CN101381711B - Method for producing inulase by solid fermentation - Google Patents
Method for producing inulase by solid fermentation Download PDFInfo
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- CN101381711B CN101381711B CN2008101524225A CN200810152422A CN101381711B CN 101381711 B CN101381711 B CN 101381711B CN 2008101524225 A CN2008101524225 A CN 2008101524225A CN 200810152422 A CN200810152422 A CN 200810152422A CN 101381711 B CN101381711 B CN 101381711B
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Abstract
The invention relates to a method for producing inulinase through solid state fermentation, namely using a solid state bioreactor to produce the inulinase through fermentation. The method comprises the following steps: (1) inoculating Kluyveromyces fragilis strains onto the agar slant of yeast powder, peptone and inulin, cultivating the strains for four to six days at a constant temperature of 30DEG C, and adding 0.75 percent (W/W) of normal saline to wash mycelium so as to obtain bacteria suspension the spore content of which is between 10<5> and 10<6>; (2) inoculating the bacteria suspension to the solid culture medium after sterilization to be cultivated into solid seeds or cultivated into liquid seeds through liquid expansion again; (3) adding the liquid seeds in the solid culture medium by 5ml to 10ml per 100 grams or adding the solid seeds in the solid culture medium by 5mg to 10mg per 100 grams, sufficiently stirring and mixing the mixture to obtain the thickness of between 0.5 and 3.0 centimeters, the humidity of between 50 percent and 80 percent, introducing 0.2 to 1.5V/(V. min) of sterile air, keeping the temperature between 25 and 45 DEG C, and making the mixture keep stand for 4 to 6 days to ferment; and (4) diluting the products, collecting the supernatant containing the inulinase through centrifugation, and drying and pulverizing the products to obtain an inulinase preparation. The method effectively improves the yield of the inulinase, shortens the fermentation cycle, reduces pollution discharge, and is advantageous to environmental protection.
Description
Technical field
The present invention relates to the method for fermentative production inulinase, particularly a kind of method of producing inulase by solid fermentation.
Background technology
Inulin (inulin) is to separate from feverfew and gain the name the earliest, and it mainly is stored in the root and stem tuber of crops such as jerusalem artichoke.Inulin is that a kind of d one fructose by the furans configuration is polymerized through the dehydration of β-2,1 glycosidic link, and is connected with a glucosyl residue at reducing end under neutral.The polymerization degree is very good functional food ingredients from the mixture of many kinds of Polylevulosan of 2-60, also is simultaneously the good raw material of producing products such as oligofructose, high fructose syrup, crystal diabetin.
Inulinase (inulinase) is can hydrolysis β-2, a class lytic enzyme of 1-d one Polylevulosan fructose glycosidic bond, and formal name used at school β-2,1-d one levanase is named β one levanase or 2 again, 1-d one fructan-hydrolying enzyme.The microorganism of secretion inulinase distributes very wide at occurring in nature, the multiple microorganism in soil, water and the animal digestive tract can both secrete.Inulinase can become high purity fructose or oligofructose to hydrolysis of inulin by a step enzyme method under certain temperature condition.And fructose or oligofructose are sweeting agent, functional food ingredient and the additives more better than sucrose, as seen produce inulinase and become particularly important.Generally adopt liquid fermenting to produce inulinase traditionally, but the inulinase that adopts the deep fermentation method to produce yields poorly, the product enzymic fermentation cycle requires complicated than production unit long and that relate to, therefore investment is big, the tooling cost height, be unsuitable for suitability for industrialized production, the discharging of waste water simultaneously causes environmental pollution more serious.
Summary of the invention
The objective of the invention is to overcome above-mentioned weak point, produce the existing problem of inulinase at present liquid submerged fermentation method, providing a kind of is the method for the producing inulase by solid fermentation of substratum main raw material with cereal, be intended to simplify its production technique, shorten fermentation period, improve inulinase output, reduce investment, reduce cost, benefit production application and fundamentally solve the sewage discharge problem, help environmental protection.
