CN109971688B - 低杂高产结冷胶产生菌及其应用 - Google Patents
低杂高产结冷胶产生菌及其应用 Download PDFInfo
- Publication number
- CN109971688B CN109971688B CN201910378308.2A CN201910378308A CN109971688B CN 109971688 B CN109971688 B CN 109971688B CN 201910378308 A CN201910378308 A CN 201910378308A CN 109971688 B CN109971688 B CN 109971688B
- Authority
- CN
- China
- Prior art keywords
- gellan gum
- yield
- strain
- low
- fermentation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 229920002148 Gellan gum Polymers 0.000 title claims abstract description 77
- 239000000216 gellan gum Substances 0.000 title claims abstract description 74
- 235000010492 gellan gum Nutrition 0.000 title claims abstract description 74
- 239000012535 impurity Substances 0.000 title claims abstract description 17
- 238000000855 fermentation Methods 0.000 claims abstract description 35
- 230000004151 fermentation Effects 0.000 claims abstract description 35
- 229920001397 Poly-beta-hydroxybutyrate Polymers 0.000 claims abstract description 22
- 229920000331 Polyhydroxybutyrate Polymers 0.000 claims abstract description 22
- DMASLKHVQRHNES-UPOGUZCLSA-N (3R)-beta,beta-caroten-3-ol Chemical compound C([C@H](O)CC=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C DMASLKHVQRHNES-UPOGUZCLSA-N 0.000 claims abstract description 13
- 239000004212 Cryptoxanthin Substances 0.000 claims abstract description 13
- DMASLKHVQRHNES-ITUXNECMSA-N beta-cryptoxanthin Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2=C(C)CCCC2(C)C DMASLKHVQRHNES-ITUXNECMSA-N 0.000 claims abstract description 13
- 235000002360 beta-cryptoxanthin Nutrition 0.000 claims abstract description 13
- 235000019244 cryptoxanthin Nutrition 0.000 claims abstract description 13
- 241000790234 Sphingomonas elodea Species 0.000 claims abstract description 4
- 239000001963 growth medium Substances 0.000 claims description 23
- 238000000034 method Methods 0.000 claims description 15
- 238000011218 seed culture Methods 0.000 claims description 9
- 239000002054 inoculum Substances 0.000 claims description 4
- 238000000746 purification Methods 0.000 abstract description 15
- 238000004519 manufacturing process Methods 0.000 abstract description 9
- 239000006227 byproduct Substances 0.000 abstract description 6
- 230000002503 metabolic effect Effects 0.000 abstract description 6
- 238000004321 preservation Methods 0.000 abstract description 4
- 239000000758 substrate Substances 0.000 abstract description 4
- 150000004676 glycans Chemical class 0.000 description 16
