CN105886573A - Method for preparing trehalose by continuous exoenzyme biological process - Google Patents
Method for preparing trehalose by continuous exoenzyme biological process Download PDFInfo
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- CN105886573A CN105886573A CN201610323539.XA CN201610323539A CN105886573A CN 105886573 A CN105886573 A CN 105886573A CN 201610323539 A CN201610323539 A CN 201610323539A CN 105886573 A CN105886573 A CN 105886573A
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- trehalose
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- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 title claims abstract description 136
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 title claims abstract description 108
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 title claims abstract description 108
- 238000000034 method Methods 0.000 title claims abstract description 43
- 230000031018 biological processes and functions Effects 0.000 title abstract 2
- 102000004190 Enzymes Human genes 0.000 claims abstract description 144
- 108090000790 Enzymes Proteins 0.000 claims abstract description 144
- 210000004027 cell Anatomy 0.000 claims abstract description 45
- 108010045348 trehalose synthase Proteins 0.000 claims abstract description 37
- 241000894006 Bacteria Species 0.000 claims abstract description 26
- 238000002360 preparation method Methods 0.000 claims abstract description 15
- 230000028023 exocytosis Effects 0.000 claims abstract description 11
- 238000002425 crystallisation Methods 0.000 claims abstract description 8
- 230000008025 crystallization Effects 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims description 96
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 58
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 58
- 238000000855 fermentation Methods 0.000 claims description 51
- 230000004151 fermentation Effects 0.000 claims description 51
- 229920001542 oligosaccharide Polymers 0.000 claims description 48
- 208000031888 Mycoses Diseases 0.000 claims description 46
- -1 Glycosyl trehalose Chemical compound 0.000 claims description 41
- 238000007445 Chromatographic isolation Methods 0.000 claims description 30
- 239000000758 substrate Substances 0.000 claims description 29
- 229920002472 Starch Polymers 0.000 claims description 28
- 235000019698 starch Nutrition 0.000 claims description 28
- 239000008107 starch Substances 0.000 claims description 28
- 244000063299 Bacillus subtilis Species 0.000 claims description 25
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 25
- 230000003301 hydrolyzing effect Effects 0.000 claims description 25
- 230000015572 biosynthetic process Effects 0.000 claims description 22
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 20
- 238000006243 chemical reaction Methods 0.000 claims description 20
- 238000003786 synthesis reaction Methods 0.000 claims description 20
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 102000003960 Ligases Human genes 0.000 claims description 16
- 108090000364 Ligases Proteins 0.000 claims description 16
- 239000012141 concentrate Substances 0.000 claims description 16
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 15
- 241000195474 Sargassum Species 0.000 claims description 15
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 14
- 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 claims description 13
- 239000008103 glucose Substances 0.000 claims description 13
- 230000003248 secreting effect Effects 0.000 claims description 13
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 12
- 238000010612 desalination reaction Methods 0.000 claims description 12
- 230000000694 effects Effects 0.000 claims description 12
- 238000001704 evaporation Methods 0.000 claims description 12
- 230000008020 evaporation Effects 0.000 claims description 12
- 238000004064 recycling Methods 0.000 claims description 12
- 239000011347 resin Substances 0.000 claims description 12
- 229920005989 resin Polymers 0.000 claims description 12
- 230000006698 induction Effects 0.000 claims description 11
- 229920001282 polysaccharide Polymers 0.000 claims description 11
- 239000005017 polysaccharide Substances 0.000 claims description 11
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 10
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 10
- 238000001323 two-dimensional chromatography Methods 0.000 claims description 10
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 9
- 238000011953 bioanalysis Methods 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000000376 reactant Substances 0.000 claims description 8
- 241000195493 Cryptophyta Species 0.000 claims description 7
- 239000001888 Peptone Substances 0.000 claims description 7
- 108010080698 Peptones Proteins 0.000 claims description 7
- 229930006000 Sucrose Natural products 0.000 claims description 7
- 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 claims description 7
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 7
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 7
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 7
- 150000004676 glycans Chemical class 0.000 claims description 7
- 238000005342 ion exchange Methods 0.000 claims description 7
- 238000002386 leaching Methods 0.000 claims description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 7
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 7
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 7
- 235000019319 peptone Nutrition 0.000 claims description 7
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 7
- 239000005720 sucrose Substances 0.000 claims description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 6
- 241000235061 Pichia sp. Species 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 235000013619 trace mineral Nutrition 0.000 claims description 6
- 239000011573 trace mineral Substances 0.000 claims description 6
- 238000010792 warming Methods 0.000 claims description 6
- 229910052564 epsomite Inorganic materials 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- 229920000856 Amylose Polymers 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 4
- 239000000919 ceramic Substances 0.000 claims description 4
- 235000016709 nutrition Nutrition 0.000 claims description 4
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 3
- 239000007836 KH2PO4 Substances 0.000 claims description 3
- 229910004619 Na2MoO4 Inorganic materials 0.000 claims description 3
- 101150086142 PMT4 gene Proteins 0.000 claims description 3
- 229910052925 anhydrite Inorganic materials 0.000 claims description 3
- 229960004543 anhydrous citric acid Drugs 0.000 claims description 3
- 229960002685 biotin Drugs 0.000 claims description 3
- 235000020958 biotin Nutrition 0.000 claims description 3
- 239000011616 biotin Substances 0.000 claims description 3
- 229910052927 chalcanthite Inorganic materials 0.000 claims description 3
- 229910052602 gypsum Inorganic materials 0.000 claims description 3
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 3
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 3
- 229910052603 melanterite Inorganic materials 0.000 claims description 3
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 3
- 239000011684 sodium molybdate Substances 0.000 claims description 3
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 3
- 229910001868 water Inorganic materials 0.000 claims description 3
- 239000011592 zinc chloride Substances 0.000 claims description 3
- 241000235648 Pichia Species 0.000 claims description 2
- 230000003321 amplification Effects 0.000 claims description 2
- 230000008859 change Effects 0.000 claims description 2
- 230000001939 inductive effect Effects 0.000 claims description 2
- 238000002372 labelling Methods 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- 239000013049 sediment Substances 0.000 claims 2
- 241000193755 Bacillus cereus Species 0.000 claims 1
- 238000001228 spectrum Methods 0.000 claims 1
- 210000002275 spiral lamina Anatomy 0.000 claims 1
- 238000000926 separation method Methods 0.000 abstract description 15
- 238000004519 manufacturing process Methods 0.000 abstract description 14
- 108010093096 Immobilized Enzymes Proteins 0.000 abstract description 4
- 238000000746 purification Methods 0.000 abstract description 4
- 238000013375 chromatographic separation Methods 0.000 abstract description 3
- 238000011097 chromatography purification Methods 0.000 abstract 1
- 230000009466 transformation Effects 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 20
- 235000013305 food Nutrition 0.000 description 9
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 8
- 238000010276 construction Methods 0.000 description 8
- 230000008569 process Effects 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 238000005374 membrane filtration Methods 0.000 description 6
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000005189 flocculation Methods 0.000 description 5
