CN101423810B - Streptomyces chatanoogensis and culture method - Google Patents
Streptomyces chatanoogensis and culture method Download PDFInfo
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Abstract
The invention provides a Streptomyces chattanoogensis L10 with a preserving registration number of CGMCC No.2644 and a nucleotide sequence of SEQ ID NO.1. A method for culturing the Streptomyces chattanoogensis L10 comprises the following steps: firstly, inoculating Streptomyces chattanoogensis L10 strains to a slant culture medium to perform solid culture; secondly, inoculating mycelia to a seed culture medium to perform secondary culture; and thirdly, inoculating the mycelia to a fermentation medium to perform liquid culture. The strains provided by the invention can generate natamycin and can be applied to preparing broad-spectrum antifungal agents and preparing biological food preservatives and antibacterial additives.
Description
Technical field
The invention belongs to microorganism field, relate to a strain streptomyces chatanoogensis bacterial strain L10 and a cultural method.
Background technology
Tennecetin is a kind of efficient, broad-spectrum antifungal agents, belongs to macrolide antibiotics, and it can effectively suppress nearly all yeast and the growth of mould, stops the formation of aflatoxin in the filamentous fungus.And tennecetin is extremely low to the toxicity of mammalian cell, is difficult to be absorbed by the stomach of animal or human body, because tennecetin is insoluble in water and grease, the major part of absorption is discharged with ight soil.
The solubleness of tennecetin is low, and available its handled to increase the quality guaranteed period of food food surfaces, but do not influence flavours in food products and mouthfeel.Its actual using dosage is 10
-6The order of magnitude has low dosage, high-level efficiency, and the characteristics that the anti-microbial effect time is long are a kind of efficient, safe natural biological antiseptic agent and antiseptic-germicides.U.S. FDA (Food and drug administrition) official approval tennecetin can be used as food preservatives; U.S. FDA suggestion tennecetin uses as foodstuff additive, also is classified as the row of GRAS product.China foodstuff additive council in 1997 estimates tennecetin and advises that approval uses, and has now listed foodstuff additive in and has used standard, and its trade name is mould gram (Natamycin
TM).
Tennecetin not only in seasonings, bake and cheese food product etc. as the food preservatives widespread use, its bacteriostatic action is stronger about 50 times than Sorbic Acid, and is difficult for causing that the target fungi forms resistance.But also be applied to be used for treating fungoid skin infections diseases such as fungal keratitis, pin moss in eye drop or the ointment etc. as high-efficient antibacterial agent.A kind of natural biological food preservatives and antimicrobial additive that more than 30 country uses have been become at present.At industrial brown yellow spore streptomycete (Streptomyces gilvosporeus) the fermentation growth tennecetins that adopt more.
Summary of the invention
An object of the present invention is to provide a strain streptomyces chatanoogensis bacterial strain, this bacterial strain can produce tennecetin.Streptomyces provided by the present invention is streptomyces chatanoogensis (Streptomyces chattanoogensis) L10, it has been preserved in China Microbial Culture Preservation Commission common micro-organisms center (being called for short CGMCC) on August 27th, 2006, preservation registration number is: CGMCC No.2644, nucleotide sequence with SEQ ID NO 1 has more the nucleotide sequence with SEQ ID NO 2.
L10 bacterial strain provided by the invention is after the ISP2 nutrient agar is cultivated 7～10 days, and the gentle living hyphal development of substrate mycelium is good, and fibrillae of spores is gentle bent, and sophisticated spore chain spore number is more than 10, and spore is oval, surperficial tool thorn.Can assimilation utilize D-glucose, D-fructose, maltose, sucrose, glycerine, inositol and D-N.F,USP MANNITOL, can not utilize L-arabinose, D-wood sugar, L-rhamnosyl, raffinose, sorbyl alcohol and sodium-acetate.Gelatine liquefication, milk solidifies.The starch hydrolysis.Do not produce class melanochrome and H2S.
Another object of the present invention provides the cultural method of this streptomyces chatanoogensis, realizes by following steps:
(1) solid culture: streptomyces chatanoogensis L10 (CGMCC No.2644) inoculation in slant medium, was cultivated 7 days in 25 ℃ of incubators, and placed 10 days down in normal temperature;
(2) liquid culture: behind the slant culture, mycelium is inoculated in the seed culture medium, on 30 ℃ of shaking tables, cultivated 24 hours, rotating speed 250rpm, first first order seed was cultivated 24 hours, secondary seed was cultivated 24 hours again, the condition of two-stage seed culture is identical, and then is seeded in the fermention medium, inoculum size 10% (V/V), cultivated 96 hours, promptly.