The technical solution adopted in the present invention is for achieving the above object: a kind of method of producing inulase by solid fermentation, promptly adopt solid bio-reactor fermentative production inulinase, and comprise the steps:
(1) preparation of spore suspension: Crewe is tieed up the saccharomyces fragilis bacterial classification inoculation to being mixed and added routinely by yeast powder-peptone-inulin on the slant medium that agar forms, described slant medium is formed and is comprised (g/L): yeast powder 10, peptone 20, inulin 15, pH value nature; In 30 ℃ of constant temperature culture 4-6 days, add the physiological saline washing mycelium of 0.75% (W/W), obtain bacteria suspension, suspension miospore content is 10
5-10
6Individual, stand-by;
(2) above-mentioned bacteria suspension is used to be seeded to and cultivates into solid-state seed on the solid medium after the sterilization or liquid once more enlarged culturing becomes liquid seeds; Liquid seed culture medium is formed (g/L): yeast powder 5-15, peptone 15-30, inulin 10-20, pH value nature; With liquid seed culture medium in 121 ℃, 20min sterilization cooling again; Inoculum size according to volume ratio 4-6% inserts spore suspension in the above-mentioned refrigerative liquid seed culture medium then, in 28-30 ℃, rotating speed is to cultivate 24h on the shaking table of 150-200rpm, obtains liquid seeds;
(3) fermentation obtains product: solid-state fermentation culture medium is formed (g/L): cereal 200.0-400.0, corn steep liquor 1.0-5.0, KH
2PO
42.0-5.0, MgSO
4.7H
2O0.2-0.6, MnSO
4.7H
2O0.02-0.04, ZnSO
4.7H
2O0.02-0.03, CaCl
2.7H
2O0.03-0.05, surplus is a water; Initial pH value 5.5-6.0, with fermention medium through 121 ℃, 20min sterilization cooling again; Then under aseptic condition, pressing the 5-10ml/100g solid medium adds liquid seeds or presses 5-10mg/100g solid medium adding solid seed, fully mix back thickness 0.5-3.0cm, humidity 50%-80%, feed the sterile air of 0.2-1.5V/ (V.min), temperature is 25-45 ℃, standing for fermentation 4-6 days;
(4) aftertreatment: water with fermentation ends after the product of gained dilute in the 1:1 ratio, centrifugal collection contains the supernatant liquor of inulinase, is crude enzyme liquid, and crude enzyme liquid is carried out enzyme activity determination, enzymic activity reaches 100-200u/ml; Carry out routinely making the inulinase preparation after the xeraphium essence.
Described cereal comprises wheat, barley, oat, millet, rice, corn.
The invention has the beneficial effects as follows: traditionally, it is very long that liquid submerged fermentation is produced the required cycle of inulinase, and inulinase is active unstable, and quantity of wastewater effluent is very big, industrial operate very difficult.Use the inventive method, solid state fermentation effectively improves the output of inulinase, shortens fermentation period, and simplified process equipment reduces cost, and benefits suitability for industrialized production and uses; Adopt producing inulase by solid fermentation, significantly reduce the sewage that liquid fermenting produces, fundamentally effectively solve the sewage discharge problem, be very beneficial for environment protection.
Embodiment
Below in conjunction with preferred embodiment, to details are as follows according to embodiment provided by the invention:
Embodiment 1
(1) Crewe is tieed up the saccharomyces fragilis bacterial classification inoculation to mixing and add routinely by yeast powder-peptone-inulin on the slant medium that agar forms, the agar add-on is 1.5-2%, in 30 ℃ of constant temperature culture 4 days, the physiological saline washing mycelium that adds 0.75% (W/W), obtain bacteria suspension, suspension miospore content is about 1 * 10
5Individual, stand-by.Described slant medium is formed and is comprised (1L): yeast powder 10g, peptone 20g, inulin 15g, pH value nature (do not need to transfer pH, keep state of nature).
(2) getting the 10ml bacteria suspension inserts the 500ml that the 100ml seed culture medium is housed and shakes in the bottle.
Shake-flask seed substratum (1L): yeast powder 10g, peptone 20g, inulin 10g, pH value nature is with 121 ℃ of liquid seed culture mediums, 20min sterilization cooling again; Be to cultivate 24h on the shaking table of 150rpm in 30 ℃, rotating speed, obtain liquid seeds.