- 230000016615 flocculation Effects 0.000 description 5
- 238000005194 fractionation Methods 0.000 description 5
- 150000002482 oligosaccharides Chemical class 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000004088 simulation Methods 0.000 description 5
- 238000001035 drying Methods 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 229920001353 Dextrin Polymers 0.000 description 3
- 239000004375 Dextrin Substances 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 235000019425 dextrin Nutrition 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 240000005708 Eugenia stipitata Species 0.000 description 2
- 235000006149 Eugenia stipitata Nutrition 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- 230000008030 elimination Effects 0.000 description 2
- 238000003379 elimination reaction Methods 0.000 description 2
- 235000013373 food additive Nutrition 0.000 description 2
- 239000002778 food additive Substances 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000009434 installation Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
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- 239000006228 supernatant Substances 0.000 description 2
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 241000143060 Americamysis bahia Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241001474374 Blennius Species 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 108010001394 Disaccharidases Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 241001267425 Meiothermus rosaceus Species 0.000 description 1
- 206010054949 Metaplasia Diseases 0.000 description 1
- 244000131316 Panax pseudoginseng Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 241000203722 Pimelobacter Species 0.000 description 1
- 241000589776 Pseudomonas putida Species 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 108010016634 Seed Storage Proteins Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 241000203780 Thermobifida fusca Species 0.000 description 1
- 241000589596 Thermus Species 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000010836 blood and blood product Substances 0.000 description 1
- 229940125691 blood product Drugs 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 235000021474 generally recognized As safe (food) Nutrition 0.000 description 1
- 235000021473 generally recognized as safe (food ingredients) Nutrition 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 230000015689 metaplastic ossification Effects 0.000 description 1
- 238000011169 microbiological contamination Methods 0.000 description 1
- YKQOSKADJPQZHB-RGYSVOEGSA-N n-[(2s)-4-amino-1-[[(2s,3r)-1-[[(2s)-4-amino-1-oxo-1-[[(6r,9s,12r,15r,18s,21s)-6,9,18-tris(2-aminoethyl)-3-[(1r)-1-hydroxyethyl]-12,15-bis(2-methylpropyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptazacyclotricos-21-yl]amino]butan-2-yl]amino]-3-hydr Chemical compound CCC(C)CCCC(=O)N[C@@H](CCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)C([C@@H](C)O)NC(=O)[C@@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O YKQOSKADJPQZHB-RGYSVOEGSA-N 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 229940124272 protein stabilizer Drugs 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 150000003625 trehaloses Chemical class 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- 230000003462 zymogenic effect Effects 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- C12P19/12—Disaccharides
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a method for preparing trehalose by a continuous exoenzyme biological process. The complete cell immobilized or secretive trehalose synthase gene engineering bacterium is utilized to continuously and efficiently produce trehalose and perform continuous chromatographic separation, purification and crystallization so as to prepare the trehalose. By establishing the high-continuity enzyme production-trehalose preparation production technique, the technique implements transformation by using the complete cell surface display or exocytosis trehalose synthase, or malt-oligosaccharide-base trehalose synthetase-malt-oligosaccharide-base trehalose hydrolase fused enzyme, thereby avoiding the cost on cell crushing and separation purification. The enzyme solution or immobilized enzyme is convenient to acquire and simple to operate.
Description
Technical field
The present invention relates to a kind of method that continuous exoenzyme bioanalysis prepares trehalose, utilize full cell fixation particularly to one
Or secretion trehalose synthase gene engineering bacteria continuous high-efficient produces trehalose continuous chromatography separation, trehalose is prepared in purification, crystallization
Method, belong to fermentation engineering field.
Background technology
Trehalose is with α by two glucose molecules, α-1, the irreducibility disaccharidase that 1 glycosidic bond is constituted, be widely present in microorganism,
In the organisms such as seaweeds, mushroom class, beans, shrimps.Owing to there is non-reducing end in trehalose, extreme environment good stability,
Being widely used in the industries such as food, medicine, cosmetics, agricultural, ectogenic trehalose is big to organism and biology equally
Molecule has nonspecific protective effect.Trehalose has the biological characteristics being different from other carbohydrate of uniqueness, as protected
The components such as probationer nurse object cell protein, fat, saccharide, nucleic acid are without prejudice, improve the arid of species, high temperature, dehydration,
Freezing, hyperosmosis and toxicant tolerance etc..In food service industry, can be used as food additive and sweeting agent, keep food
Product shelf-life；The plasma protein stabilizer as the bioactive substance such as blood products, vaccine is substituted in pharmaceuticals industry；?
As super sun-proof Moisture factor in cosmetics, for skin protection at product and lip pomade etc.；For Seed storage in crop breeding.
2000, FAO (Food and Agriculture Organization of the United Nation) and food additive joint committee of World Health Organization (WHO) (JECFA) confirmed the every of trehalose
Intake is allowed to be not required to limit day.In the same year, food and drug administration (FDA) authorizes trehalose GRAS and (generally acknowledges peace
Status entirely), and ratify to enter U.S. food field.In March, 2005, ministry of Health of China approval trehalose is new resource food.
Traditional trehalose production method is to extract in yeast body, and yeast grows in the presence of a harsh environment, internal can accumulate a large amount of
Trehalose, but this production method, separation and Extraction process is complicated, yields poorly, and cost is high.Woods primaryization institute in 1994
It is found that malt oligosaccharide based mycose synthetase (MTSase) and malto-oligosaccharides base hydrolysis of trehalose enzyme (MTHase), double
Enzyme cooperates with glucidtemns and produces trehalose.Nineteen ninety-five, Japan woods primaryization institute from Pimelobacter,
Isolated and purified trehalose synthase in tri-kinds of microorganism belonging to genus of Psceuclomonus and Thermus, it can convert maltose and is
Trehalose.Both the above enzyme process is prepared the method for trehalose and is constantly studied and improve, and has become trehalose industry metaplasia
The main method produced.In order to accelerate the industrialization process of trehalose, improve the production efficiency of trehalose, reduce production cost, real
The domestic extensive industrialization process of existing trehalose.China the most in succession screens and obtains product malt oligosaccharide based mycose synthetase
(MTSase) and malto-oligosaccharides base hydrolysis of trehalose enzyme (MTHase) and produce the microbial strains of trehalose synthase, and right
Part enzyme achieves heterogenous expression, establishes and is prepared trehalose by Permeabilized cells, product enzyme recombinant bacterium and utilized chromatographic isolation pure
Change Technology and the method for trehalose.
Chinese patent literature CN103205475A (application number 201310128939.1) discloses a kind of novel Fructus Hordei Germinatus oligose Ji Hai
Algae sugar synzyme and the application in trehalose produces of the malt oligosaccharide based mycose hydrolytic enzyme.Used by this patent, double enzymes are respectively at large intestine
Obtaining heterogenous expression in bacillus, after IPTG or lactose-induced, smudge cells, centrifugal supernatant of collecting obtains crude enzyme liquid.Will
Double enzymes are respectively according to adding 100-300U malt oligosaccharide based mycose synthetase, 300-900U Fructus Hordei Germinatus widow in every liter of glucidtemns
Glycosyl hydrolysis of trehalose enzyme carries out conversion reaction, reacts 25-35h, prepare sea under conditions of temperature 50-65 DEG C, pH5.3-5.5
Algae sugar, the conversion ratio of trehalose is 76.5-85.4%.