The described slant medium of step (1) is: yeast extract 0.4%, and malt extract 1%, glucose 0.4%, agarose 1.5～2%, all the other are water, and the pH value transfers to 6.0, and described percentage composition is the quality percentage composition.
The described seed culture medium of step (2) is: glucose 1.75%, and peptone 1.5%, NaCl 1.0%, and all the other are water, and described percentage composition is the quality percentage composition, the pH nature.
The described fermention medium of step (2) is: raw soya bean cake powder 2.8%, and yeast powder 0.7%, glucose 6%, all the other are water, described percentage composition is the quality percentage composition, the pH nature.
A further object of the present invention provides the application of this bacterial strain in producing tennecetin.
Usefulness of the present invention (advantage or characteristics) is: the invention provides a kind of new streptomyces chatanoogensis, called after: Streptomyces chattanoogensis L10, and the cultural method of this bacterium.Bacterial strain provided by the invention can produce tennecetin, can use in preparation broad-spectrum antifungal agents, the biological food preservatives of preparation and antimicrobial additive.
Description of drawings
Fig. 1 is the form photo of streptomyces chatanoogensis L10 fibrillae of spores under opticmicroscope (1000 *).
Fig. 2 is the form photo of streptomyces chatanoogensis L10 spore under scanning electronic microscope (7700 *).
Fig. 3 is the systematic evolution tree with the 16S rDNA sequence construct of streptomyces chatanoogensis L10 and relevant bacterial classification.
Fig. 4 is the evolutionary tree that makes up with the rpoB of streptomyces chatanoogensis L10 and relevant bacterial classification (RNA polymerase β subunit) gene order.
Embodiment
The present invention is further described with specific embodiment in conjunction with the accompanying drawings.Experimental technique among the following embodiment is ordinary method if no special instructions.
Employed substratum among the embodiment:
1. slant medium: yeast extract 0.4%, malt extract 1%, glucose 0.4%, agarose 1.5～2%, all the other are water, and the pH value transfers to 6.0, and described percentage composition is the quality percentage composition.
2. seed culture medium: glucose 1.75%, peptone 1.5%, NaCl 1.0%, and all the other are water, and described percentage composition is the quality percentage composition, the pH nature.
3. fermention medium: raw soya bean cake powder 2.8%, yeast powder 0.7%, glucose 6%, all the other are water, described percentage composition is the quality percentage composition.The pH nature.
Embodiment 1
The Microbiological Characteristics of L10 is identified:
(1) morphological specificity of thalline
Bacterial strain L10 is after the ISP2 nutrient agar is cultivated 7～10d, and the gentle living hyphal development of substrate mycelium is good, and fibrillae of spores is gentle bent or crooked, and sophisticated spore chain spore number (is being seen accompanying drawing 1) more than 10, and spore is oval, surperficial tool thorn (seeing accompanying drawing 2).Appearance features in various substratum the results are shown in Table 1; The physio-biochemical characteristics of L10 see Table 2.
(2) table 1: the cultural characters of bacterial strain L10:
(3) table 2: the physio-biochemical characteristics of bacterial strain L10:
Annotate: the weak positive findings of " W " expression, "+" expression positive findings, "-" expression negative findings
(4) 16S rDNA sequence and rpoB sequential system evolutionary analysis
The 16S rDNA sequence of the approximate total length of bacterial strain L10 shows that with correlated series Blast comparative result among the GenBank it belongs to streptomyces shown in SEQ ID NO 1 in the sequence table; The sequence similarity of the 1459bp that 1459bp and streptomyces chatanoogensis bacterial strain (Streptomyces chattanoogensis) NBRC 12754 preserve in database in its known 16S rDNA sequence (1521bp) reaches 100%, ((Streptomyces lydicus) NBRC 13058 similaritys are 99.93% (1480/1481bp) with the streptomyces lydicus bacterial strain, with streptomyces chatanoogensis bacterial strain (Streptomyces chattanoogensis) DSM 40002 similaritys be 99.79% (1481/1484bp), with brown yellow spore streptomycete bacterial strain (Streptomyces gilvosporeus) ATCC13326 similarity be 99.23% (1421/1432bp), with Natal streptomycete bacterial strain (Streptomyces natalensis) NBRC 13367 similaritys be 99.32% (1474/1484bp), see accompanying drawing 3 with the systematic evolution tree of the 16S rDNA sequence construct of streptomyces chatanoogensis L10 and relevant bacterial classification.
The part rpoB gene order of bacterial strain L10 is shown in SEQ ID NO 2 in the sequence table, with the Blast comparative result show its with brown yellow spore streptomycete bacterial strain (Streptomyces gilvosporeus) ATCC13326 similarity be 99.14% (347/350bp), with streptomyces chatanoogensis bacterial strain (Streptomyceschattanoogensis) ATCC13358 similarity be 98.37% (301/306bp).The evolutionary tree that makes up with rpoB (the RNA polymerase β subunit) gene order of streptomyces chatanoogensis L10 and relevant bacterial classification sees accompanying drawing 4.Bacterial strain L10 relatively sees Table 3 with the form and the physio-biochemical characteristics of contiguous several streptomyces strains.
Table 3: the form and the physio-biochemical characteristics of bacterial strain L10 and contiguous several streptomyces strains compare:
Data?for?reference?strains?were?taken?from?Shirling?&?Gottlieb(1972)，Karwowski?et?al(1992)andLin?et?al?(1993)
Attention: "+" expression positive findings, "-" expression negative findings; " ND " represent that this index is detection or does not have data; " Smooth " represent that spore surface is smooth; " Spiny " expression spore surface band spinule; " Spirales " represent fibrillae of spores twist, " Flexuous " expression fibrillae of spores is gentle curved.
Comprehensive its morphology, cultural characters, Physiology and biochemistry, 16S rDNA sequence and rpoB The sequencing results, it is accredited as a mutation of streptomyces chatanoogensis (Streptomyces chattanoogensis), and with its called after streptomyces chatanoogensis (Streptomyces chattanoogensis) L10.Streptomyces chatanoogensis (Streptomyces chattanoogensis) L10 is preserved in China Microbial Culture Preservation Commission common micro-organisms center (being called for short CGMCC) on August 27th, 2008, and preservation registration number is CGMCC No.2644.
Embodiment 2
The training method of L10 bacterial strain is:
(1) solid culture: bacterial classification inoculation in slant medium, was cultivated 7 days in 25 ℃ of incubators; And under normal temperature, placed 10 days.
(2) liquid culture: behind the slant culture, mycelium is inoculated in the seed culture medium, cultivates 24 hours rotating speed 250rpm on 30 ℃ of shaking tables.Be inoculated in the secondary seed medium after first order seed cultivates 24 and cultivate with identical condition.Secondary seed is cultivated after 24 hours and is seeded in the fermention medium, and inoculum size 10% (V/V) was cultivated 96 hours, promptly.
The sequence that the present invention relates to
＜110〉Zhejiang University, Zhejiang Zhenyuan Pharmaceutical Co., Ltd
＜120〉a strain streptomyces chatanoogensis bacterial strain and a cultural method
<160>2
<210>1
<211>1521
<212>DNA
＜213〉streptomyces chatanoogensis (Streptomyces chattanoogensis) L10
<400>1
agagtttgat?cctggctcag?gacgaacgct?ggcggcgtgc?ttaacacatg?caagtcgaac 60
gatgaacctc?cttcgggagg?ggattagtgg?cgaacgggtg?agtaacacgt?gggcaatctg 120
cccttcactc?tgggacaagc?cctggaaacg?gggtctaata?ccggatacga?cacggggtcg 180
catgacctcc?gtgtggaaag?ctccggcggt?gaaggatgag?cccgcggcct?atcagcttgt 240
tggtggggtg?atggcctacc?aaggcgacga?cgggtagccg?gcctgagagg?gcgaccggcc 300
acactgggac?tgagacacgg?cccagactcc?tacgggaggc?agcagtgggg?aatattgcac 360
aatgggcgaa?agcctgatgc?agcgacgccg?cgtgagggat?gacggccttc?gggttgtaaa 420
cctctttcag?cagggaagaa?gcgagagtga?cggtacctgc?agaagaagcg?ccggctaact 480
acgtgccagc?agccgcggta?atacgtaggg?cgcaagcgtt?gtccggaatt?attgggcgta 540
aagagctcgt?aggcggcttg?tcacgtcgga?tgtgaaagcc?cggggcttaa?ccccgggtct 600
gcattcgata?cgggcaggct?agagttcggt?aggggagatc?ggaattcctg?gtgtagcggt 660
gaaatgcgca?gatatcagga?ggaacaccgg?tggcgaaggc?ggatctctgg?gccgatactg 720
acgctgagga?gcgaaagcgt?ggggagcgaa?caggattaga?taccctggta?gtccacgccg 780
taaacgttgg?gaactaggtg?tgggcgacat?tccacgtcgt?ccgtgccgca?gctaacgcat 840
taagttcccc?gcctggggag?tacggccgca?aggctaaaac?tcaaaggaat?tgacgggggc 900
ccgcacaagc?agcggagcat?gtggcttaat?tcgacgcaac?gcgaagaacc?ttaccaaggc 960
ttgacataca?ccggaaaacc?ctggagacag?ggtccccctt?gtggtcggtg?tacaggtggt 1020
gcatggctgt?cgtcagctcg?tgtcgtgaga?tgttgggtta?agtcccgcaa?cgagcgcaac 1080
ccttgttctg?tgttgccagc?atgcccttcg?gggtgatggg?gactcacagg?agactgccgg 1140
ggtcaactcg?gaggaaggtg?gggacgacgt?caagtcatca?tgccccttat?gtcttgggct 1200
gcacacgtgc?tacaatggcc?ggtacaatga?gctgcgatac?cgcgaggtgg?agcgaatctc 1260
aaaaagccgg?tctcagttcg?gattggggtc?tgcaactcga?ccccatgaag?tcggagttgc 1320
tagtaatcgc?agatcagcat?tgctgcggtg?aatacgttcc?cgggccttgt?acacaccgcc 1380
cgtcacgtca?cgaaagtcgg?taacacccga?agccggtggc?ccaacccctt?gtgggaggga 1440
atcgtcgaag?gtgggactgg?cgattgggac?gaagtcgtaa?caaggtagcc?gtaccggaag 1500
gtgcggctgg?atcacctcct?t 1521
<210>2
<211>352
<212>DNA
＜213〉streptomyces chatanoogensis (Streptomyces chattanoogensis) L10
<400>2
cgatcgggca?catgcggccg?tagtgcgagg?ggtgcacgtc?acggacgtcc?aggccggccc 60
gctcacggga?gagaccaccc?gggcccagcg?ccaacagacg?gcgcttgtgg?gtcagacccg 120
acagcgggtt?ggtctggtcc?atgaattggg?acagctggct?ggtgccgaag?aactccttga 180
tggaggcgac?gaccggccgg?atgttgatca?gggtctgcgg?cgtgatcgcc?tcgacgtcct 240
gggtggtcat?gcgctcgcgc?acgacgcgct?ccatacgggc?gagacccgta?cggacctggt 300
tctggatgag?ctcgccgacg?ttgcgcagac?ggcggttgcc?gaagtggtcg?aa 352
Claims (6)
1. streptomyces chatanoogensis, it is characterized in that: described streptomyces chatanoogensis called after: streptomyces chatanoogensis (Streptomyces chattanoogensis) L10, it has been preserved in China Microbial Culture Preservation Commission common micro-organisms center on August 27th, 2006, preservation registration number is: CGMCCNo.2644, the nucleotide sequence of the 16S rDNA of described streptomyces chatanoogensis is SEQ ID NO 1, and the nucleotide sequence of the rpoB gene of described streptomyces chatanoogensis is SEQ ID NO 2.
2. the cultural method of streptomyces chatanoogensis according to claim 1 is characterized in that realizing by following steps:
(1) solid culture: the inoculation of streptomyces chatanoogensis L10 in slant medium, was cultivated 7 days in 25 ℃ of incubators, and placed 10 days down in normal temperature;
(2) liquid culture: behind the slant culture, mycelium is inoculated in the seed culture medium, on 30 ℃ of shaking tables, cultivated 24 hours, rotating speed 250rpm, first order seed is cultivated to be inoculated in the secondary seed medium after 24 hours and is cultivated with identical condition, and secondary seed is cultivated after 24 hours and is seeded in the fermention medium inoculum size 10% (V/V), cultivated 96 hours, promptly.
3. the cultural method of streptomyces chatanoogensis according to claim 2, it is characterized in that: the described slant medium of step (1) is: yeast extract 0.4%, malt extract 1%, glucose 0.4%, agarose 1.5～2%, all the other are water, and the pH value transfers to 6.0, and described percentage composition is the quality percentage composition.
4. the cultural method of streptomyces chatanoogensis according to claim 2, it is characterized in that: the described seed culture medium of step (2) is: glucose 1.75%, and peptone 1.5%, NaCl 1.0%, all the other are water, and described percentage composition is the quality percentage composition.
5. the cultural method of streptomyces chatanoogensis according to claim 2, it is characterized in that: the described fermention medium of step (2) is: raw soya bean cake powder 2.8%, yeast powder 0.7%, glucose 6%, all the other are water, and described percentage composition is the quality percentage composition.
6. the application of a kind of streptomyces chatanoogensis according to claim 1 in producing tennecetin.
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