CN103045561B - Solid state fermentation production method for beta-D-fructofuranosidase - Google Patents
Solid state fermentation production method for beta-D-fructofuranosidase Download PDFInfo
- Publication number
- CN103045561B CN103045561B CN201210545255.7A CN201210545255A CN103045561B CN 103045561 B CN103045561 B CN 103045561B CN 201210545255 A CN201210545255 A CN 201210545255A CN 103045561 B CN103045561 B CN 103045561B
- Authority
- CN
- China
- Prior art keywords
- solid
- state fermentation
- liquid
- beta
- culture medium
- 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.)
- Expired - Fee Related
Links
- 238000010563 solid-state fermentation Methods 0.000 title claims abstract description 68
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 20
- 108010051210 beta-Fructofuranosidase Proteins 0.000 title abstract description 9
- 239000007788 liquid Substances 0.000 claims abstract description 86
- 239000001963 growth medium Substances 0.000 claims abstract description 58
- 238000002360 preparation method Methods 0.000 claims abstract description 52
- 108090000790 Enzymes Proteins 0.000 claims abstract description 45
- 102000004190 Enzymes Human genes 0.000 claims abstract description 45
- 241000609240 Ambelania acida Species 0.000 claims abstract description 28
- 244000068988 Glycine max Species 0.000 claims abstract description 28
- 235000010469 Glycine max Nutrition 0.000 claims abstract description 28
- 240000007594 Oryza sativa Species 0.000 claims abstract description 28
- 235000007164 Oryza sativa Nutrition 0.000 claims abstract description 28
- 239000010905 bagasse Substances 0.000 claims abstract description 28
- 239000010903 husk Substances 0.000 claims abstract description 28
- 235000009566 rice Nutrition 0.000 claims abstract description 28
- 238000000855 fermentation Methods 0.000 claims abstract description 23
- 230000004151 fermentation Effects 0.000 claims abstract description 23
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 38
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 38
- 239000002609 medium Substances 0.000 claims description 28
- 238000011218 seed culture Methods 0.000 claims description 27
- 238000012258 culturing Methods 0.000 claims description 26
- 230000000050 nutritive effect Effects 0.000 claims description 26
- 239000007787 solid Substances 0.000 claims description 26
- 239000008247 solid mixture Substances 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- 239000008367 deionised water Substances 0.000 claims description 19
- 229910021641 deionized water Inorganic materials 0.000 claims description 19
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 19
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 19
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 19
- 239000004317 sodium nitrate Substances 0.000 claims description 19
- 229940001516 sodium nitrate Drugs 0.000 claims description 19
- 235000010344 sodium nitrate Nutrition 0.000 claims description 19
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 18
- RFSUNEUAIZKAJO-ARQDHWQXSA-N beta-D-fructofuranoseose Natural products OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 18
- 230000001954 sterilising effect Effects 0.000 claims description 17
- 239000000463 material Substances 0.000 claims description 16
- 235000013379 molasses Nutrition 0.000 claims description 16
- -1 beta-D-fructofuranose glycosides Chemical class 0.000 claims description 15
- 229930182470 glycoside Natural products 0.000 claims description 15
- 240000008042 Zea mays Species 0.000 claims description 13
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 13
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 13
- 239000007979 citrate buffer Substances 0.000 claims description 13
- 235000005822 corn Nutrition 0.000 claims description 13
- 239000000706 filtrate Substances 0.000 claims description 13
- 239000002054 inoculum Substances 0.000 claims description 13
- 238000002386 leaching Methods 0.000 claims description 13
- 238000005360 mashing Methods 0.000 claims description 13
- 239000002504 physiological saline solution Substances 0.000 claims description 13
- 235000020183 skimmed milk Nutrition 0.000 claims description 13
- 238000003756 stirring Methods 0.000 claims description 13
- 238000005406 washing Methods 0.000 claims description 13
- 241000228245 Aspergillus niger Species 0.000 claims description 9
- 229930006000 Sucrose Natural products 0.000 claims description 6
- 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 6
- 239000005720 sucrose Substances 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 239000011790 ferrous sulphate Substances 0.000 claims description 3
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 3
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 3
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 14
- 239000002994 raw material Substances 0.000 abstract description 5
- 230000008901 benefit Effects 0.000 abstract description 4
- 230000008569 process Effects 0.000 abstract description 2
- 230000007547 defect Effects 0.000 abstract 1
- FTSSQIKWUOOEGC-RULYVFMPSA-N fructooligosaccharide Chemical compound OC[C@H]1O[C@@](CO)(OC[C@@]2(OC[C@@]3(OC[C@@]4(OC[C@@]5(OC[C@@]6(OC[C@@]7(OC[C@@]8(OC[C@@]9(OC[C@@]%10(OC[C@@]%11(O[C@H]%12O[C@H](CO)[C@@H](O)[C@H](O)[C@H]%12O)O[C@H](CO)[C@@H](O)[C@@H]%11O)O[C@H](CO)[C@@H](O)[C@@H]%10O)O[C@H](CO)[C@@H](O)[C@@H]9O)O[C@H](CO)[C@@H](O)[C@@H]8O)O[C@H](CO)[C@@H](O)[C@@H]7O)O[C@H](CO)[C@@H](O)[C@@H]6O)O[C@H](CO)[C@@H](O)[C@@H]5O)O[C@H](CO)[C@@H](O)[C@@H]4O)O[C@H](CO)[C@@H](O)[C@@H]3O)O[C@H](CO)[C@@H](O)[C@@H]2O)[C@@H](O)[C@@H]1O FTSSQIKWUOOEGC-RULYVFMPSA-N 0.000 abstract 1
- 229940107187 fructooligosaccharide Drugs 0.000 abstract 1
- 238000001514 detection method Methods 0.000 description 12
- 101000765308 Aspergillus niger N-(5'-phosphoribosyl)anthranilate isomerase Proteins 0.000 description 11
- ODEHMIGXGLNAKK-OESPXIITSA-N 6-kestotriose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@@H]1[C@@H](O)[C@H](O)[C@](CO)(O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)O1 ODEHMIGXGLNAKK-OESPXIITSA-N 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 235000011073 invertase Nutrition 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000006098 transglycosylation Effects 0.000 description 2
- 238000005918 transglycosylation reaction Methods 0.000 description 2
- 244000291564 Allium cepa Species 0.000 description 1
- 235000002732 Allium cepa var. cepa Nutrition 0.000 description 1
- 240000005528 Arctium lappa Species 0.000 description 1
- 235000003130 Arctium lappa Nutrition 0.000 description 1
- 235000008078 Arctium minus Nutrition 0.000 description 1
- 241000186063 Arthrobacter Species 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 235000017166 Bambusa arundinacea Nutrition 0.000 description 1
- 235000017491 Bambusa tulda Nutrition 0.000 description 1
- 240000008892 Helianthus tuberosus Species 0.000 description 1
- 235000003230 Helianthus tuberosus Nutrition 0.000 description 1
- 229920001202 Inulin Polymers 0.000 description 1
- 244000082204 Phyllostachys viridis Species 0.000 description 1