Chinese patent literature CN103468624A (application number 201310296116.X) discloses a kind of malt oligosaccharide based mycose
Synzyme and malt oligosaccharide based mycose hydrolytic enzyme combine the method preparing trehalose.Used by the method, double enzymes are simultaneously escherichia coli
Carrying out heterogenous expression, after collecting thalline, pair enzyme enzyme liquid that simultaneously obtains broken, centrifugal is for converted starch hydrolyzed solution, and conversion ratio reaches
85%.
Trehalose synthase disclosed in Chinese patent literature CN1563372 (application number 200410013007.3) likes hot tearing selected from brown
Spore bacterium (Thermobifida fusca DSM 43792), the optimum temperature of this trehalose synthase is 20-30 DEG C, and optimum pH is
6.5-7.0.This trehalose synthase gene obtains heterogenous expression in escherichia coli, after IPTG induction 17h, and centrifuge washing,
With broken liquid smudge cells, centrifugal supernatant of collecting obtains trehalose synthase, 25 DEG C of substrate maltose slurries converting 30%, converts
Rate reaches 60%.
Chinese patent literature CN1648242 (application number 200410093874.2) disclose a kind of toluene using 1%-5% or
The method that the microbial cell that meiothermus rosaceus is belonged to by chloroform carries out permeabilized treatment.This patented method avoid the broken of cell and by
Level purification procedures.Cell is after permeabilized treatment, and overall structure is complete, and cell wall and cell membrane penetration sexually revise, the end
Thing can enter cell.Permeabilized cells makes the pH value toleration of trehalose synthase and heat stability be improved.Chinese patent
Document CN101831474A (application number 201010137964.2) discloses a kind of pseudomonasputida Permeabilized cells and converts wheat
The method of trehalose prepared by bud sugar.The method carries out Cell Permeabilization process with the beta-mercaptoethanol of final concentration 0.1%-1%.China
Patent documentation CN103146779A (application number 201310112536.8) discloses a kind of process through surfactant and produces trehalose
Synthase recombination bacillus coli prepares Permeabilized cells and converts the method that trehalose prepared by maltose, and trehalose production rate reaches
55%-60%.
Chinese patent literature CN103266152A (application number 201310182033.8) discloses a kind of immobilization trehalose synthesis
Enzyme produces the method for trehalose.The method is the strain culturing producing trehalose synthase to be obtained after thalline, through centrifugation,
The broken crude enzyme liquid that obtains, after addition ammonium sulfate precipitation remove impurity, it is thus achieved that trehalose synthetase enzyme liquid, then utilizes glutaraldehyde and shell to gather
Sugar carries out cross-linking embedding and prepares immobilized enzyme, uses immobilized enzyme conversion to prepare trehalose.
Chinese patent literature CN105039455A (application number 201510036540.X) discloses a kind of scale and produces sea continuously
The method of algae sugar.PCS cell substrates is utilized to be fixed on force ventilation shaft, the recombination bacillus coli that inoculation to construct is good,
After cultivation, cell is fixed on PCS cell substrates, then adds 1.0g/L-1.5g/L colistine sulfate and changes fixation cell
Born of the same parents' permeability, and the maltose nutritional solution of 25%, produce trehalose through Cell of Anmrobe preparation.
Chinese patent literature CN104418919A (application number 201310378497.6) discloses the production method of a kind of trehalose,
The method utilizes chromatographic separation technology, and the mixed sugar liquid after prepared by conversion maltose or starch trehalose carries out isolated and purified.Will
Mixed liquor hydrogenates at high temperature under high pressure, and maltose is hydrogenated and is reduced to maltose alcohol, is separated with trehalose by maltose alcohol.This point
From technology by two set chromatographic separation devices, finally realize the separation of polysaccharide, glucose, maltose and trehalose.Chinese patent
Document CN104017027A (application number 201410259263.4) discloses one and utilizes sequential simulated mobile chromatographic isolation sea
Algae sugar and the method for glucose.
Up to the present, two enzymes method route has been realized in trehalose industrialized production, and single enzyme process is because of its conversion ratio relatively two enzymes method
It is lower slightly, so its industrialization produces still is continuing exploration.As it has been described above, the enzyme work produced by fermentation for original zymogenic bacteria kind
Power is relatively low, and fermentation period is the longest, and energy consumption is big so that fermentation costs is the highest, the limiting factor that production efficiency is low, by building
Recombination bacillus coli can strengthen double enzyme and the expression of single enzyme, but it is intracellular expression that escherichia coli produce enzyme, needs after broken
Obtaining, and resolvase has shortcomings such as can not reclaiming, utilization rate is low, it is isolated and purified to be difficult to, use cost is high, this not only increases
Add broken cost, and there is food safety limiting factor.Enzyme Permeabilized cells is produced in immobilization can reduce a large amount of broken of cell
Broken, improve the stability of enzyme, eliminate and extract and the process of purifying enzyme, but, the interpolation of chemical reagent adds preparation cost,
In industrialization produces continuously, utilization rate of equipment and installations is low, and the production cycle is long.
For production and the separation problem of current trehalose, inventing a kind of safe and efficient trehalose production process route is very
There is practical significance.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, it is provided that a kind of method that continuous exoenzyme bioanalysis prepares trehalose.The method is
Industrial can large-scale popularization application scale, continuous prodution trehalose lay a good foundation, it is simple that the method has technique, efficiency
Height, the feature that the suitability is strong.
Technical scheme is as follows:
A kind of method that continuous exoenzyme bioanalysis prepares trehalose, comprises the steps:
(1) by few to the product trehalose synthase with maltose or soluble starch as substrate or the Fructus Hordei Germinatus of exocytosis or surface display
Glycosyl trehalose synthase-malt oligosaccharide based mycose hydrolytic enzyme merges the bacterial strain of enzyme and carries out activation culture, then through amplification culture,
Prepared cell concentration is the bacterium solution of 50～80g/L；
(2) bacterium solution that step (1) prepares is inoculated in fermentation medium and carries out fermentation culture, the exocytosis table of inducible enzyme
Reach or spore surface is shown or full cell surface display, then filter, prepare full cell surface display thalline, surface display spore
Or crude enzyme liquid, by crude enzyme liquid after membrance concentration, prepare and concentrate enzyme liquid；
(3) by concentration enzyme liquid, full cell surface display thalline or surface display spore and concentration that step (2) prepares be 250～
The maltose solution of 350g/L reacts, and is 15～55 DEG C in temperature, under conditions of pH7.5～8.5, reacts 24h～36h,
Reactant liquor, through filtering, prepares sugar liquid；
Or, by concentration enzyme liquid, full cell surface display thalline or surface display spore and concentration that step (2) prepares be 150～
The soluble starch of 250g/L reacts, and is 15～55 DEG C in temperature, under conditions of pH5.0～6.0, reacts 24h～36h,
Reactant liquor, through filtering, prepares sugar liquid；
(4) sugar liquid that step (3) prepares is warming up to 75～80 DEG C, enzyme denaturing alive 0.4～0.6h, filters through kieselguhr, dimension
Hold temperature 70～75 DEG C carry out activated carbon decolorizing, be then cooled to 40～50 DEG C, again filter after, carry out ion exchange, system
Obtain desalination sugar liquid；
(5) the desalination sugar liquid that step (4) prepares being concentrated into sugar concentration is 50%～55% (mass percent), carries out one
Secondary chromatographic isolation, Polysaccharide removing and glucose, being then concentrated into sugar concentration is 50%～55% (mass percent), through secondary
Chromatographic isolation, prepares Sargassum sugar liquid and Fructus Hordei Germinatus sugar liquid, Fructus Hordei Germinatus sugar liquid recycling, the concentrated crystallization of Sargassum sugar liquid, prepares sea
Algae sugar.