- 229920001282 polysaccharide Polymers 0.000 description 16
- 239000005017 polysaccharide Substances 0.000 description 16
- 241000192656 Nostoc Species 0.000 description 9
- 239000000843 powder Substances 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 8
- 238000002703 mutagenesis Methods 0.000 description 8
- 231100000350 mutagenesis Toxicity 0.000 description 8
- 238000012216 screening Methods 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
- PLUBXMRUUVWRLT-UHFFFAOYSA-N Ethyl methanesulfonate Chemical compound CCOS(C)(=O)=O PLUBXMRUUVWRLT-UHFFFAOYSA-N 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 5
- 241000736131 Sphingomonas Species 0.000 description 5
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 5
- 229960000723 ampicillin Drugs 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 238000009630 liquid culture Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical group O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 235000013373 food additive Nutrition 0.000 description 4
- 239000002778 food additive Substances 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000012065 filter cake Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 3
- 230000001376 precipitating effect Effects 0.000 description 3
- 238000003825 pressing Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- WHBMMWSBFZVSSR-UHFFFAOYSA-N 3-hydroxybutyric acid Chemical compound CC(O)CC(O)=O WHBMMWSBFZVSSR-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 108091005658 Basic proteases Proteins 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 239000012880 LB liquid culture medium Substances 0.000 description 2
- 102000016943 Muramidase Human genes 0.000 description 2
- 108010014251 Muramidase Proteins 0.000 description 2
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 2
- 125000002252 acyl group Chemical group 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000000701 coagulant Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 238000005265 energy consumption Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 229960000274 lysozyme Drugs 0.000 description 2
- 239000004325 lysozyme Substances 0.000 description 2
- 235000010335 lysozyme Nutrition 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- XJCPMUIIBDVFDM-UHFFFAOYSA-M nile blue A Chemical compound [Cl-].C1=CC=C2C3=NC4=CC=C(N(CC)CC)C=C4[O+]=C3C=C(N)C2=C1 XJCPMUIIBDVFDM-UHFFFAOYSA-M 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000001052 yellow pigment Substances 0.000 description 2