(3) under aseptic condition, press the 5ml/100g solid medium and add liquid seeds.Solid-state fermentation culture medium is formed (1kg): cereal 300.0g, corn steep liquor 3.0g, KH
2PO
44.0g, MgSO
4.7H
2O0.3g, MnSO
4.7H
2O0.02g, ZnSO
4.7H
2O0.02g, CaCl
2.7H
2O0.03g, surplus is a water; Initial pH value 5.5 is cooled to 28 ℃ with fermention medium again through 121 ℃, 20min sterilization; Fully mix back thickness 1.5cm, humidity 60%, the sterile air of feeding 1.0V/ (V.min), temperature is 28 ℃, standing for fermentation 4 days.The selection of cereal comprises wheat, barley, oat, millet, rice, corn.
(4) aftertreatment: water with fermentation ends after the product of gained dilute in 1: 1 ratio, centrifugal collection contains the supernatant liquor of inulinase, is crude enzyme liquid, and crude enzyme liquid is carried out enzyme activity determination, enzymic activity can reach 128.54u/ml.Below make the inulinase preparation after the xeraphium essence routinely.
Embodiment 2
(1) Crewe is tieed up the saccharomyces fragilis bacterial classification inoculation to mixing and add routinely by yeast powder-peptone-inulin on the slant medium that agar forms, in 30 ℃ of constant temperature culture 5 days, add the physiological saline washing mycelium of 0.75% (W/W), obtain bacteria suspension, suspension miospore content is about 1.5 * 10
5Individual, stand-by.Described slant medium is formed and is comprised (1L): yeast powder 10g, peptone 20g, inulin 15g, pH value nature.
(2) getting the 8m1 bacteria suspension inserts in the 100g solid medium.Solid-state fermentation culture medium is formed (1kg): cereal 350.0g, corn steep liquor 2.0g, KH
2PO
42.0g, MgSO
4.7H
2O0.3g, MnSO
4.7H
2O0.03g, ZnSO
4.7H
2O0.02g, CaCl
2.7H
2O0.03g, surplus is a water; Initial pH value 5.5 is cooled to 30 ℃ with fermention medium again through 121 ℃, 20min sterilization; Fully mix back thickness 1.5cm, humidity 60% feeds the sterile air of 1.2V/ (V.min), and temperature is 30 ℃, and standing for fermentation 2 days is as the solid seed.
(3) under aseptic condition, press the 8g/100g solid medium and add the solid seed.Solid-state fermentation culture medium is formed (1kg): cereal 350.0g, corn steep liquor 2.0g, KH
2PO
42.0g, MgSO
4.7H
2O0.3g, MnSO
4.7H
2O0.03g, ZnSO
4.7H
2O0.02g, CaCl
2.7H
2O0.03g, surplus is a water; Initial pH value 5.5 is cooled to 30 ℃ with fermention medium again through 121 ℃, 20min sterilization; Fully mix back thickness 2.0cm, humidity 70%, the sterile air of feeding 1.0V/ (V.min), temperature is 30 ℃, standing for fermentation 5 days.The selection of cereal comprises wheat, barley, oat, millet, rice, corn.
(4) aftertreatment: water with fermentation ends after the product of gained dilute in 1: 1 ratio, centrifugal collection contains the supernatant liquor of inulinase, is crude enzyme liquid, and crude enzyme liquid is carried out enzyme activity determination, enzymic activity can reach 137.26u/ml.Make the inulinase preparation after the drying powder essence.
Embodiment 3
(1) Crewe is tieed up the saccharomyces fragilis bacterial classification inoculation to mixing and add routinely by yeast powder-peptone-inulin on the slant medium that agar forms, in 30 ℃ of constant temperature culture 5 days, add the physiological saline washing mycelium of 0.75% (W/W), obtain bacteria suspension, suspension miospore content is about 2.0 * 10
5Individual, stand-by.Described slant medium is formed and is comprised (1L): yeast powder 10g, peptone 20g, inulin 15g, pH value nature.