According to currently preferred, in described step (1) with soluble Amylose be substrate trehalose synthesis enzyme as Fructus Hordei Germinatus
Oligosaccharide based mycose synthetase merges enzyme with malt oligosaccharide based mycose hydrolytic enzyme, and its expression strain is surface display Fructus Hordei Germinatus oligose base
The Pichi strain that trehalose synthetase merges enzyme with malt oligosaccharide based mycose hydrolytic enzyme (sees Chinese patent literature
Construction method disclosed in CN105039191A (application number 201510571619.2) is prepared) or secreting, expressing Fructus Hordei Germinatus widow
The bacillus subtilis that glycosyl trehalose synthetase merges enzyme with malt oligosaccharide based mycose hydrolytic enzyme (sees Chinese patent literature
Construction method disclosed in CN105039374A (application number 201510486544.8) is prepared)；With maltose as substrate
The enzyme of trehalose synthesis is trehalose synthase, and its expression strain is the bacillus subtilis (ginseng that spore surface shows trehalose synthase
See that the construction method disclosed in Chinese patent literature CN105132450A (application number 201510571328.3) is prepared) or
The bacillus subtilis of secreting, expressing trehalose synthase (sees Chinese patent literature CN105039381A (application number
201510431599.9 the construction method disclosed in) is prepared)；
According to currently preferred, in described step (2), when the Fructus Hordei Germinatus being substrate trehalose synthesis with soluble Amylose
Oligosaccharide based mycose synthetase and malt oligosaccharide based mycose hydrolytic enzyme fusion enzyme are when Pichi strain surface display, and step is such as
Under:
The bacterium solution by volume percentage ratio 8 step (1) prepared～the ratio of 12% are inoculated in fermentation medium, send out for 28～32 DEG C
After ferment cultivates 20～28h, use methanol induction 80～120h, make Pichia sp. start surface display malt oligosaccharide based mycose
Synzyme merges enzyme with malt oligosaccharide based mycose hydrolytic enzyme, and described methanol concentration in fermentation liquid controls 0.3～0.7g/L；
Described fermentation medium (RSM) every liter of component is as follows:
KH2PO442.9g, (NH4)2SO45g, CaSO40.6g, K2SO414.3g, anhydrous citric acid 1.92g,
MgSO4·7H2O 11.7g, glycerol 40mL, PTM4 trace element solution 2.0mL；
Above-mentioned every liter of component of PMT4 trace element solution is as follows:
CuSO4·5H2O 2.0g, NaI 0.08g, MnSO4·H2O 3.0g, Na2MoO4·2H2O 0.2g, H3BO30.02g,
CaSO4·2H2O 0.5g, CoCl20.5g, ZnCl27g, FeSO4·7H2O 22g, biotin 0.2g, mass concentration is 98%
H2SO41mL, pH are 6.0～6.5.
In above-mentioned fermentation medium, glycerol and MgSO4·7H2O is needed to be sterilized separately by this area conventional formulation method.
According to currently preferred, in described step (2), when the Fructus Hordei Germinatus being substrate trehalose synthesis with soluble Amylose
Oligosaccharide based mycose synthetase and malt oligosaccharide based mycose hydrolytic enzyme merge enzyme in bacillus subtilis during secreting, expressing, step
As follows:
The bacterium solution by volume percentage ratio 8 step (1) prepared～the ratio of 12% are inoculated in fermentation medium, send out for 35～38 DEG C
After ferment cultivates 18～24h, add starch induction 32～40h, make bacillus subtilis start secreting, expressing Fructus Hordei Germinatus oligose base Sargassum
Sugar synzyme merges enzyme with malt oligosaccharide based mycose hydrolytic enzyme, and described starch concentration in fermentation liquid is 15～25g/L；
Described fermentation medium every liter component is as follows:
Sucrose 20g, peptone 7.5g, yeast leaching powder 5g, ammonium sulfate 5g, dipotassium hydrogen phosphate 3g, potassium dihydrogen phosphate 4g,
Magnesium sulfate 0.8g, pH7.0～7.5.
It is further preferred that described starch is soluble starch.
According to currently preferred, in described step (2), when existing with maltose for the trehalose synthase of substrate trehalose synthesis
During bacillus subtilis spore surface display, step is as follows:
The bacterium solution by volume percentage ratio 8 step (1) prepared～the ratio of 12% are inoculated in fermentation medium, send out for 35～38 DEG C
After ferment cultivates 48h, control nutritional labeling, make bacillus subtilis start to sprout formation spore, make spore surface show trehalose
Synthase；
Described fermentation medium every liter component is as follows:
Sucrose 20g, peptone 7.5g, yeast leaching powder 5g, ammonium sulfate 5g, dipotassium hydrogen phosphate 3g, potassium dihydrogen phosphate 4g,
Magnesium sulfate 0.8g, pH7.0～7.5.
According to currently preferred, in described step (2), when existing with maltose for the trehalose synthase of substrate trehalose synthesis
In bacillus subtilis during secreting, expressing, step is as follows:
The bacterium solution by volume percentage ratio 8 step (1) prepared～the ratio of 12% are inoculated in fermentation medium, send out for 35～38 DEG C
After ferment cultivates 18～24h, add maltose induction 24～36h, make bacillus subtilis start secreting, expressing trehalose synthase,
Described maltose concentration of substrate in fermentation liquid is 25～30g/L；
According to currently preferred, in described step (2), membrance concentration filters for employing ceramic membrane filter system, filter membrane
Aperture is 0.22 μm, collects and concentrates enzyme liquid；
According to currently preferred, in described step (3), enzyme is 150～200U/g with the reaction ratio of maltose；Complete thin
Cellular surface shows that enzyme and the reaction ratio of maltose, in terms of the enzyme unit alive of surface display enzyme, are 150～200U/g；Described enzyme with
The reaction ratio of soluble starch is 150～200U/g；The reaction ratio of full cell surface display enzyme and soluble starch is with surface
Show the enzyme unit meter alive of enzyme, be 150～200U/g.
According to currently preferred, in described step (4), heat up and use spiral-plate heat exchanger to heat up；Activated carbon decolorizing
Activated carbon decolorizing post is used to decolour；Filter and use piping filter to filter.
At 75～80 DEG C, enzyme denaturing alive 0.4～0.6h, it is possible not only to inactivate residual enzyme or albumen, accelerates albumen flocculation, and
Sugar liquid mobility can be strengthened, shorten the operating time of subsequent step.
According to currently preferred, in described step (5), concentrate as using triple effect Vacuum plate type evaporation concentrator to concentrate,
Vacuum is-0.09pa；One time chromatographic isolation uses drift Lay spy Ca2+Type resin carries out chromatographic isolation；Two dimensional chromatography separates and uses
The Ca of Finix2+Type resin carries out chromatographic isolation；Described chromatographic isolation temperature is 60 DEG C, and flowing is deionized water mutually.
According to currently preferred, in described step (5), Fructus Hordei Germinatus sugar liquid recycling is dense through the evaporation of triple effect Vacuum plate type
It is 60wt% that contracting device is concentrated into sugar concentration, carries out reuse in step (2) or step (3).