- 235000015334 Phyllostachys viridis Nutrition 0.000 description 1
- 101710184309 Probable sucrose-6-phosphate hydrolase Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 102400000472 Sucrase Human genes 0.000 description 1
- 101710112652 Sucrose-6-phosphate hydrolase Proteins 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 241000364021 Tulsa Species 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000011425 bamboo Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Substances OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 208000002925 dental caries Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 description 1
- 229940029339 inulin Drugs 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000010808 liquid waste Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 230000003534 oscillatory effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to a preparation method for beta-D-fructofuranosidase, EC 3.1.2.26 short for FFase, in particular to a solid state fermentation production method for the beta-D-fructofuranosidase. The method is characterized by comprising the following steps: (1) preparing spore liquid; (2) performing enlarging cultivation on seeds; (3) performing solid state fermentation to produce the beta-D-fructofuranosidase; and (4) performing aftertreatment to obtain the beta-D-fructofuranosidase. By the method, the defect of liquid fermentation of the FFase is overcome. The invention provides the solid state fermentation production method for the FFase, which provides a high-quality enzyme source for large-scale industrialized production of fructo-oligosaccharide. The solid state fermentation production method for the beta-D-fructofuranosidase has the advantages that (1) a solid state fermentation culture medium adopts bagasse, rice husk, soybean cake and the like as raw materials and the raw materials are low in price; and (2) the fermentation period is short, the process equipment is simple, the cost is reduced and the industrialized production is promoted.
Description
Technical field
The present invention relates to the preparation method of beta-D-fructofuranose glycosides enzyme (β-D-fructofuranosidase, EC 3.1.2.26, is abbreviated as FFase), especially a kind of solid state fermentation production method of beta-D-fructofuranose glycosides enzyme.
Background technology
Oligofructose (fructooligosaccharides) is a kind of novel protective foods, be in functional oligose, have much representational a kind of.Oligofructose is on the fructosyl of sucrose, to connect 1 ~ 3 fructosyl and a series of fructooligosaccharideses of forming by β-(2,1) glycosidic link, mainly comprises kestose (GF2), GF3 (GF3), GF4 (GF4).In daily edible vegetables and fruit, contain this type of oligosaccharides, especially in onion, burdock, awns bamboo shoot and wheat class, content is higher.Oligofructose has bifidus bacillus in low heat value, preventing dental caries, propagation enteron aisle, regulates the physiological functions such as immunity of organisms, is widely used in food, feed and medicine industry, is a kind of important health additive.
Current industrial production oligofructose method mainly contains two kinds: the one, and take jerusalem artichoke as raw material extraction inulin, then obtain through enzymic hydrolysis.Second method is to take sucrose as raw material, adopts beta-D-fructofuranose glycosides enzyme by a series of Transglycosylations of enzyme-catalyzed change, to obtain oligofructose as biological catalyst.Can be by beta-D-fructofuranose glycosides enzyme immobilization be realized to continuous production in a kind of rear method, have that level of automation is high, operational stability good, the advantage such as enzyme energy Reusability, production cost are low and being favored, the most of oligofructose products on domestic and international market are all adopted in this way and are produced.
Beta-D-fructofuranose glycosides enzyme (β-D-fructofuranosidase, EC 3.1.2.26, is abbreviated as FFase) claim again sucrase or saccharase, can catalytic hydrolysis β-D-Fructose glycosidic bond, and also can catalysis Transglycosylation.It is present in organic sphere widely, and according to the literature, the FFase catalytic activity in plant is very weak, productive rate is low, and is subject to season limit, and higher than the catalytic activity of plant from the FFase of microorganism, and high temperature resistant, in reaction process, be subject to the opportunities for contamination of miscellaneous bacteria few, easy to use.The microorganism that can produce FFase mainly has aspergillus niger, aspergillus oryzae, yeast and Arthrobacter etc.Production by biological FFase turns the height of glycosyl vigor and the complexity of fermentation preparation is that decision enzyme transforming process is prepared the whether economic key factor of oligofructose technique.
But, at present to the microbial source main liquid submerged fermentation method that adopts of FFase fermentation preparation in aspergillus source particularly, shortcoming is brought a lot of difficulties to actual industrial production to have fermentation period long (generally taking about 7 days), enzymatic production level is low, wastewater discharge is large, production cost is high etc.
Summary of the invention
The solid state fermentation production method that the object of this invention is to provide the beta-D-fructofuranose glycosides enzyme that a kind of unit matrix enzyme activity is high, fermentation period is short, technique is simple, production cost is low.
A solid state fermentation production method for beta-D-fructofuranose glycosides enzyme, its special feature is, comprises the steps:
(1) preparation of spore liquid: first prepare test tube slant substratum, aspergillus niger is inoculated on the test tube slant substratum preparing, cultivate 3～5 days to growing spore at 25-32 ℃, then add physiological saline washing mycelium on inclined-plane, obtain spore liquid;
(2) enlarged culturing of seed: prepare liquid seed culture medium, spore liquid access liquid seed culture medium step (1) being obtained according to the inoculum size of volume ratio 2%～5%, on 25～32 ℃, the shaking table of rotating speed 180～220rpm, enlarged culturing is 2～4 days, obtains seed liquor;
(3) Produced by Solid-state Fermentation beta-D-fructofuranose glycosides enzyme: preparation solid-state fermentation culture medium, the solid-state fermentation culture medium that seed liquor access is prepared, fully be uniformly mixed, then standing for fermentation 3～5 days at 1～3 centimetre of bed thickness, humidity 45%～80%, leavening temperature 25-35 ℃;
(4) aftertreatment obtains beta-D-fructofuranose glycosides enzyme: get the solid culture that step (3) obtains, the citrate buffer that adds 50 mmole pH 5.5 of its 6-10 times weight, with tissue mashing machine, pulverize, stirring and leaching is at least 4 hours again, through centrifugal or filtration, make solid-liquid separation, the supernatant liquor obtaining or filtrate be thick beta-D-fructofuranose glycosides enzyme enzyme liquid.