(2) getting the 5ml bacteria suspension inserts the 500ml that the 100ml seed culture medium is housed and shakes in the bottle.Shake-flask seed substratum (1L): yeast powder 10g, peptone 25g, inulin 15g, pH value nature.With 121 ℃ of liquid seed culture mediums, 20min sterilization cooling again; Be to cultivate 24h on the shaking table of 200rpm in 30 ℃, rotating speed, obtain liquid seeds.
(3) under aseptic condition, press the 10ml/100g solid medium and add liquid seeds solid-state fermentation culture medium composition (1kg): cereal 350.0g, corn steep liquor 4.0g, KH
2PO
43.0g, MgSO
4.7H
2O0.4g, MnSO
4.7H
2O0.03g, ZnSO
4.7H
2O0.02g, CaCl
2.7H
2O0.04g, surplus is a tap water; Initial pH value 6.0 is cooled to 28 ℃ with fermention medium again through 121 ℃, 20min sterilization; Fully mix back thickness 1.5cm, humidity 80%, the sterile air of feeding 1.5V/ (V.min), temperature is 28 ℃, standing for fermentation 6 days.The selection of cereal comprises wheat, barley, oat, millet, rice, corn.
(4) aftertreatment: water with fermentation ends after the product of gained dilute in 1: 1 ratio, centrifugal collection contains the supernatant liquor of inulinase, is crude enzyme liquid, and crude enzyme liquid is carried out enzyme activity determination, enzymic activity can reach 125.48u/ml.Make the inulinase preparation after the drying powder essence.
Embodiment 4
(1) Crewe is tieed up the saccharomyces fragilis bacterial classification inoculation to mixing and add routinely by yeast powder-peptone-inulin on the slant medium that agar forms, in 30 ℃ of constant temperature culture 4 days, add the physiological saline washing mycelium of 0.75% (W/W), obtain bacteria suspension, suspension miospore content is about 1.0 * 10
5Individual, stand-by.Described slant medium is formed and is comprised (1L): yeast powder 10g, peptone 20g, inulin 15g, pH value nature.
(2) getting the 10ml bacteria suspension inserts in the 100g solid medium.Solid-state fermentation culture medium is formed (1kg): cereal 375.0g, corn steep liquor 4.0g, KH
2PO
43.0g, MgSO
4.7H
2O0.4g, MnSO
4.7H
2O0.03g, ZnSO
4.7H
2O0.02g, CaCl
2.7H
2O0.04g, surplus is a tap water; Initial pH value 6.0 is cooled to 30 ℃ with fermention medium again through 121 ℃, 20min sterilization; Fully mix back thickness 1.5cm, humidity 70% feeds the sterile air of 1.2V/ (V.min), and temperature is 30 ℃, and standing for fermentation 2 days is as the solid seed.
(3) under aseptic condition, press the 10g/100g solid medium and add the solid seed.Solid-state fermentation culture medium is formed (1kg): cereal 375.0g, corn steep liquor 3.0g, KH
2PO
43.0g, MgSO
4.7H
2O0.3g, MnSO
4.7H
2O0.03g, ZnSO
4.7H
2O0.02g, CaCl
2.7H
2O0.03g, surplus is a tap water; Initial pH value 6.0 is cooled to 30 ℃ with fermention medium again through 121 ℃, 20min sterilization; Fully mix back thickness 2.0cm, humidity 80%, the sterile air of feeding 1.5V/ (V.min), temperature is 30 ℃, standing for fermentation 5 days.The selection of cereal comprises wheat, barley, oat, millet, rice, corn.
(4) aftertreatment: water with fermentation ends after the product of gained dilute in 1: 1 ratio, centrifugal collection contains the supernatant liquor of inulinase, is crude enzyme liquid, and crude enzyme liquid is carried out enzyme activity determination, enzymic activity can reach 132.94u/ml.Make the inulinase preparation after the drying powder essence.
Above-mentioned detailed description of the method for this producing inulase by solid fermentation being carried out with reference to embodiment; be illustrative rather than determinate; can exemplify out several embodiment according to institute's limited range; therefore in the variation and the modification that do not break away under the general plotting of the present invention, should belong within protection scope of the present invention.
