added metal ions on the stability was examined, and the results are shown in fig. 9:
as can be seen from FIG. 10, K is within 0.01mol/L+、Ca2+、Mg2+No obvious influence on the stability of melanin; fe3+、Zn2+、Cu2+Has a large influence on the stability of melanin, wherein Fe3+Has a color-enhancing effect, probably due to Fe3+Coordinate with melanin to form a new substance having absorption value at 400nm and OD400The increase is obvious; and Cu2+、Zn2+When present, melanin showed OD due to precipitation400Reduce, probably, the chelation of melanin with these metal ions when Cu is present2+、Zn2+At a concentration of 0.01mol/L, melanin was completely precipitated, indicating that the stability of melanin was different in the presence of different metal ions.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
< 110 > Applicant name: jiaxing school
Streptomyces clavuligerus with purity of less than 120 and application method thereof in production of melanin
＜160＞3
＜210＞1
＜211＞27
＜212＞DNA
Less than 213 melanin-producing bacterial colony obtained by culture, separation and purification from Jiaxing park soil
＜400＞1
AGAGTTTGATCCTGGCTCAGAACGAAC
＜210＞2
＜211＞28
＜212＞DNA
Less than 213 melanin-producing bacterial colony obtained by culture, separation and purification from Jiaxing park soil
＜400＞2
TACGGTTACCTTGTTACGACTTCACCCC
＜210＞3
＜211＞613
＜212＞RNA
Less than 213 melanin-producing bacterial colony obtained by culture, separation and purification from Jiaxing park soil
＜400＞3
1 cagaccaggt cttacggtta ccttgttacg acttcacccc aatcgccagt cccaccttcg
61 acagctccct cccacaaggg gttgggccac cggcttcggg tgttaccgac tttcgtgacg
121 tgacgggcgg tgtgtacaag gcccgggaac gtattcaccg cagcaatgct gatctgcgat
181 tactagcaac tccgacttca tggggtcgag ttgcagaccc caatccgaac tgagaccggc
241 tttttgagat tcgctctacc tcacggtttc gcagctcatt gtaccggcca ttgtagcacg
301 tgtgcagccc aagacataag gggcatgatg acttgacgtc gtccccacct tcctccgagt
361 tgaccccggc ggtctcctgt gagtccccat caccccgaag ggcatgctgg caacacagga
421 caagggttgc gctcgttgcg ggacttaacc caacatctca cgacacgagc tgacgacagc
481 catgcaccac ctgtataccg accacaaggg gggcactatc tctaatgctt tccggtatat
541 gtcaagcctt ggtaaggttc ttcgcgttgc gtcgaattaa gccacatgct ccgctacttg
601 tgcgggcccc cg
Claims (6)
1. An application method of streptomyces clavuligerus in melanin production is characterized in that the preservation number of the streptomyces clavuligerus strain is CGMCC No. 8934; the application method of the streptomyces clavuligerus in the production of melanin comprises the following steps:
step one, the streptomyces clavuligerus preserved by freezing is in Gao' sFirstly, strain activation is carried out on the synthetic first culture medium, and the synthetic first culture medium of Gao's: soluble starch 2g, KNO30.1g，K2HPO40.05g，NaCl 0.05g，MgSO4·7H2O 0.05g，FeSO4·7H2O0.01 g, pH7.2-7.4; the activation conditions were: the temperature is 37 ℃ and the time is 48 h; selecting a single colony on an activation plate, inoculating the single colony in a seed culture solution, and performing enrichment culture;
step two, transferring the seed liquid to a fermentation medium for fermentation culture, wherein the g/L of the fermentation medium comprises: 1g/L of maltose, 5g/L of beef extract, 5g/L of sodium chloride, 0.1g/L of calcium chloride, 2g/L of L-tyrosine and pH7.0; obtaining fermentation liquor; the conditions of fermentation culture are as follows: the temperature is 25-30 ℃, and the time is 6-10 days; the fermentation culture is shaking culture, and the rotating speed is 180 r/min-220 r/min;
separating and drying the fermentation liquor to obtain a melanin product; centrifuging the fermentation liquor to obtain a supernatant, concentrating the supernatant, adjusting the pH value to 1-2, standing for one day, centrifuging, and taking a precipitate to obtain a melanin crude product; and (3) soaking the melanin crude product in a hydrochloric acid solution of 5-7 mol/L for 4-5 h, washing with absolute ethyl alcohol and acetone in sequence, and drying at normal temperature to constant weight to obtain a melanin pure product.
2. The method of using Streptomyces clavuligerus for producing melanin according to claim 1, wherein in step one, the propagation culture conditions are as follows: the temperature is 30-40 ℃, and the time is 12-36 h; the proliferation culture is shaking culture with the rotating speed of 150 r/min-200 r/min.
3. The method of using Streptomyces clavuligerus for producing melanin according to claim 1, wherein in step one, the propagation culture conditions are as follows: the temperature is 35-37 ℃, and the time is 24-28 h; the proliferation culture is shaking culture with the rotating speed of 150 r/min-200 r/min.
4. The application method of streptomyces clavuligerus in the production of melanin as claimed in claim 1, wherein the inoculation amount of the seed liquid in the second step is 5-15%; the temperature of fermentation culture is 28 ℃, and the time is 7 days; the fermentation culture is shake culture at a rotation speed of 200 r/min.
5. The method for using Streptomyces clavuligerus in the production of melanin according to claim 1, wherein the inoculation amount of the seed solution in the second step is 10%; the temperature of fermentation culture is 28 ℃, and the time is 7 days; the fermentation culture is shake culture at a rotation speed of 200 r/min.
6. The method of claim 1, wherein the yield of melanin is up to 0.85 g/L.
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| Title |
|---|
| GenBank Accession：JN088281.1;Liu,X.X. et al.;《GenBank》;20110619;第1页 * |
| Streptomyces clavuligerus relA-null mutants overproduce clavulanic acid and cephamycin C:negative regulation of secondary metabolism by (p)ppGpp.;Gomez-Escribano JP et al.;《Microbiology》;20080331;第154卷(第Pt3期);第744-755页 * |
| 产克拉维酸的棒状链霉菌突变株的选育;王艳萍等;《食品与发酵工业》;20070531;第33卷(第5期);第9-12页 * |
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