In step (3), the preparation method of solid-state fermentation culture medium is evenly sprayed on solid mixture by the nutritive medium of 1.5～3 times of weight, then 121 ℃ of sterilizings 20 minutes, wherein the composition of nutritive medium was counted with g/L, cane molasses 10～30, corn steep liquor 5～15, skim-milk 3～10, SODIUMNITRATE 1～3, dipotassium hydrogen phosphate 2～5, magnesium sulfate 0.5, Repone K 0.5, these materials are first dissolved in 800 ml deionized water, and then constant volume to 1 liter; Solid mixture is mixed and is formed by bagasse, rice husk, soybean cake three, and its composition is bagasse according to weight ratio: rice husk: soybean cake is 3～6:1～3:0.5～1.5.
In step (1), the composition of test tube slant medium is counted with g/L, sucrose 30, SODIUMNITRATE 3, dipotassium hydrogen phosphate 1, magnesium sulfate 0.5, Repone K 0.5, ferrous sulfate 0.01, agar 15, these materials are first dissolved in 800 ml deionized water, and then constant volume to 1 liter.
In step (1), aspergillus niger is aspergillus niger AS 3.877.
In step (2), the composition of liquid seed culture medium is counted with g/L, cane molasses 20, peptone 10, yeast extract paste 5, SODIUMNITRATE 2, dipotassium hydrogen phosphate 3, magnesium sulfate 0.5, Repone K 0.5, these materials are first dissolved in 800 ml deionized water, and then constant volume to 1 liter.
When in step (3), seed liquor accesses the solid-state fermentation culture medium preparing, the inoculative proportion of seed liquor is 5～15 milliliters/100 grams solid-state fermentation culture medium.
The inventive method has overcome the shortcoming of FFase liquid state fermentation, and the solid state fermentation production method of a kind of FFase is provided, and by the large-scale commercial production that the method for solid state fermentation is oligofructose, provides high-quality enzyme source.The advantage of the inventive method is: it is raw material that (1) solid-state fermentation culture medium be take bagasse, rice husk, soybean cake etc., and cost of material is cheap; (2) fermentation period is short, and processing unit is simple, reduces costs, and benefits suitability for industrialized production; (3) liquid waste water greatly reduces compared with liquid fermenting, fundamentally effectively solves sewage discharge problem, is very beneficial for environment protection.
Embodiment
The detection method adopting in the following example is:
FFase turns the mensuration of fructosyl vigor, Sodium phosphate dibasic-citric acid solution preparation 25%(w/w with 50mM, pH 5.5) sucrose solution is as substrate, add thick FFase enzyme liquid (to make to react after 1 hour in right amount, in system, the content of kestose is no more than the 10%(w/w of total sugar content) be advisable), be placed in 55 ℃ of water-bath oscillatory reactions 1 hour, then in boiling water bath, heat 10 minutes, stop enzyme reaction.With the kestose in high effective liquid chromatography for measuring reaction system, by the amount of kestose, calculate the fructosyl vigor that turns.An enzyme activity unit is defined as: per minute produces the required enzyme amount of 1umoL kestose under these conditions.
Chromatographic condition: chromatographic column, Hypersil-NH
2(4.6 * 250 * 5, Dalian is according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S.); The sensitivity of differential detector, 1/8; Moving phase, acetonitrile: water=68:32; Flow velocity, 1mL/min; Column temperature, 30 ℃.
The aspergillus niger adopting in the present invention and the following example is aspergillus niger AS 3.877, it is the open bacterial strain for asking for, buying of Chinese common micro-organisms culture presevation administrative center (CGMCC), bacterium numbering is 3.877, and Chinese bacterium is called aspergillus niger, and Latin formal name used at school is Aspergillus niger.
Embodiment 1:
Solid state fermentation is produced FFase.
(1) preparation of spore liquid: 30g sucrose, 3g SODIUMNITRATE, 1g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.5g Repone K, 0.01g ferrous sulfate, 15g agar are dissolved in 800 ml deionized water, be settled to again 1000 milliliters, pH nature, 121 ℃ of sterilizings 20 minutes, 30 degree that tilt before cooled and solidified are made test tube slant substratum.Aspergillus niger AS 3.877 is inoculated on the test tube slant substratum preparing, cultivates for 25 ℃ and within 5 days, grow spore, the physiological saline washing mycelium to adding 0.9% on inclined-plane, obtains spore liquid.
(2) enlarged culturing of seed: 20g cane molasses, 10g peptone, 5g yeast extract paste, 2g SODIUMNITRATE, 3g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.5g Repone K are dissolved in 800 ml deionized water, be settled to again 1000 milliliters, pH nature, 121 ℃ of sterilizings 20 minutes, prepare liquid seed culture medium.Inoculum size according to volume ratio 2% accesses liquid seed culture medium by spore liquid, and on 25 ℃, the shaking table of rotating speed 180rpm, enlarged culturing is 2 days, obtains seed liquor.
(3) Produced by Solid-state Fermentation FFase: the nutritive medium of 1.5 times of weight is evenly sprayed on solid mixture, 121 ℃ of sterilizings 20 minutes, preparation solid-state fermentation culture medium.Described nutritive medium consists of (g/L): cane molasses 10, and corn steep liquor 5, skim-milk 3, SODIUMNITRATE 1, dipotassium hydrogen phosphate 2, magnesium sulfate 0.5, Repone K 0.5, these materials are first dissolved in 800 ml deionized water, and then constant volume to 1 liter.Described solid mixture is mixed and is formed by bagasse, rice husk, soybean cake three, and its weight proportion of composing is bagasse: rice husk: soybean cake is 3:1:0.5.The solid-state fermentation culture medium that seed liquor is prepared according to the ratio access of 5 milliliters/100 grams of solid mediums, fully be uniformly mixed, then 1 centimetre of bed thickness, (be that seed liquor first adds in solid fermentation substratum, then be fully uniformly mixed, after mixing, this mixture is placed in fermenting container, make 1 centimetre of its height, lower with), standing for fermentation 3 days at 25 ℃ of humidity 45%, leavening temperature.