Above-mentioned chromatographic fractionation system can use this area conventional chromatogram piece-rate system, preferably Chinese patent literature CN203483914U
Simulation moving bed described in (application number: CN201320504144.1).
Beneficial effect
1, the present invention establishes the strong product enzyme of a set of seriality first and prepares trehalose production technology, and this technique is with full cell table
Face displaying or the trehalose synthase of exocytosis or malt oligosaccharide based mycose synthetase-malt oligosaccharide based mycose hydrolytic enzyme are merged
Enzyme converts, it is to avoid broken and isolated and purified cost, enzyme liquid or the immobilized enzyme of cell are convenient to be obtained, easy and simple to handle；
2, the present invention produces strain secretes trehalose synthase, the Fructus Hordei Germinatus oligose of Pichia sp. surface display by bacillus subtilis
Base trehalose synthetase, malt oligosaccharide based mycose hydrolytic enzyme merge enzyme, and the Sargassum of bacillus subtilis spore surface display
Sugar synthase, conversion is prepared trehalose, is not only solved the cell breakage because the intracellular expression of enzyme brings, enzyme purification cost difficulty
Topic；Also solve the recycling difficulty of chemical reagent cost and enzyme used in Permeabilized cells preparation process and stablizing of enzyme
Property difference etc. problem；
3, the present invention uses exocytosis and full cell fixation enzyme to produce trehalose, can reduce microbiological contamination probability and by-product is raw
Become, improve utilization rate of equipment and installations, reduce the production cost of trehalose further.
4, the present invention is by double set the most isolated and purified trehaloses of isolated and purified system and maltose, and maltose can obtain very well
Recovery convert again, enhance overall maltose or soluble dextrins prepare the conversion ratio of trehalose；
5, the present invention uses bacillus subtilis, Pichia sp. are as food safety bacterial strain, can be used for preparing food stage sea
Algae sugar, surface display enzyme used, with yeast cells or spore as fixation support, can repeatedly recycle.
Detailed description of the invention
The present invention is explained further below in conjunction with example, but the present invention is not limited in any form by case study on implementation.
Ion exchange system described in embodiment is that the film purchased from Xiamen Shi Da company is continuously from friendship system；
Embodiment 1
A kind of method that continuous exoenzyme bioanalysis prepares trehalose, comprises the steps:
(1) by exocytosis with soluble dextrins be substrate trehalose synthesis bacterium producing multi enzyme preparation 37 DEG C, in LB culture medium
Activation 10h, is then seeded to LB culture medium with the ratio of percent by volume 10%, and 37 DEG C are enlarged cultivating 20h, prepare
Cell concentration is the bacterium solution of 50～80g/L；
With soluble dextrins for substrate trehalose synthesis enzyme as malt oligosaccharide based mycose synthetase and Fructus Hordei Germinatus in described step (1)
Oligosaccharide based mycose hydrolytic enzyme merges enzyme, and its expression strain is that the bacillus subtilis of secreting, expressing fusion enzyme (sees Chinese patent
Construction method disclosed in document CN105039374A (application number 201510486544.8) is prepared)；
(2) bacterium solution that step (1) prepares is inoculated in fermentation medium, 37 DEG C of fermentation culture 24h, starch abduction delivering
Exocytosis, then membrane filtration separating thallus, filter filtrate through ceramic membrane filter system, and filter sizes is 0.22 μm,
Prepare and concentrate enzyme liquid；
In described step (2), fermentation medium every liter component is as follows:
Sucrose 20g, peptone 7.5g, yeast leaching powder 5g, ammonium sulfate 5g, dipotassium hydrogen phosphate 3g, potassium dihydrogen phosphate 4g,
Magnesium sulfate 0.8g, pH7.0.
Described induction starch is soluble starch, and its concentration in fermentation liquid is 15～25g/L；
(3) by step (2) prepare concentrate enzyme liquid react with the soluble starch solution that concentration is 200g/L, enzyme and
The reaction ratio of soluble starch is 200U/g, is 25 DEG C in temperature, under conditions of pH8.0, reacts 24h, and reactant liquor passes through
Elimination is dezymotized, and prepares sugar liquid；
After testing, sugar liquid is mainly composed of trehalose, a small amount of glucose, maltose and maltose polysaccharide, soluble starch
Conversion ratio is more than 84%；
(4) sugar liquid that step (3) prepares is warming up to 75～80 DEG C, enzyme denaturing 0.5h alive, accelerates its flocculation, strengthens sugar liquid
Mobility, filters through kieselguhr, maintains temperature 70～75 DEG C to carry out activated carbon decolorizing, is then cooled to 40～50 DEG C, again
After piping filter filters, carry out ion exchange, prepare electrical conductivity less than 30 μ s/cm desalination sugar liquid；
(5) the desalination sugar liquid that step (4) prepares being concentrated into sugar concentration is 55% (mass percent), carries out a chromatograph
Separating, Polysaccharide removing and glucose, separation temperature 60 DEG C, after separation, mixed sugar (maltose+trehalose) purity reaches 90%
Above；Then being concentrated into sugar concentration is 55% (mass percent), separates through two dimensional chromatography, prepares Sargassum sugar liquid and maltose
Liquid, aqueous trehalose purity reaches 87%, and Fructus Hordei Germinatus sugar liquid recycling, it is 80% that Sargassum sugar liquid is concentrated into sugar content, cooling knot
Brilliant 24h, after centrifugation, 60 DEG C obtain trehalose crystallization through vibrations fluid bed drying, and trehalose purity reaches 99%.
In described step (5), concentrating as using triple effect Vacuum plate type evaporation concentrator to concentrate, vacuum is-0.09pa；
One time chromatographic isolation uses drift Lay spy Ca2+Type resin carries out chromatographic isolation；Two dimensional chromatography separates the Ca using Finix2+Type resin
Carry out chromatographic isolation；Described chromatographic isolation temperature is 60 DEG C, and flowing is deionized water mutually.
In described step (5), Fructus Hordei Germinatus sugar liquid recycling for being concentrated into sugar concentration through triple effect Vacuum plate type evaporation concentrator is
60wt%, carries out reuse in step (2) or step (3).
Above-mentioned chromatographic fractionation system uses Chinese patent literature CN203483914U (application number: CN201320504144.1) institute
The simulation moving bed stated.
Embodiment 2
A kind of method that continuous exoenzyme bioanalysis prepares trehalose, comprises the steps:
(1) by exocytosis with maltose for substrate prepare the bacterium producing multi enzyme preparation of trehalose 37 DEG C, LB culture medium activates
10h, is then seeded to LB culture medium with the ratio of percent by volume 10%, and 37 DEG C are enlarged cultivating 20h, prepare thalline
Concentration is the bacterium solution of 50～80g/L；
In described step (1) with maltose be substrate trehalose synthesis enzyme as trehalose synthase, its expression strain for secretion table
The bacillus subtilis reaching trehalose synthase (sees Chinese patent literature CN105039381A (application number 201510431599.9)
Disclosed in construction method be prepared)；
(2) bacterium solution that step (1) prepares is inoculated in fermentation medium, 37 DEG C of fermentation culture 24h, maltose induction table
Reaching exocytosis, then membrane filtration separating thallus, filtrate filtered through ceramic membrane filter system, filter sizes is 0.22 μm,
Prepare and concentrate enzyme liquid；
In described step (2), fermentation medium every liter component is as follows:
Sucrose 20g, peptone 7.5g, yeast leaching powder 5g, ammonium sulfate 5g, dipotassium hydrogen phosphate 3g, potassium dihydrogen phosphate 4g,
Magnesium sulfate 0.8g, pH7.0.