Claims (1)
1. the method for a producing inulase by solid fermentation adopts solid bio-reactor fermentative production inulinase, comprises the steps:
(1) preparation of bacteria suspension: Crewe is tieed up the saccharomyces fragilis bacterial classification inoculation to being mixed and added routinely by yeast powder-peptone-inulin on the slant medium that agar forms, described slant medium is formed and is comprised that unit is g/L: yeast powder 10, peptone 20, inulin 15, do not need to transfer pH, keep state of nature; In 30 ℃ of constant temperature culture 4-6 days, the adding weight percent was 0.75% physiological saline washing mycelium, obtains bacteria suspension, and suspension miospore content is 10
5-10
6Individual, stand-by;
(2) above-mentioned bacteria suspension is used to be seeded to and cultivates into solid-state seed on the solid medium after the sterilization or liquid once more enlarged culturing becomes liquid seeds; It is g/L that liquid seed culture medium is formed unit: yeast powder 5-15, and peptone 15-30, inulin 10-20 does not need to transfer pH, keeps state of nature; With liquid seed culture medium in 121 ℃, 20min sterilization cooling again; Inoculum size according to volume ratio 4-6% inserts bacteria suspension in the above-mentioned refrigerative liquid seed culture medium then, in 28-30 ℃, rotating speed is to cultivate 24h on the shaking table of 150-200rpm, obtains liquid seeds;
(3) fermentation obtains product: it is g/L that solid-state fermentation culture medium is formed unit: cereal 200.0-400.0, corn steep liquor 1.0-5.0, KH
2PO
42.0-5.0, MgSO
47H
2O 0.2-0.6, MnSO
47H
2O0.02-0.04, ZnSO
47H
2O 0.02-0.03, CaCl
27H
2O 0.03-0.05, surplus is a water; Initial pH value 5.5-6.0, with fermention medium through 121 ℃, 20min sterilization cooling again; Then under aseptic condition, pressing the 5-10ml/100g solid medium adds liquid seeds or presses 5-10mg/100g solid medium adding solid seed, fully mix back thickness 0.5-3.0cm, humidity 50%-80%, feed the sterile air of 0.2-1.5V/ (Vmin), temperature is 25-45 ℃, standing for fermentation 4-6 days;
(4) aftertreatment: water with fermentation ends after the product of gained dilute in 1: 1 ratio, centrifugal collection contains the supernatant liquor of inulinase, is crude enzyme liquid, and crude enzyme liquid is carried out enzyme activity determination, enzymic activity reaches 100-200u/ml; Carry out routinely making the inulinase preparation after the xeraphium essence.
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| CN101381711B true CN101381711B (en) | 2011-01-26 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1252438A (en) * | 1998-10-26 | 2000-05-10 | 中国农业科学院饲料研究所 | Inulinase generating saccharomycetes strain and its application in producing high fructure syrup |
| CN1341709A (en) * | 2001-09-11 | 2002-03-27 | 湖北大学 | Yeast gene engineering bacterium and endoinulase preparation and its application method |
| CN1495252A (en) * | 2002-07-12 | 2004-05-12 | 王建华 | Prduction method of exoinulase |
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2008
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1252438A (en) * | 1998-10-26 | 2000-05-10 | 中国农业科学院饲料研究所 | Inulinase generating saccharomycetes strain and its application in producing high fructure syrup |
| CN1341709A (en) * | 2001-09-11 | 2002-03-27 | 湖北大学 | Yeast gene engineering bacterium and endoinulase preparation and its application method |
| CN1495252A (en) * | 2002-07-12 | 2004-05-12 | 王建华 | Prduction method of exoinulase |
Non-Patent Citations (7)
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| 杨慧敏 等.微生物菊粉酶发酵生产研究.贵州农业科学32 3.2004,32(3),83-84. |
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| 郑忠辉 等.菊粉酶高活力菌株的筛选和发酵条件的研究.厦门大学学报(自然科学版)32 3.1993,32(3),360-364. * |
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