- KEQXNNJHMWSZHK-UHFFFAOYSA-L 1,3,2,4$l^{2}-dioxathiaplumbetane 2,2-dioxide Chemical compound [Pb+2].[O-]S([O-])(=O)=O KEQXNNJHMWSZHK-UHFFFAOYSA-L 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 1
- 108010013639 Peptidoglycan Proteins 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- QLACRIKFZRFWRU-UHFFFAOYSA-N [4-oxo-4-(4-oxobutan-2-yloxy)butan-2-yl] 3-hydroxybutanoate Chemical compound CC(O)CC(=O)OC(C)CC(=O)OC(C)CC=O QLACRIKFZRFWRU-UHFFFAOYSA-N 0.000 description 1
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 description 1
- 229940100228 acetyl coenzyme a Drugs 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 235000021466 carotenoid Nutrition 0.000 description 1
- 150000001747 carotenoids Chemical class 0.000 description 1
- 239000000679 carrageenan Substances 0.000 description 1
- 235000010418 carrageenan Nutrition 0.000 description 1
- 229920001525 carrageenan Polymers 0.000 description 1
- 229940113118 carrageenan Drugs 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000003311 flocculating effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000003349 gelling agent Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000019710 soybean protein Nutrition 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 239000000230 xanthan gum Substances 0.000 description 1
- 229920001285 xanthan gum Polymers 0.000 description 1
- 235000010493 xanthan gum Nutrition 0.000 description 1
- 229940082509 xanthan gum Drugs 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种低杂高产结冷胶产生菌,为Sphingomonas elodea LG010,其保藏号为:CCTCC NO:2016143。本发明还提供了该低杂高产结冷胶产生菌的用途:生产不含念珠藻黄素和聚‑β‑羟基丁酸的结冷胶。本发明从源头去除与结冷胶竞争底物并影响提纯的代谢副产物,并有效改善发酵效率、简化提纯步骤,降低生产成本。
Description
技术领域
本发明涉及低杂高产结冷胶产生菌及其应用。
背景技术
微生物多糖作为发酵工业产品,不仅性能优良且具备许多其它同类多糖所欠缺的特殊功用,比动植物多糖应用更广泛,且其生产不受地理环境、气候、自然灾害等因素的影响,生产周期短,产量及质量稳定,性价比较高。在很大程度上能满足人们对天然无公害食品的需求。
结冷胶是一种新型的微生物多糖,于1978年被发现,是伊乐鞘氨醇单胞菌在有氧条件下发酵产生的线性阴离子杂多糖。先后在日本、美国、欧盟及我国获得批准作为食品添加剂应用于各类食品中。其理化性质及凝胶性能优越,逐步作为新型乳化剂、悬浮剂、增稠剂、稳定剂、凝胶剂、缓释剂、成膜材料等日益应用于食品、医药、化工等领域,甚至已逐步取代卡拉胶、琼脂等同类食品添加剂,且市场需求量在逐年增加,其年增长率在30%以上。由于其生产所用的原料(如蔗糖)价格低廉且易得,而自身的市场价格却很高,因此有极高的商业利润和市场前景。
虽然国内市场的需求量增长很快,结冷胶在市场上占的份额越来越大,但是结冷胶行业还存在以下困境。
首先,虽然具更优秀的理化性质以及更广泛的应用价值,但结冷胶发酵时的糖胶转化率(每克发酵底物糖产出的多糖胶比例)只有40%-50%左右,显著低于另外一种微生物胞外多糖黄原胶(60-80%),因此造成结冷胶产量较低。其次,菌株发酵产结冷胶的同时能产生代谢副产物黄色类胡萝卜色素—念珠藻黄素,使发酵液变黄,给纯化带来不便。并且念珠藻黄素合成的前体乙酰辅酶A也是结冷胶合成的前体之一,因此,念珠藻黄素的合成也在一定程度上抑制了结冷胶的合成。第三,除了黄色色素外,结冷胶生产所需的高碳氮比培养基同样也利于主要由碳骨架组成的聚-β-羟基丁酸(PHB)的大量合成,其合成量可达总生物量的15-25%。PHB与结冷胶竞争有限的碳源,且降低结冷胶成品的透明度,影响应用,因此也需提纯过程中去除。由于发酵液的粘性高,结冷胶粘连在细胞表面,以粘性聚合物的形式构成网络结构,使得黄色念珠藻黄素和PHB等杂质的去除存在较大难度。在结冷胶的提取和纯化过程中,一般需要2倍发酵液体积的乙醇或异丙醇用于去除胶体中的黄色素等杂质并沉淀多糖,并且还需硅藻土板框压滤去除PHB。这种提取方法需要消耗大量的有机溶剂和硅藻土,增加纯化步骤和能耗,极大的降低了生产效率,增加了生产成本。