(4) aftertreatment obtains FFase: get solid culture 10g, add the citrate buffer of 80 milliliter of 50 mmole pH 5.5, with tissue mashing machine, pulverize, then stirring and leaching 4 hours, to filter, filtrate is thick FFase enzyme liquid.According to detection method, recording enzyme lives as 236.48U/mL.
Embodiment 2:
Solid state fermentation is produced FFase.
(1) preparation of the preparation test tube slant substratum of spore liquid is with embodiment 1.Aspergillus niger AS 3.877 is inoculated on the test tube slant substratum preparing, cultivates for 26 ℃ and within 5 days, grow spore, on inclined-plane, add physiological saline washing mycelium, obtain spore liquid.
(2) preparation of the enlarged culturing liquid seed culture medium of seed is with embodiment 1.Inoculum size according to volume ratio 2.25% accesses liquid seed culture medium by spore liquid, and on 26 ℃, the shaking table of rotating speed 185rpm, enlarged culturing is 2 days, obtains seed liquor.
(3) Produced by Solid-state Fermentation FFase is evenly sprayed at the nutritive medium of 1.75 times of weight on solid mixture, 121 ℃ of sterilizings 20 minutes, and preparation solid-state fermentation culture medium.Described nutritive medium consists of (g/L): cane molasses 12.5, and corn steep liquor 7.5, skim-milk 4, SODIUMNITRATE 1.25, dipotassium hydrogen phosphate 2.25, magnesium sulfate 0.5, Repone K 0.5, these materials are first dissolved in 800 ml deionized water, and then constant volume to 1 liter.Described solid mixture is mixed and is formed by bagasse, rice husk, soybean cake three, and its weight proportion of composing is bagasse: rice husk: soybean cake is 3.25:1.25:0.75.The solid-state fermentation culture medium that seed liquor is prepared according to the ratio access of 7.5 milliliters/100 grams of solid mediums, is fully uniformly mixed, then standing for fermentation 3 days at 26 ℃ of 1.25 centimetres of bed thickness, humidity 50%, leavening temperature.
(4) aftertreatment obtains FFase and gets solid culture 10g, adds the citrate buffer of 80 milliliter of 50 mmole pH 5.5, with tissue mashing machine, pulverize, then stirring and leaching 4.5 hours, to filter, filtrate is thick FFase enzyme liquid.According to detection method, recording enzyme lives as 274.32U/mL.
Embodiment 3:
Solid state fermentation is produced FFase.
(1) preparation of the preparation test tube slant substratum of spore liquid is with embodiment 1.Aspergillus niger AS 3.877 is inoculated on the test tube slant substratum preparing, cultivates for 27 ℃ and within 5 days, grow spore, on inclined-plane, add physiological saline washing mycelium, obtain spore liquid.
(2) preparation of the enlarged culturing liquid seed culture medium of seed is with embodiment 1.Inoculum size according to volume ratio 2.5% accesses liquid seed culture medium by spore liquid, and on 27 ℃, the shaking table of rotating speed 190rpm, enlarged culturing is 2 days, obtains seed liquor.
(3) Produced by Solid-state Fermentation FFase is evenly sprayed at the nutritive medium of 2 times of weight on solid mixture, 121 ℃ of sterilizings 20 minutes, and preparation solid-state fermentation culture medium.Described nutritive medium consists of (g/L): cane molasses 15, and corn steep liquor 10, skim-milk 5, SODIUMNITRATE 1.5, dipotassium hydrogen phosphate 2.5, magnesium sulfate 0.5, Repone K 0.5, these materials are first dissolved in 800 ml deionized water, and then constant volume to 1 liter.Described solid mixture is mixed and is formed by bagasse, rice husk, soybean cake three, and its weight proportion of composing is bagasse: rice husk: soybean cake is 3.5:1.5:1.0.The solid-state fermentation culture medium that seed liquor is prepared according to the ratio access of 10 milliliters/100 grams of solid mediums, is fully uniformly mixed, then standing for fermentation 3 days at 27 ℃ of 1.5 centimetres of bed thickness, humidity 55%, leavening temperature.
(4) aftertreatment obtains FFase and gets solid culture 10g, adds the citrate buffer of 80 milliliter of 50 mmole pH 5.5, with tissue mashing machine, pulverize, then stirring and leaching 5 hours, to filter, filtrate is thick FFase enzyme liquid.According to detection method, recording enzyme lives as 355.25U/mL.
Embodiment 4:
Solid state fermentation is produced FFase.
(1) preparation of the preparation test tube slant substratum of spore liquid is with embodiment 1.Aspergillus niger AS 3.877 is inoculated on the test tube slant substratum preparing, cultivates for 28 ℃ and within 4 days, grow spore, on inclined-plane, add physiological saline washing mycelium, obtain spore liquid.
(2) preparation of the enlarged culturing liquid seed culture medium of seed is with embodiment 1.Inoculum size according to volume ratio 2.75% accesses liquid seed culture medium by spore liquid, and on 28 ℃, the shaking table of rotating speed 195rpm, enlarged culturing is 3 days, obtains seed liquor.
(3) Produced by Solid-state Fermentation FFase is evenly sprayed at the nutritive medium of 2.25 times of weight on solid mixture, 121 ℃ of sterilizings 20 minutes, and preparation solid-state fermentation culture medium.Described nutritive medium consists of (g/L): cane molasses 17.5, and corn steep liquor 10, skim-milk 6, SODIUMNITRATE 1.75, dipotassium hydrogen phosphate 2.75, magnesium sulfate 0.5, Repone K 0.5, these materials are first dissolved in 800 ml deionized water, and then constant volume to 1 liter.Described solid mixture is mixed and is formed by bagasse, rice husk, soybean cake three, and its weight proportion of composing is bagasse: rice husk: soybean cake is 3.75:1.75:1.0.The solid-state fermentation culture medium that seed liquor is prepared according to the ratio access of 10 milliliters/100 grams of solid mediums, is fully uniformly mixed, then standing for fermentation 4 days at 28 ℃ of 1.75 centimetres of bed thickness, humidity 60%, leavening temperature.
(4) aftertreatment obtains FFase and gets solid culture 10g, adds the citrate buffer of 80 milliliter of 50 mmole pH 5.5, with tissue mashing machine, pulverize, then stirring and leaching 5 hours, to filter, filtrate is thick FFase enzyme liquid.According to detection method, recording enzyme lives as 420.45U/mL.
Embodiment 5:
Solid state fermentation is produced FFase.