The described induction maltose final concentration of 25g/L-30g/L in fermentation liquid；
(3) the enzyme liquid that concentrates step (2) prepared reacts with the maltose that concentration is 300g/L, and enzyme is anti-with maltose
Answering ratio is 150U/g, is 25 DEG C in temperature, under conditions of pH8.0, reacts 24h, and reactant liquor is dezymotized through elimination, prepares
Sugar liquid；
After testing, sugar liquid is mainly composed of trehalose, maltose, a small amount of glucose and maltose polysaccharide, the conversion of maltose
Rate is more than 65%；
(4) sugar liquid that step (3) prepares is warming up to 75～80 DEG C, enzyme denaturing 0.4h alive, accelerates its flocculation, strengthens sugar liquid
Mobility, filters through kieselguhr, maintains temperature 70～75 DEG C to carry out activated carbon decolorizing, is then cooled to 40～50 DEG C, again
After piping filter filters, carry out ion exchange, prepare electrical conductivity less than 30 μ s/cm desalination sugar liquid；
(5) the desalination sugar liquid that step (4) prepares being concentrated into sugar concentration is 50% (mass percent), carries out a chromatograph
Separating, Polysaccharide removing and glucose, separation temperature 60 DEG C, after separation, mixed sugar (maltose+trehalose) purity reaches 90%
Above；Then being concentrated into sugar concentration is 50% (mass percent), separates through two dimensional chromatography, prepares Sargassum sugar liquid and maltose
Liquid, aqueous trehalose purity reaches 87%, and Fructus Hordei Germinatus sugar liquid recycling, it is 80% that Sargassum sugar liquid is concentrated into sugar content, cooling knot
Brilliant 24h, after centrifugation, 60 DEG C obtain trehalose crystallization through vibrations fluid bed drying, and trehalose purity reaches 99%.
In described step (5), concentrating as using triple effect Vacuum plate type evaporation concentrator to concentrate, vacuum is-0.09pa；
One time chromatographic isolation uses drift Lay spy Ca2+Type resin carries out chromatographic isolation；Two dimensional chromatography separates the Ca using Finix2+Type resin
Carry out chromatographic isolation；Described chromatographic isolation temperature is 60 DEG C, and flowing is deionized water mutually.
In described step (5), Fructus Hordei Germinatus sugar liquid recycling for being concentrated into sugar concentration through triple effect Vacuum plate type evaporation concentrator is
60wt%, carries out reuse in step (2) or step (3).
Above-mentioned chromatographic fractionation system uses Chinese patent literature CN203483914U (application number: CN201320504144.1) institute
The simulation moving bed stated.
Embodiment 3
A kind of method that continuous exoenzyme bioanalysis prepares trehalose, comprises the steps:
(1) by be substrate trehalose synthesis with maltose spore surface show enzyme bacterial strain, at 37 DEG C, in LB culture medium
Middle activation 8h, is then seeded in fermentation medium with the ratio of 10%, 37 DEG C, after high density fermentation cultivates 48h, enters
In the poor nutritional stage, bacillus subtilis starts to produce spore, after continuing fermentation 8h, and micro-filtrate membrane filtration separation spore, it is thus achieved that
The spore of surface display trehalose synthase；
In described step (1) with maltose be substrate trehalose synthesis enzyme as trehalose synthase, its expression strain is surface exhibition
Show that the bacillus subtilis of trehalose synthase (sees Chinese patent literature CN105132450A (application number 201510571328.3)
Disclosed in construction method be prepared)；
(2) bacterium solution that step (1) prepares is inoculated in fermentation medium, 37 DEG C of fermentation culture 48h, enters spore and generate
Stage, after continuing fermentation 10h, membrane filtration separation spore, prepare the bacillus subtilis spore of surface display trehalose synthase；
In described step (2), fermentation medium every liter component is as follows:
Sucrose 20g, peptone 7.5g, yeast leaching powder 5g, ammonium sulfate 5g, dipotassium hydrogen phosphate 3g, potassium dihydrogen phosphate 4g,
Magnesium sulfate 0.8g, pH7.0.
(3) spore and the maltose that concentration is 300g/L of surface display trehalose synthase step (2) prepared is carried out instead
Should, enzyme and maltose ratio are 180U/g, are 25 DEG C in temperature, under conditions of pH8.0, react 26h, and reactant liquor is through filtering
Remove spore, prepare sugar liquid；
After testing, sugar liquid is mainly composed of trehalose, maltose, a small amount of glucose and maltose polysaccharide, the conversion of maltose
Rate is more than 64%；
(4) sugar liquid that step (3) prepares is warming up to 75～80 DEG C, enzyme denaturing 0.6h alive, accelerates its flocculation, strengthens sugar liquid
Mobility, filters through kieselguhr, maintains temperature 70～75 DEG C to carry out activated carbon decolorizing, is then cooled to 40～50 DEG C, again
After piping filter filters, carry out ion exchange, prepare electrical conductivity less than 30 μ s/cm desalination sugar liquid；
(5) the desalination sugar liquid that step (4) prepares being concentrated into sugar concentration is 53% (mass percent), carries out a chromatograph
Separating, Polysaccharide removing and glucose, separation temperature 60 DEG C, after separation, mixed sugar (maltose+trehalose) purity reaches 90%
Above；Then being concentrated into sugar concentration is 53% (mass percent), separates through two dimensional chromatography, prepares Sargassum sugar liquid and maltose
Liquid, aqueous trehalose purity reaches 87%, and Fructus Hordei Germinatus sugar liquid recycling, it is 80% that Sargassum sugar liquid is concentrated into sugar content, cooling knot
Brilliant 24h, after centrifugation, 60 DEG C obtain trehalose crystallization through vibrations fluid bed drying, and trehalose purity reaches 99%.
In described step (5), concentrating as using triple effect Vacuum plate type evaporation concentrator to concentrate, vacuum is-0.09pa；
One time chromatographic isolation uses drift Lay spy Ca2+Type resin carries out chromatographic isolation；Two dimensional chromatography separates the Ca using Finix2+Type resin
Carry out chromatographic isolation；Described chromatographic isolation temperature is 60 DEG C, and flowing is deionized water mutually.
In described step (5), Fructus Hordei Germinatus sugar liquid recycling for being concentrated into sugar concentration through triple effect Vacuum plate type evaporation concentrator is
60wt%, carries out reuse in step (2) or step (3).
Above-mentioned chromatographic fractionation system uses Chinese patent literature CN203483914U (application number: CN201320504144.1) institute
The simulation moving bed stated.