规模相对较小,结冷胶产率较低,能耗成本高、产物提取繁杂、产品澄清纯化困难等难题,造成生产成本相对较高,限制了结冷胶企业的进一步发展。因此,解决当前国内结冷胶生产企业困境的当务之急是提高结冷胶产量、简化提纯步骤从而降低生产成本。尽管当前许多基因工程手段提高产量降低代谢副产物的方法,但结冷胶作为一种食品添加剂,使用基因工程菌株进行生产,尤其是在食品安全问题极为突出的当下,容易引起消费者的顾虑。因此,采用非基因工程方法对菌株进行改造是解决这一问题的关键。
发明内容
本发明要解决的技术问题是提供一株不产代谢副产物代谢念珠藻黄素和聚-β-羟丁酸结冷胶产生菌——伊乐鞘氨醇单胞菌,并且此菌株发酵时结冷胶产量提高,此菌株用于结冷胶工业生产不仅可以增加产量而且可以简化纯化步骤,降低纯化成本。
为了解决上述技术问题,本发明提供一种低杂高产结冷胶产生菌,为Sphingomonas elodeaLG010,其保藏号为:CCTCC NO:2016143。
本发明还同时提供了上述低杂高产结冷胶产生菌的用途:生产结冷胶。
作为本发明的低杂高产结冷胶产生菌的用途的改进:生产不含念珠藻黄素和聚-β-羟基丁酸(PHB)的结冷胶。
作为本发明的低杂高产结冷胶产生菌的用途的进一步改进:结冷胶的制备方法为:
1)、种子培养:将斜面保存的菌株转接入种子培养基,于30℃,200rpm振荡培养24h,得为种子液;
2)发酵:将种子液以15%体积比的接种量接入发酵培养基中,于30℃,220rpm振荡培养60h;得含结冷胶的发酵液。
本发明是利用伊乐鞘氨醇单胞菌通过甲基磺酸乙酯(EMS)结合高效筛选方法经多轮突变得到不产念珠藻黄素和聚-β-羟丁酸,同时结冷胶产量提高的菌株。
本发明还涉及基于此菌株的优化的发酵培养基和纯化工艺。
方法一、发酵培养过程如下:
种子培养:将一接种环的斜面保存的菌株转接入50~100mL种子培养基(盛于250~500mL三角瓶),于30℃,200rpm振荡培养24h,即为种子液;
发酵:将种子液以15%体积比的接种量接入100mL发酵培养基中(盛于500mL三角瓶),于30℃,220rpm振荡培养60h;得含结冷胶的发酵液。
所涉及的培养基组成:
种子培养基:蔗糖10g/L;酵母膏3g/L;蛋白胨3g/L;磷酸二氢钾0.6g/L;磷酸氢二钾0.6g/L;硫酸镁0.6g/L;溶剂为蒸馏水;pH7.2;
发酵培养基:蔗糖40g/L;酵母粉0.45g/L;大豆蛋白粉5.5g/L;磷酸二氢钾0.8g/L;磷酸氢二钾1.2g/L;硫酸镁0.6g/L;硫酸亚铁0.002g/L硫酸锰0.00005g/L;硫酸锌0.00006g/L;氯化钴0.00005g/L;溶剂为普通自来水;pH7.2。
方法二、结冷胶的纯化工艺如下:
将上述含结冷胶的发酵液调pH值为6.0,升温至50℃~70℃(优选60℃)保温30min~2h(优选1h)后,降温至40℃~50℃,调pH为7.0,加入溶菌酶和碱性蛋白酶保温1h~3h(优选2h)去除蛋白质;去除蛋白质后的发酵液加入占发酵液体积的5%的CaCl2溶液(浓度通常为20%,w/v)及发酵液体积20%的乙醇的进行絮凝沉淀,搅拌,板框压滤、所得滤饼干燥(90℃),制得不含念珠藻黄素及PHB的结冷胶(高酰基结冷胶)。
出胶率%(多糖产率%,1g/100ml)=每百毫升发酵液制备获得的结冷胶干重(g)。
本发明的有益效果主要体现在:提供一种不产念珠藻黄素及PHB等代谢副产物且高产结冷胶的伊乐鞘氨醇单胞菌;从源头去除与结冷胶竞争底物并影响提纯的代谢副产物,并有效改善发酵效率、简化提纯步骤,降低生产成本。此菌株发酵时结冷胶产量提高,此菌株用于结冷胶工业生产不仅可以增加产量而且可以简化纯化步骤,降低纯化成本。
具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
实施例1、不含念珠藻黄素的结冷胶产生菌的筛选(EMS诱变及高效筛选):
1)、第一轮诱变处理:
伊乐鞘氨醇单胞菌ATCC 31461YM培养基保藏的斜面转接LB液体培养基,30℃培养16h,然后按照LB液体培养基1%的体积比加入EMS(甲基磺酸乙酯),避光室温放置1h,再转接新鲜YM液体培养基,30℃培养6h。
YM液体培养基的组成:蛋白胨5g/L,麦芽浸粉3g/L,葡萄糖10g/L,酵母粉3g/L,溶剂为水,pH7.0。
YM固体培养基的组成:蛋白胨5g/L,麦芽浸粉3g/L,葡萄糖10g/L,酵母粉3g/L,15g/L琼脂,溶剂为水,pH7.0。
2)、第一轮诱变处理所得的样品经梯度稀释涂布于YM平板(YM固体培养基)中,30℃培养72h,肉眼观察筛选的白色单菌落,经YM平板划线分离纯化后单菌落继续为白色的则为不产念珠藻黄素的菌株;
3)、收集所有不产念珠藻黄素的菌株,发酵(按照上述方法一)、测定(按照上述方法二)结冷胶的多糖产率。
实施例2、不含念珠藻黄素和PHB的结冷胶产生菌的筛选:
1)、第二轮诱变处理:
以实施例1所得的结冷胶多糖产率最高且不产念珠藻黄素的菌株为第二轮诱变的出发菌株,转接YM液体培养基,30℃培养16h,然后按照YM液体培养基1%的体积比加入EMS,避光室温放置1h,再转接新鲜YM培养基,30℃培养6h。
2)、第二轮诱变处理所得的样品经梯度稀释涂布于加入荧光显色剂5μg/mL尼罗蓝尼罗蓝的YM固体培养基中,30℃培养72h。其中尼罗蓝可以与PHB结合而使含PHB的菌株在紫外下产生荧光,而无荧光或者荧光极弱的菌株则为不产PHB的缺陷型菌株。在此筛选平板中紫外下无荧光或者荧光极弱的菌落则是念珠藻黄素和PHB均不产的菌株;