(1) preparation of the preparation test tube slant substratum of spore liquid is with embodiment 1.Aspergillus niger AS3.877 is inoculated on the test tube slant substratum preparing, cultivates for 28 ℃ and within 4 days, grow spore, on inclined-plane, add physiological saline washing mycelium, obtain spore liquid.
(2) preparation of the enlarged culturing liquid seed culture medium of seed is with embodiment 1.Inoculum size according to volume ratio 3% accesses liquid seed culture medium by spore liquid, and on 28 ℃, the shaking table of rotating speed 200rpm, enlarged culturing is 3 days, obtains seed liquor.
(3) Produced by Solid-state Fermentation FFase is evenly sprayed at the nutritive medium of 2.5 times of weight on solid mixture, 121 ℃ of sterilizings 20 minutes, and preparation solid-state fermentation culture medium.Described nutritive medium consists of (g/L): cane molasses 20, and corn steep liquor 10, skim-milk 5, SODIUMNITRATE 2, dipotassium hydrogen phosphate 3, magnesium sulfate 0.5, Repone K 0.5, these materials are first dissolved in 800 ml deionized water, and then constant volume to 1 liter.Described solid mixture is mixed and is formed by bagasse, rice husk, soybean cake three, and its weight proportion of composing is bagasse: rice husk: soybean cake is 4:2:1.The solid-state fermentation culture medium that seed liquor is prepared according to the ratio access of 10 milliliters/100 grams of solid mediums, is fully uniformly mixed, then standing for fermentation 4 days at 28 ℃ of 2 centimetres of bed thickness, humidity 60%, leavening temperature.
(4) aftertreatment obtains FFase and gets solid culture 10g, adds the citrate buffer of 80 milliliter of 50 mmole pH 5.5, with tissue mashing machine, pulverize, then stirring and leaching 5 hours, to filter, filtrate is thick FFase enzyme liquid.According to detection method, recording enzyme lives as 521.65U/mL.
Embodiment 6:
Solid state fermentation is produced FFase.
(1) preparation of the preparation test tube slant substratum of spore liquid is with embodiment 1.Aspergillus niger AS3.877 is inoculated on the test tube slant substratum preparing, cultivates for 28 ℃ and within 4 days, grow spore, on inclined-plane, add physiological saline washing mycelium, obtain spore liquid.
(2) preparation of the enlarged culturing liquid seed culture medium of seed is with embodiment 1.Inoculum size according to volume ratio 3.5% accesses liquid seed culture medium by spore liquid, and on 28 ℃, the shaking table of rotating speed 200rpm, enlarged culturing is 3 days, obtains seed liquor.
(3) Produced by Solid-state Fermentation FFase is evenly sprayed at the nutritive medium of 2.5 times of weight on solid mixture, 121 ℃ of sterilizings 20 minutes, and preparation solid-state fermentation culture medium.Described nutritive medium consists of (g/L): cane molasses 20, and corn steep liquor 12.5, skim-milk 5, SODIUMNITRATE 2, dipotassium hydrogen phosphate 3.5, magnesium sulfate 0.5, Repone K 0.5, these materials are first dissolved in 800 ml deionized water, and then constant volume to 1 liter.Described solid mixture is mixed and is formed by bagasse, rice husk, soybean cake three, and its weight proportion of composing is bagasse: rice husk: soybean cake is 4.5:2.5:1.The solid-state fermentation culture medium that seed liquor is prepared according to the ratio access of 10 milliliters/100 grams of solid mediums, is fully uniformly mixed, then standing for fermentation 4 days at 28 ℃ of 2 centimetres of bed thickness, humidity 65%, leavening temperature.
(4) aftertreatment obtains FFase and gets solid culture 10g, adds the citrate buffer of 80 milliliter of 50 mmole pH 5.5, with tissue mashing machine, pulverize, then stirring and leaching 5.5 hours, to filter, filtrate is thick FFase enzyme liquid.According to detection method, recording enzyme lives as 486.13U/mL.
Embodiment 7:
Solid state fermentation is produced FFase.
(1) preparation of the preparation test tube slant substratum of spore liquid is with embodiment 1.Aspergillus niger AS 3.877 is inoculated on the test tube slant substratum preparing, cultivates for 28 ℃ and within 4 days, grow spore, on inclined-plane, add physiological saline washing mycelium, obtain spore liquid.
(2) preparation of the enlarged culturing liquid seed culture medium of seed is with embodiment 1.Inoculum size according to volume ratio 3.75% accesses liquid seed culture medium by spore liquid, and on 28 ℃, the shaking table of rotating speed 200rpm, enlarged culturing is 3 days, obtains seed liquor.
(3) Produced by Solid-state Fermentation FFase is evenly sprayed at the nutritive medium of 2.5 times of weight on solid mixture, 121 ℃ of sterilizings 20 minutes, and preparation solid-state fermentation culture medium.Described nutritive medium consists of (g/L): cane molasses 22.5, and corn steep liquor 12.5, skim-milk 7, SODIUMNITRATE 2, dipotassium hydrogen phosphate 3.5, magnesium sulfate 0.5, Repone K 0.5, these materials are first dissolved in 800 ml deionized water, and then constant volume to 1 liter.Described solid mixture is mixed and is formed by bagasse, rice husk, soybean cake three, and its weight proportion of composing is bagasse: rice husk: soybean cake is 4.75:2.5:1.25.The solid-state fermentation culture medium that seed liquor is prepared according to the ratio access of 12.5 milliliters/100 grams of solid mediums, is fully uniformly mixed, then standing for fermentation 4 days at 28 ℃ of 2 centimetres of bed thickness, humidity 65%, leavening temperature.
(4) aftertreatment obtains FFase and gets solid culture 10g, adds the citrate buffer of 80 milliliter of 50 mmole pH 5.5, with tissue mashing machine, pulverize, then stirring and leaching 5.5 hours, to filter, filtrate is thick FFase enzyme liquid.According to detection method, recording enzyme lives as 465.20U/mL.
Embodiment 8:
Solid state fermentation is produced FFase.
(1) preparation of the preparation test tube slant substratum of spore liquid is with embodiment 1.Aspergillus niger AS 3.877 is inoculated on the test tube slant substratum preparing, cultivates 4 days to growing spore for 29 ℃, on inclined-plane, add physiological saline washing mycelium, obtain spore liquid.
(2) preparation of the enlarged culturing liquid seed culture medium of seed is with embodiment 1.Inoculum size according to volume ratio 4% accesses liquid seed culture medium by spore liquid, and on 29 ℃, the shaking table of rotating speed 200rpm, enlarged culturing is 3 days, obtains seed liquor.
(3) Produced by Solid-state Fermentation FFase is evenly sprayed at the nutritive medium of 2.5 times of weight on solid mixture, 121 ℃ of sterilizings 20 minutes, and preparation solid-state fermentation culture medium.Described nutritive medium consists of (g/L): cane molasses 25, and corn steep liquor 12.5, skim-milk 7, SODIUMNITRATE 2.5, dipotassium hydrogen phosphate 4, magnesium sulfate 0.5, Repone K 0.5, these materials are first dissolved in 800 ml deionized water, and then constant volume to 1 liter.Described solid mixture is mixed and is formed by bagasse, rice husk, soybean cake three, and its weight proportion of composing is bagasse: rice husk: soybean cake is 5:2.75:1.25.The solid-state fermentation culture medium that seed liquor is prepared according to the ratio access of 12.5 milliliters/100 grams of solid mediums, is fully uniformly mixed, then standing for fermentation 4 days at 29 ℃ of 2.5 centimetres of bed thickness, humidity 70%, leavening temperature.
(4) aftertreatment obtains FFase and gets solid culture 10g, adds the citrate buffer of 80 milliliter of 50 mmole pH 5.5, with tissue mashing machine, pulverize, then stirring and leaching 6 hours, to filter, filtrate is thick FFase enzyme liquid.According to detection method, recording enzyme lives as 440.25U/mL.
Embodiment 9:
Solid state fermentation is produced FFase.
(1) preparation of the preparation test tube slant substratum of spore liquid is with embodiment 1.Aspergillus niger AS 3.877 is inoculated on the test tube slant substratum preparing, cultivates for 30 ℃ and within 4 days, grow spore, on inclined-plane, add physiological saline washing mycelium, obtain spore liquid.
(2) preparation of the enlarged culturing liquid seed culture medium of seed is with embodiment 1.Inoculum size according to volume ratio 4.5% accesses liquid seed culture medium by spore liquid, and on 30 ℃, the shaking table of rotating speed 210rpm, enlarged culturing is 3 days, obtains seed liquor.
(3) Produced by Solid-state Fermentation FFase is evenly sprayed at the nutritive medium of 2.75 times of weight on solid mixture, 121 ℃ of sterilizings 20 minutes, and preparation solid-state fermentation culture medium.Described nutritive medium consists of (g/L): cane molasses 27.5, and corn steep liquor 12.5, skim-milk 8, SODIUMNITRATE 2.75, dipotassium hydrogen phosphate 4.5, magnesium sulfate 0.5, Repone K 0.5, these materials are first dissolved in 800 ml deionized water, and then constant volume to 1 liter.Described solid mixture is mixed and is formed by bagasse, rice husk, soybean cake three, and its weight proportion of composing is bagasse: rice husk: soybean cake is 5.5:2.75:1.25.The solid-state fermentation culture medium that seed liquor is prepared according to the ratio access of 12.5 milliliters/100 grams of solid mediums, is fully uniformly mixed, then standing for fermentation 4 days at 32 ℃ of 2.75 centimetres of bed thickness, humidity 75%, leavening temperature.
(4) aftertreatment obtains FFase and gets solid culture 10g, adds the citrate buffer of 80 milliliter of 50 mmole pH 5.5, with tissue mashing machine, pulverize, then stirring and leaching 7 hours, to filter, filtrate is thick FFase enzyme liquid.According to detection method, recording enzyme lives as 365.45U/mL.
Embodiment 10:
Solid state fermentation is produced FFase.
(1) preparation of the preparation test tube slant substratum of spore liquid is with embodiment 1.Aspergillus niger AS 3.877 is inoculated on the test tube slant substratum preparing, cultivates for 32 ℃ and within 5 days, grow spore, on inclined-plane, add physiological saline washing mycelium, obtain spore liquid.
(2) preparation of the enlarged culturing liquid seed culture medium of seed is with embodiment 1.Inoculum size according to volume ratio 5% accesses liquid seed culture medium by spore liquid, and on 32 ℃, the shaking table of rotating speed 220rpm, enlarged culturing is 3 days, obtains seed liquor.
(3) Produced by Solid-state Fermentation FFase is evenly sprayed at the nutritive medium of 3 times of weight on solid mixture, 121 ℃ of sterilizings 20 minutes, and preparation solid-state fermentation culture medium.Described nutritive medium consists of (g/L): cane molasses 27.5, and corn steep liquor 15, skim-milk 9, SODIUMNITRATE 3, dipotassium hydrogen phosphate 5, magnesium sulfate 0.5, Repone K 0.5, these materials are first dissolved in 800 ml deionized water, and then constant volume to 1 liter.Described solid mixture is mixed and is formed by bagasse, rice husk, soybean cake three, and its weight proportion of composing is bagasse: rice husk: soybean cake is 6:2.5:1.5.The solid-state fermentation culture medium that seed liquor is prepared according to the ratio access of 15 milliliters/100 grams of solid mediums, is fully uniformly mixed, then standing for fermentation 5 days at 32 ℃ of 2.75 centimetres of bed thickness, humidity 75%, leavening temperature.
(4) aftertreatment obtains FFase and gets solid culture 10g, adds the citrate buffer of 80 milliliter of 50 mmole pH 5.5, with tissue mashing machine, pulverize, then stirring and leaching 7 hours, to filter, filtrate is thick FFase enzyme liquid.According to detection method, recording enzyme lives as 205.85U/mL.
Embodiment 11:
Solid state fermentation is produced FFase.
(1) preparation of the preparation test tube slant substratum of spore liquid is with embodiment 1.Aspergillus niger AS 3.877 is inoculated on the test tube slant substratum preparing, cultivates for 32 ℃ and within 5 days, grow spore, on inclined-plane, add physiological saline washing mycelium, obtain spore liquid.
(2) preparation of the enlarged culturing liquid seed culture medium of seed is with embodiment 1.Inoculum size according to volume ratio 5% accesses liquid seed culture medium by spore liquid, and on 32 ℃, the shaking table of rotating speed 220rpm, enlarged culturing is 4 days, obtains seed liquor.