Embodiment 4
A kind of method that continuous exoenzyme bioanalysis prepares trehalose, comprises the steps:
(1) by be substrate trehalose synthesis with soluble starch the bacterial strain of full cell surface display enzyme, at 30 DEG C, at RSM
Activating 12h in culture medium, be then seeded in RSM fermentation medium with the ratio of 10%, 30 DEG C, high density fermentation is cultivated
After 24h, add the methanol making concentration of substrate be 0.5% and induce, induce 120h, micro-filtrate membrane filtration separating thallus, it is thus achieved that
Surface display malt oligosaccharide based mycose synthetase-malt oligosaccharide based mycose hydrolytic enzyme merges the full cell of enzyme；
In described step (1) with soluble starch be substrate trehalose synthesis enzyme as malt oligosaccharide based mycose synthetase-Fructus Hordei Germinatus
Oligosaccharide based mycose hydrolytic enzyme merges enzyme, and its expression strain is surface display malt oligosaccharide based mycose synthetase-Fructus Hordei Germinatus oligose base
Hydrolysis of trehalose enzyme merges the Pichia yeast of enzyme and (sees Chinese patent literature CN105039191A (application number
201510571619.2 the construction method disclosed in) is prepared)；
(2) bacterium solution that step (1) prepares is inoculated in fermentation medium, 30 DEG C of fermentation culture 24h, adds concentration of substrate
It is the methanol of 0.5%, maintains concentration to enter induction period, after continuing fermentation 120h, membrane filtration separating thallus, prepare surface exhibition
Show that malt oligosaccharide based mycose synthetase-malt oligosaccharide based mycose hydrolytic enzyme merges the full cell of Pichia sp. of enzyme；
In described step (2), fermentation medium (RSM) every liter of component is as follows:
KH2PO442.9g, (NH4)2SO45g, CaSO40.6g, K2SO414.3g, anhydrous citric acid 1.92g,
MgSO4·7H2O 11.7g, glycerol 40mL, PTM4 trace element solution 2.0mL；
Above-mentioned every liter of component of PMT4 trace element solution is as follows:
CuSO4·5H2O 2.0g, NaI 0.08g, MnSO4·H2O 3.0g, Na2MoO4·2H2O 0.2g, H3BO30.02g,
CaSO4·2H2O 0.5g, CoCl20.5g, ZnCl27g, FeSO4·7H2O 22g, biotin 0.2g, mass concentration is 98%
H2SO41mL, pH are 6.0～6.5.
In above-mentioned fermentation medium, glycerol and MgSO4·7H2O is needed to be sterilized separately by this area conventional formulation method.
(3) surface display malt oligosaccharide based mycose synthetase-malt oligosaccharide based mycose hydrolytic enzyme that step (2) is prepared
The full cell of Pichia sp. merging enzyme reacts with the soluble starch that concentration is 200g/L, and enzyme with soluble starch ratio is
175U/g, is 55 DEG C in temperature, under conditions of pH5.0, reacts 22h, and reactant liquor, through filtering off degerming body, prepares sugar liquid；
After testing, sugar liquid is mainly composed of trehalose, glucose, a small amount of maltose and maltose polysaccharide, the conversion of maltose
Rate is more than 84%；
(4) sugar liquid that step (3) prepares is warming up to 75～80 DEG C, enzyme denaturing 0.5h alive, accelerates its flocculation, strengthens sugar liquid
Mobility, filters through kieselguhr, maintains temperature 70～75 DEG C to carry out activated carbon decolorizing, is then cooled to 40～50 DEG C, again
After piping filter filters, carry out ion exchange, prepare electrical conductivity less than 30 μ s/cm desalination sugar liquid；
(5) the desalination sugar liquid that step (4) prepares being concentrated into sugar concentration is 50%～55% (mass percent), carries out one
Secondary chromatographic isolation, Polysaccharide removing and glucose, separation temperature 60 DEG C, after separation, mixed sugar (maltose+trehalose) purity reaches
To more than 90%；Then being concentrated into sugar concentration is 50%～55% (mass percent), separates through two dimensional chromatography, prepares Sargassum
Sugar liquid and Fructus Hordei Germinatus sugar liquid, aqueous trehalose purity reaches 87%, and Fructus Hordei Germinatus sugar liquid recycling, Sargassum sugar liquid is concentrated into sugar content and is
80%, decrease temperature crystalline 24h, after centrifugation, 60 DEG C obtain trehalose crystallization through vibrations fluid bed drying, and trehalose purity reaches
To 99%.
In described step (5), concentrating as using triple effect Vacuum plate type evaporation concentrator to concentrate, vacuum is-0.09pa；
One time chromatographic isolation uses drift Lay spy Ca2+Type resin carries out chromatographic isolation；Two dimensional chromatography separates the Ca using Finix2+Type resin
Carry out chromatographic isolation；Described chromatographic isolation temperature is 60 DEG C, and flowing is deionized water mutually.
In described step (5), Fructus Hordei Germinatus sugar liquid recycling for being concentrated into sugar concentration through triple effect Vacuum plate type evaporation concentrator is
60wt%, carries out reuse in step (2) or step (3).
Above-mentioned chromatographic fractionation system uses Chinese patent literature CN203483914U (application number: CN201320504144.1) institute
The simulation moving bed stated.
Claims (10)
1. the method that continuous exoenzyme bioanalysis prepares trehalose, comprises the steps:
(1) by few to the product trehalose synthase with maltose or soluble starch as substrate or the Fructus Hordei Germinatus of exocytosis or surface display
Glycosyl trehalose synthase-malt oligosaccharide based mycose hydrolytic enzyme merges the bacterial strain of enzyme and carries out activation culture, then through amplification culture,
Prepared cell concentration is the bacterium solution of 50～80g/L；
(2) bacterium solution that step (1) prepares is inoculated in fermentation medium and carries out fermentation culture, the exocytosis table of inducible enzyme
Reach or spore surface is shown or full cell surface display, then filter, prepare full cell surface display thalline, surface display spore
Or crude enzyme liquid, by crude enzyme liquid after membrance concentration, prepare and concentrate enzyme liquid；
(3) by concentration enzyme liquid, full cell surface display thalline or surface display spore and concentration that step (2) prepares be 250～
The maltose solution of 350g/L reacts, and is 15～55 DEG C in temperature, under conditions of pH7.5～8.5, reacts 24h～36h,
Reactant liquor, through filtering, prepares sugar liquid；
Or, by concentration enzyme liquid, full cell surface display thalline or surface display spore and concentration that step (2) prepares be 150～
The soluble starch of 250g/L reacts, and is 15～55 DEG C in temperature, under conditions of pH5.0～6.0, reacts 24h～36h,
Reactant liquor, through filtering, prepares sugar liquid；
(4) sugar liquid that step (3) prepares is warming up to 75～80 DEG C, enzyme denaturing alive 0.4～0.6h, filters through kieselguhr, dimension
Hold temperature 70～75 DEG C carry out activated carbon decolorizing, be then cooled to 40～50 DEG C, again filter after, carry out ion exchange, system
Obtain desalination sugar liquid；
(5) the desalination sugar liquid that step (4) prepares being concentrated into sugar concentration is 50%～55% (mass percent), carries out one
Secondary chromatographic isolation, Polysaccharide removing and glucose, being then concentrated into sugar concentration is 50%～55% (mass percent), through secondary
Chromatographic isolation, prepares Sargassum sugar liquid and Fructus Hordei Germinatus sugar liquid, Fructus Hordei Germinatus sugar liquid recycling, the concentrated crystallization of Sargassum sugar liquid, prepares sea
Algae sugar.