3)、收集所有念珠藻黄素和PHB均不产的菌株,发酵(按照上述方法一)、测定(按照上述方法二)结冷胶的多糖产率。
实施例3、不含念珠藻黄素和PHB且高产结冷胶的产生菌的筛选:
1)、第三轮诱变处理:
以实施例2所得的多糖产率最高且不产念珠藻黄素和PHB的菌株为第三轮诱变的出发菌株,转接YM液体培养基,30℃培养16h,然后按照YM液体培养基1%的体积比加入EMS,避光室温放置1h,转接新鲜YM培养基,30℃培养6h;
2)、第三轮诱变处理所得的样品经梯度稀释涂布于添加100μg/mL氨苄青霉素的YM高产筛选平板中,30℃培养5天。
添加100μg/mL氨苄青霉素的YM高产筛选平板的配方:
蛋白胨5g/L,麦芽浸粉3g/L,葡萄糖10g/L,酵母粉3g/L,结冷胶15g/L,氨苄青霉素100μg/mL,溶剂为水,pH7.0。
3)、挑选筛选平板中长出的具有氨苄青霉素抗性,菌落生成处培养基不下沉的单菌落,这些单菌落为潜在的高产菌株。
说明:结冷胶的生物合成与肽聚糖的合成途径有共通之处,而氨苄青霉素耐药性菌株可能具有更强的结冷胶合成性能。另外结冷胶产生菌能分泌结冷胶降解酶降解结冷胶,当以结冷胶为凝固剂的平板筛选时,普通菌株因凝固剂结冷胶被降解而造成菌落生长处的培养基内凹,而高产菌株可能具有更强的结冷胶产生性能,能补充被降解的结冷胶而使培养基维持原样,通过这种方法筛选到了结冷胶高产菌株。本发明结合上述高效筛选方法,对筛选培养基进行优化。
4)、收集所有潜在的高产菌株,发酵测定结冷胶多糖的产量。从而筛选出一株不产念珠藻黄素色素和PHB的高产菌株,发酵(按照上述方法一)、测定(按照上述方法二)结冷胶的多糖产率。
该高产菌株的保藏信息如下:
保藏名称为Sphingomonas elodea LG010,保藏单位:中国典型培养物保藏中心,保藏地址:中国武汉武汉大学,保藏编号:CCTCC NO:2016143,保藏时间2016年03月23日。
实施例4、鞘氨醇单胞菌(CCTCC NO:M 2016143)的发酵工艺及于野生型出发菌株结冷胶出胶率的比较:
将Sphingomonas elodea LG010(CCTCC NO:2016143)和野生型菌株ATCC 31461分别进行如下步骤:
1)、将菌株接种于YM培养基斜面上活化,30℃培养72h;
2)、种子培养:
将一接种环的斜面种子接入盛于250mL三角瓶的50mL种子培养基中,于30℃,200rpm振荡培养24h,即为种子液;
3)、发酵:
将种子液以15%体积比的接种量接入100mL发酵培养基中(盛于500mL三角瓶),于30℃,220rpm振荡培养60h;得含结冷胶的发酵液。
出胶率%(多糖产率%,1g/100ml)=每百毫升发酵液制备获得的结冷胶干重(g)。
CCTCC NO:M 2016143和野生型菌株ATCC 31461的出胶率见表1。
表1
菌株 | 出胶率% | 纯化后念珠藻黄素含量% | 纯化后PHB含量% |
ATCC 31461 | 1.12 | 0.062 | 5.75 |
CCTCC NO:M 2016143 | 1.67 | 无 | 无 |
注:CCTCC NO:M 2016143的转化率(每克发酵底物糖产出的多糖胶比例)为41.75%。
实施例5:高酰基结冷胶的制备
1)、发酵液预处理:
发酵液100L,用10%(v/v)的HCl调pH6,升温至60℃,保温1h;
2)、蛋白杂质去除:
步骤1)所得物降温至40℃时,10%(w/v)NaOH调pH7.0,加入50g的溶菌酶(20万U/g,庞博生物)及100g的碱性蛋白酶(2万U/g,庞博生物),保温2h;从而去除蛋白杂质;
3)、絮凝沉淀,分离:
将在步骤2)所得的蛋白去杂处理后的发酵液中加入5L浓度为20%(w/v)的CaCl2溶液和20L乙醇,搅拌1h后,板框压滤;
4)、干燥、粉碎:
将压滤所得的滤饼90℃干燥1h后粉碎至能过80目的筛,制得高酰基结冷胶成品(含水率≤5%),为乳白色粉末,测定成品高酰基结冷胶中念珠藻黄素及PHB的含量,具体含量见表1。
注:通过高效液相测定确证菌株不产念珠藻黄素色素,通过次氯酸处理菌体后离心加入浓硫酸反应后经分光光度计测定确认菌株不产PHB。
对比试验:将如下表2所述的菌株按照上述实施例4和实施例5所述方法进行检测,所得结果与CCTCC NO:M 2016143的对比如下表2。
表2
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
Claims (3)
1.低杂高产结冷胶产生菌,其特征在于:为Sphingomonas elodea LG010,其保藏号为:CCTCC NO:2016143。
2.如权利要求1所述的低杂高产结冷胶产生菌的用途,其特征在于:生产不含念珠藻黄素和聚-β-羟基丁酸的结冷胶。
3.根据权利要求2所述的低杂高产结冷胶产生菌的用途,其特征在于结冷胶的制备方法为:
1)、种子培养:将斜面保存的菌株转接入种子培养基,于30℃,200rpm振荡培养24h,得为种子液;
2)发酵:将种子液以15%体积比的接种量接入发酵培养基中,于30℃,220rpm振荡培养60h;得含结冷胶的发酵液。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910378308.2A CN109971688B (zh) | 2019-05-08 | 2019-05-08 | 低杂高产结冷胶产生菌及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910378308.2A CN109971688B (zh) | 2019-05-08 | 2019-05-08 | 低杂高产结冷胶产生菌及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN109971688A CN109971688A (zh) | 2019-07-05 |