(3) Produced by Solid-state Fermentation FFase is evenly sprayed at the nutritive medium of 3 times of weight on solid mixture, 121 ℃ of sterilizings 20 minutes, and preparation solid-state fermentation culture medium.Described nutritive medium consists of (g/L): cane molasses 30, and corn steep liquor 15, skim-milk 10, SODIUMNITRATE 3, dipotassium hydrogen phosphate 5, magnesium sulfate 0.5, Repone K 0.5, these materials are first dissolved in 800 ml deionized water, and then constant volume to 1 liter.Described solid mixture is mixed and is formed by bagasse, rice husk, soybean cake three, and its weight proportion of composing is bagasse: rice husk: soybean cake is 6:3:1.5.The solid-state fermentation culture medium that seed liquor is prepared according to the ratio access of 15 milliliters/100 grams of solid mediums, is fully uniformly mixed, then standing for fermentation 5 days at 35 ℃ of 3 centimetres of bed thickness, humidity 80%, leavening temperature.
(4) aftertreatment obtains FFase and gets solid culture 10g, adds the citrate buffer of 80 milliliter of 50 mmole pH 5.5, with tissue mashing machine, pulverize, then stirring and leaching 7 hours, to filter, filtrate is thick FFase enzyme liquid.According to detection method, recording enzyme lives as 128.36U/mL.
Above-mentioned detailed description of the method for solid state fermentation production FFase being carried out with reference to embodiment; illustrative and not restrictive; can list several embodiment according to institute's limited range; therefore in any variation and the modification that do not depart under overall thought of the present invention, within should belonging to protection scope of the present invention.
Claims (1)
1. a solid state fermentation production method for beta-D-fructofuranose glycosides enzyme, is characterized in that, comprises the steps:
(1) preparation of spore liquid: 30g sucrose, 3g SODIUMNITRATE, 1g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.01g ferrous sulfate, 15g agar are dissolved in 800 ml deionized water, be settled to again 1000 milliliters, pH nature, 121 ℃ of sterilizings 20 minutes, 30 degree that tilt before cooled and solidified are made test tube slant substratum; Aspergillus niger AS3.877 is inoculated on the test tube slant substratum preparing, cultivates for 28 ℃ and within 4 days, grow spore, on inclined-plane, add physiological saline washing mycelium, obtain spore liquid;
(2) enlarged culturing of seed: 20g cane molasses, 10g peptone, 5g yeast extract paste, 2g SODIUMNITRATE, 3g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.5g Repone K are dissolved in 800 ml deionized water, be settled to again 1000 milliliters, pH nature, 121 ℃ of sterilizings 20 minutes, prepare liquid seed culture medium; Inoculum size according to volume ratio 3% accesses liquid seed culture medium by spore liquid, and on 28 ℃, the shaking table of rotating speed 200rpm, enlarged culturing is 3 days, obtains seed liquor;
(3) Produced by Solid-state Fermentation beta-D-fructofuranose glycosides enzyme: the nutritive medium of 2.5 times of weight is evenly sprayed on solid mixture, 121 ℃ of sterilizings 20 minutes, preparation solid-state fermentation culture medium; Described nutritive medium consists of (g/L): cane molasses 20, and corn steep liquor 10, skim-milk 5, SODIUMNITRATE 2, dipotassium hydrogen phosphate 3, magnesium sulfate 0.5, Repone K 0.5, these materials are first dissolved in 800 ml deionized water, and then constant volume to 1 liter; Described solid mixture is mixed and is formed by bagasse, rice husk, soybean cake three, its weight proportion of composing is bagasse: rice husk: soybean cake is 4:2:1, the solid-state fermentation culture medium that seed liquor is prepared according to the ratio access of 10 milliliters/100g solid medium, fully be uniformly mixed, then standing for fermentation 4 days at 28 ℃ of 2 centimetres of bed thickness, humidity 60%, leavening temperature;
(4) aftertreatment obtains beta-D-fructofuranose glycosides enzyme: get solid culture 10g, the citrate buffer that adds 80 milliliter of 50 mmole pH5.5, pulverizes with tissue mashing machine, then stirring and leaching 5 hours, filter, filtrate is thick beta-D-fructofuranose glycosides enzyme enzyme liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210545255.7A CN103045561B (en) | 2012-12-17 | 2012-12-17 | Solid state fermentation production method for beta-D-fructofuranosidase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210545255.7A CN103045561B (en) | 2012-12-17 | 2012-12-17 | Solid state fermentation production method for beta-D-fructofuranosidase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103045561A CN103045561A (en) | 2013-04-17 |
| CN103045561B true CN103045561B (en) | 2014-12-03 |
Family
ID=48058425
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210545255.7A Expired - Fee Related CN103045561B (en) | 2012-12-17 | 2012-12-17 | Solid state fermentation production method for beta-D-fructofuranosidase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103045561B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148243B (en) * | 2016-08-29 | 2019-03-19 | 山东百龙创园生物科技股份有限公司 | One plant of production saccharase arthrobacterium and its application |
| CN106754411B (en) * | 2016-11-29 | 2020-06-05 | 山东隆科特酶制剂有限公司 | Aspergillus niger strain with high yield of β -D-fructofuranosidase and liquid fermentation enzyme production method thereof |
| CN110172407A (en) * | 2018-12-11 | 2019-08-27 | 青岛蔚蓝生物集团有限公司 | One plant of aspergillus oryzae for producing transfructosylase and its application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1626656A (en) * | 2003-12-12 | 2005-06-15 | 中国科学院沈阳应用生态研究所 | Method for preparing composite enzyme of pectin in high vigor |
| CN1920016A (en) * | 2006-08-25 | 2007-02-28 | 天津达美科技有限公司 | Preparation method of solid-state machinery fermentation xylanase |
-
2012
- 2012-12-17 CN CN201210545255.7A patent/CN103045561B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1626656A (en) * | 2003-12-12 | 2005-06-15 | 中国科学院沈阳应用生态研究所 | Method for preparing composite enzyme of pectin in high vigor |
| CN1920016A (en) * | 2006-08-25 | 2007-02-28 | 天津达美科技有限公司 | Preparation method of solid-state machinery fermentation xylanase |
Non-Patent Citations (6)
| Title |
|---|
| Balasubramaniem Ashokkumar et al.Optimization of media for β-fructofuranosidase production by Aspergillus niger in submerged and solid state fermentation.《Process Biochemistry》.2001,第37卷(第4期),331-338. * |
| Balasubramaniem Ashokkumar et al.β-fructofuranosidase production by 2-deoxyglucose resistant mutants of Aspergillus niger in submerged and solid state fermentation.《Indian Journal of Experimental Biology》.2002,第40卷(第9期),1032-1037. * |