2. preparation method as claimed in claim 1, it is characterised in that described step in (1) with soluble Amylose is
The enzyme of substrate trehalose synthesis is that malt oligosaccharide based mycose synthetase merges enzyme with malt oligosaccharide based mycose hydrolytic enzyme, and it is expressed
Bacterial strain is the Pichia yeast that surface display malt oligosaccharide based mycose synthetase and malt oligosaccharide based mycose hydrolytic enzyme merge enzyme
Strain, or secreting, expressing malt oligosaccharide based mycose synthetase and malt oligosaccharide based mycose hydrolytic enzyme merge the bacillus subtilis of enzyme
Bacterium；
With maltose be substrate trehalose synthesis enzyme as trehalose synthase, its expression strain be spore surface show trehalose synthase
Bacillus subtilis, or the bacillus subtilis of secreting, expressing trehalose synthase.
3. preparation method as claimed in claim 1, it is characterised in that in described step (2), when forming sediment with soluble linear
Powder is that the malt oligosaccharide based mycose synthetase of substrate trehalose synthesis merges enzyme complete red with malt oligosaccharide based mycose hydrolytic enzyme
During yeast strain surface display, step is as follows:
The bacterium solution by volume percentage ratio 8 step (1) prepared～the ratio of 12% are inoculated in fermentation medium, send out for 28～32 DEG C
After ferment cultivates 20～28h, use methanol induction 80～120h, make Pichia sp. start surface display malt oligosaccharide based mycose
Synzyme merges enzyme with malt oligosaccharide based mycose hydrolytic enzyme, and described methanol concentration in fermentation liquid controls 0.3～0.7g/L；
Described fermentation medium every liter component is as follows:
KH2PO442.9g, (NH4)2SO45g, CaSO40.6g, K2SO414.3g, anhydrous citric acid 1.92g,
MgSO4·7H2O 11.7g, glycerol 40mL, PTM4 trace element solution 2.0mL；
Above-mentioned every liter of component of PMT4 trace element solution is as follows:
CuSO4·5H2O 2.0g, NaI 0.08g, MnSO4·H2O 3.0g, Na2MoO4·2H2O 0.2g, H3BO30.02g,
CaSO4·2H2O 0.5g, CoCl20.5g, ZnCl27g, FeSO4·7H2O 22g, biotin 0.2g, mass concentration is 98%
H2SO41mL, pH are 6.0～6.5.
4. preparation method as claimed in claim 1, it is characterised in that in described step (2), when forming sediment with soluble linear
Powder is that the malt oligosaccharide based mycose synthetase of substrate trehalose synthesis merges enzyme hay with malt oligosaccharide based mycose hydrolytic enzyme
In bacillus cereus during secreting, expressing, step is as follows:
The bacterium solution by volume percentage ratio 8 step (1) prepared～the ratio of 12% are inoculated in fermentation medium, send out for 35～38 DEG C
After ferment cultivates 18～24h, add starch induction 32～40h, make bacillus subtilis start secreting, expressing Fructus Hordei Germinatus oligose base Sargassum
Sugar synzyme merges enzyme with malt oligosaccharide based mycose hydrolytic enzyme, and described starch concentration in fermentation liquid is 15～25g/L；
Described fermentation medium every liter component is as follows:
Sucrose 20g, peptone 7.5g, yeast leaching powder 5g, ammonium sulfate 5g, dipotassium hydrogen phosphate 3g, potassium dihydrogen phosphate 4g,
Magnesium sulfate 0.8g, pH7.0～7.5；
It is further preferred that described starch is soluble starch.
5. preparation method as claimed in claim 1, it is characterised in that in described step (2), when with maltose as substrate
The trehalose synthase of trehalose synthesis is when bacillus subtilis spore surface display, and step is as follows:
The bacterium solution by volume percentage ratio 8 step (1) prepared～the ratio of 12% are inoculated in fermentation medium, send out for 35～38 DEG C
After ferment cultivates 48h, control nutritional labeling, make bacillus subtilis start to sprout formation spore, make spore surface show trehalose
Synthase；
Described fermentation medium every liter component is as follows:
Sucrose 20g, peptone 7.5g, yeast leaching powder 5g, ammonium sulfate 5g, dipotassium hydrogen phosphate 3g, potassium dihydrogen phosphate 4g,
Magnesium sulfate 0.8g, pH7.0～7.5.
6. preparation method as claimed in claim 1, it is characterised in that in described step (2), when with maltose as substrate
The trehalose synthase of trehalose synthesis is in bacillus subtilis during secreting, expressing, and step is as follows:
The bacterium solution by volume percentage ratio 8 step (1) prepared～the ratio of 12% are inoculated in fermentation medium, send out for 35～38 DEG C
After ferment cultivates 18～24h, add maltose induction 24～36h, make bacillus subtilis start secreting, expressing trehalose synthase,
Described maltose concentration of substrate in fermentation liquid is 25～30g/L；
Preferably, in described step (2), membrance concentration is for using ceramic membrane filter system to filter, and filter sizes is 0.22 μm,
Collect and concentrate enzyme liquid.
7. preparation method as claimed in claim 1, it is characterised in that in described step (3), enzyme and the reaction of maltose
Ratio is 150～200U/g；The reaction ratio of cell surface display enzyme and maltose is in terms of the enzyme unit alive of surface display enzyme entirely,
It is 150～200U/g；Described enzyme is 150～200U/g with the reaction ratio of soluble starch；Full cell surface display enzyme with can
The reaction ratio of soluble starch, in terms of the enzyme unit alive of surface display enzyme, is 150～200U/g.
8. preparation method as claimed in claim 1, it is characterised in that in described step (4), heats up and uses spiral lamina to change
Hot device heats up；Activated carbon decolorizing uses activated carbon decolorizing post to decolour；Filter and use piping filter to filter.
9. preparation method as claimed in claim 1, it is characterised in that in described step (5), concentrates as using triple effect true
Hollow plate formula evaporation concentrator concentrates, and vacuum is-0.09pa；One time chromatographic isolation uses drift Lay spy Ca2+Type resin carries out color
Spectrum separates；Two dimensional chromatography separates the Ca using Finix2+Type resin carries out chromatographic isolation；Described chromatographic isolation temperature is 60 DEG C,
Flowing is deionized water mutually.
10. preparation method as claimed in claim 1, it is characterised in that in described step (5), Fructus Hordei Germinatus sugar liquid reclaims again
Being utilized as being concentrated into sugar concentration through triple effect Vacuum plate type evaporation concentrator is 60wt%, carries out back in step (2) or step (3)
With.
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| CN108841897A (en) * | 2018-07-16 | 2018-11-20 | 安徽民祯生物工程有限公司 | It is a kind of for producing the rhodozyma culture method of trehalose |
| CN110643552A (en) * | 2019-11-22 | 2020-01-03 | 河南省科学院生物研究所有限责任公司 | Bacterial strain for preparing seaweed syrup by using soluble starch and application thereof |
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| CN108841898A (en) * | 2018-07-16 | 2018-11-20 | 安徽民祯生物工程有限公司 | A kind of method of permeability yeast cells production trehalose |
| CN108841897A (en) * | 2018-07-16 | 2018-11-20 | 安徽民祯生物工程有限公司 | It is a kind of for producing the rhodozyma culture method of trehalose |
| CN110643552A (en) * | 2019-11-22 | 2020-01-03 | 河南省科学院生物研究所有限责任公司 | Bacterial strain for preparing seaweed syrup by using soluble starch and application thereof |
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