CN103045561B - Solid state fermentation production method for beta-D-fructofuranosidase - Google Patents

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

A kind of solid state fermentation production method of beta-D-fructofuranose glycosides enzyme

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-NH₂(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.