| Optimization of media for β-fructofuranosidase production by Aspergillus niger in submerged and solid state fermentation;Balasubramaniem Ashokkumar et al;《Process Biochemistry》;20011210;第37卷(第4期);331-338 * |
| β-fructofuranosidase production by 2-deoxyglucose resistant mutants of Aspergillus niger in submerged and solid state fermentation;Balasubramaniem Ashokkumar et al;《Indian Journal of Experimental Biology》;20020930;第40卷(第9期);1032-1037 * |
| 杨晓兰等.黑曲霉固体发酵条件的优化.《养殖与饲料》.2012,(第12期),19-21. * |
| 黑曲霉固体发酵条件的优化;杨晓兰等;《养殖与饲料》;20121201(第12期);19-21 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103045561A (en) | 2013-04-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101225121B (en) | Unsaturated pectin oligosaccharide and compound biological preservatives | |
| CN102894447B (en) | Ganoderma lucidum polysaccharide health-care drink and production method thereof | |
| CN104087638A (en) | Method for preparing antioxidative peptide through fermentation of rice residue by use of bacillus subtilis | |
| CN101200750B (en) | Rhubarb horsetails Erwinia sp. and its application in preparation of isomaltulose | |
| CN102296032B (en) | Transglucosidase, its preparation method and immobilization method | |
| CN103045561B (en) | Solid state fermentation production method for beta-D-fructofuranosidase | |
| CN102533877B (en) | Method for preparing citric acid by fermentation | |
| Cui et al. | Production, purification and analysis of the isomalto-oligosaccharides from Chinese chestnut (Castanea mollissima Blume) and the prebiotics effects of them on proliferation of Lactobacillus | |
| CN107853588A (en) | A kind of preparation method of feature highland barley flour | |
| CN101857890A (en) | Method for biologically converting stevioside in stevia sugar into rebaudioside | |
| CN101531975B (en) | Lactobacilus fermentum and method for preparing D-tagatose by utilizing Lactobacilus fermentum | |
| Prangviset et al. | Fructose production from Jerusalem artichoke using mixed inulinases | |
| CN111534557A (en) | Selenium-rich konjac oligosaccharide and preparation method thereof | |
| CN104059857A (en) | Aspergillus and application of aspergillus in fructosyltransferase preparing | |
| CN102443611B (en) | Production method of citric acid | |
| CN104911106B (en) | A kind of method that thermophilic loose penicillium bacterial strain and its bacterial strain prepare dextranase | |
| CN100526470C (en) | Preparation method of functional sweetener D-tatai sugar | |
| CN105112387A (en) | Preparation method of lipase | |
| CN102010857B (en) | Method for producing acid xylanase from whey powder | |
| CN102168122A (en) | A method employing Enterococcus faecalis fermentation for preparing chitin from exoskeletons of crustaceans | |
| CN104711208B (en) | A kind of lactic acid bacteria with high starch capacity of decomposition | |
| JP4338356B2 (en) | Microorganism producing fructosyltransferase, and method for producing fructooligosaccharide and neoflactooligosaccharide using the same | |
| CN104774880A (en) | Preparation method of L-lactic acid by fermenting sweet sorghum straw juice | |
| CN101508966B (en) | Method for preparing manna oligosacchride with zymohydrolysis of konjaku flour | |
| CN102919522A (en) | Preparation method for feed for ruminants |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Solid state fermentation production method for beta-D-fructofuranosidase Effective date of registration: 20161208 Granted publication date: 20141203 Pledgee: Bank of Shizuishan Limited by Share Ltd. Central Branch Pledgor: NINGXIA YIZHENG BIOLOGICAL ENGINEERING Co.,Ltd. Registration number: 2016640000002 |
|
| PLDC | Enforcement, change and cancellation of contracts on pledge of patent right or utility model | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20171127 Granted publication date: 20141203 Pledgee: Bank of Shizuishan Limited by Share Ltd. Central Branch Pledgor: NINGXIA YIZHENG BIOLOGICAL ENGINEERING Co.,Ltd. Registration number: 2016640000002 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Solid state fermentation production method for beta-D-fructofuranosidase Effective date of registration: 20171128 Granted publication date: 20141203 Pledgee: Bank of Shizuishan Limited by Share Ltd. Central Branch Pledgor: NINGXIA YIZHENG BIOLOGICAL ENGINEERING Co.,Ltd. Registration number: 2017640000005 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20181203 Granted publication date: 20141203 Pledgee: Bank of Shizuishan Limited by Share Ltd. Central Branch Pledgor: NINGXIA YIZHENG BIOLOGICAL ENGINEERING Co.,Ltd. Registration number: 2017640000005 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Solid state fermentation production method for beta-D-fructofuranosidase Effective date of registration: 20181205 Granted publication date: 20141203 Pledgee: Bank of Shizuishan Limited by Share Ltd. Central Branch Pledgor: NINGXIA YIZHENG BIOLOGICAL ENGINEERING Co.,Ltd. Registration number: 2018640000004 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20191127 Granted publication date: 20141203 Pledgee: Bank of Shizuishan Limited by Share Ltd. Central Branch Pledgor: NINGXIA YIZHENG BIOLOGICAL ENGINEERING Co.,Ltd. Registration number: 2018640000004 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Solid state fermentation production method for beta-D-fructofuranosidase Effective date of registration: 20191211 Granted publication date: 20141203 Pledgee: Bank of Shizuishan Limited by Share Ltd. Central Branch Pledgor: NINGXIA YIZHENG BIOLOGICAL ENGINEERING Co.,Ltd. Registration number: Y2019640000003 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20201217 Granted publication date: 20141203 Pledgee: Bank of Shizuishan Limited by Share Ltd. Central Branch Pledgor: NINGXIA YIZHENG BIOLOGICAL ENGINEERING Co.,Ltd. Registration number: Y2019640000003 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A production method of b - d-fructofuranosidase by solid state fermentation Effective date of registration: 20201222 Granted publication date: 20141203 Pledgee: Bank of Shizuishan Limited by Share Ltd. Central Branch Pledgor: NINGXIA YIZHENG BIOLOGICAL ENGINEERING Co.,Ltd. Registration number: Y2020640000005 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20211229 Granted publication date: 20141203 Pledgee: Bank of Shizuishan Limited by Share Ltd. Central Branch Pledgor: NINGXIA YIZHENG BIOLOGICAL ENGINEERING Co.,Ltd. Registration number: Y2020640000005 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20141203 Termination date: 20211217 |