CN109971688B true CN109971688B (zh) | 2020-12-01 |
Family
ID=67073154
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910378308.2A Active CN109971688B (zh) | 2019-05-08 | 2019-05-08 | 低杂高产结冷胶产生菌及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN109971688B (zh) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106929458A (zh) * | 2009-07-31 | 2017-07-07 | Cp凯尔科美国公司 | 产生产量大大提高的phb缺陷型鞘氨糖的鞘氨醇单胞菌菌株 |
-
2019
- 2019-05-08 CN CN201910378308.2A patent/CN109971688B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106929458A (zh) * | 2009-07-31 | 2017-07-07 | Cp凯尔科美国公司 | 产生产量大大提高的phb缺陷型鞘氨糖的鞘氨醇单胞菌菌株 |
Non-Patent Citations (2)
Title |
---|
A carotenoid- and poly-β-hydroxybutyrate-free mutant strain of Sphingomonas elodea ATCC 31461 for the commercial production of gellan;Li A.等;《MSPHERE》;20191031;第4卷(第5期);文献号:e00668-19 * |
高产低杂高品质结冷胶生产菌株的构建;殷瑞 等;《2017年浙江省微生物学会青年论坛——微生物与健康专题研讨会会议资料》;20171231;第39页 * |
Also Published As
Publication number | Publication date |
---|---|
CN109971688A (zh) | 2019-07-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8685698B2 (en) | Yellow pigments generation deficient Sphingomonas strain and application thereof in gellan gum production | |
CN106554931B (zh) | 一株拜氏羧菌及其应用 | |
CN114214251B (zh) | 一种生产d-阿洛酮糖用枯草芽孢杆菌及其培养方法和应用 | |
CN101993841B (zh) | 一种黄单胞菌及用其制备黄原胶的方法 | |
CN108277184A (zh) | 产褐藻胶裂解酶的芽孢杆菌及其制备方法和应用 | |
CN108546660B (zh) | 甲壳素脱乙酰基酶高产菌株及其应用 | |
CN108753642B (zh) | 一株产褐藻胶裂解酶菌株约氏黄杆菌 | |
CN111826308B (zh) | 一株海洋沉积物来源的几丁质高效降解菌及其应用 | |
CN105713851B (zh) | 一株拜氏梭菌及其应用 | |
CN110129225A (zh) | γ~聚谷氨酸产生菌及选育、制备γ~聚谷氨酸的方法 | |
CN111019995B (zh) | 一种以丁香酚为底物发酵生成香兰素的方法 | |
CN109971688B (zh) | 低杂高产结冷胶产生菌及其应用 | |
WO2021056683A1 (zh) | 一株产脂肪酶菌株及其应用 | |
CN111471603A (zh) | 一种产β-葡萄糖苷酶的生香季也蒙毕赤酵母菌与应用 | |
CN101851591A (zh) | 一种纤维堆囊菌生产埃坡霉素b的发酵方法及发酵培养基 | |
CN105483171A (zh) | 一种提高辅酶q10工业产量的生产方法 | |
CN115369138A (zh) | 一种利用花生枯饼固态发酵制备鼠李糖脂的方法 | |
WO2008062558A1 (fr) | Levure thermo-tolérante productrice d'éthanol et procédé de production d'éthanol avec cette levure | |
CN112746026B (zh) | 一株维斯假丝酵母及其应用 | |
JP2004344084A (ja) | アルコール発酵性酵母 | |
Osumah et al. | Production of yeast using acid-hydrolyzed cassava and poultry manure extract | |
CN110283733A (zh) | 土星轮头酵母zjph1807及其应用 | |
CN109868245A (zh) | 一种枯草芽孢杆菌bsln-08及其培养方法和应用 | |
CN116286513B (zh) | 一株希氏乳杆菌FR-1012及其工业化生产γ-氨基丁酸的方法 | |
CN116179402B (zh) | 一株类胡萝卜素合成菌株及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |