CN103695392B - A kind of pomace enzyme and Synthesis and applications thereof - Google Patents
A kind of pomace enzyme and Synthesis and applications thereof Download PDFInfo
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- CN103695392B CN103695392B CN201310686847.5A CN201310686847A CN103695392B CN 103695392 B CN103695392 B CN 103695392B CN 201310686847 A CN201310686847 A CN 201310686847A CN 103695392 B CN103695392 B CN 103695392B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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Abstract
The invention discloses a kind of pomace enzyme and application thereof, belong to enzymic preparation field.Pomace enzyme is by following parts by weight composition: cellulase 10-15 part, polygalacturonase 5-10 part, zytase 3-8 part, glycerine 10-15 part, NaCl5-10 part, water 52-67 part.Wherein is the cellulase of the present invention's use by aspergillus niger (Aspergillus? does niger) Li-2013-03 fermentation obtain, deposit number CGMCC? NO.7927.The glucose yield 45-55% of the high enzymolysis pomace alive of the pomace enzyme enzyme in the present invention, than being greatly improved by the glucose yield (10%-30%) that other modes obtain, for product Mierocrystalline cellulose in pomace and pectin substance being converted into the high added values such as the available fructose of suitability for industrialized production, wood sugar, glucose and oligose provides novel method.
Description
Technical field
The invention belongs to enzymic preparation field, be specifically related to a kind of pomace enzyme and Synthesis and applications thereof.
Background technology
China is Production of fruit big country in the world, and fruit ultimate production occupies first place in the world, and along with China's fruit industry fast development, fruits output significantly rises, and proposes urgent requirement to processing industry.But along with the increase of processing fruits amount, the fruit pomace that annual generation is a large amount of.Pomace is the residuum of fresh fruit after squeeze juice extracting, primarily of pericarp, fruit stone and remaining pulp composition, containing the nutritive substance that soluble sugar, VITAMIN, mineral substance and Mierocrystalline cellulose etc. are abundant.After measured, containing total reducing sugar 15.1% in Fructus Mali pumilae dregs and peel, crude fat 6.8%, crude protein content 6.2%, remaining most of material is robust fibre.Although pomace has very important industrial value, be not utilized effectively for a long time, cause the huge waste of resource, simultaneously yet serious environment pollution, therefore develop the strategic task that apple pomace resource is current processing fruits industry.At present mainly concentrate on feed to the utilization of pomace to add and the aspect such as cellulosic production.
Chinese patent 200510041807.0 discloses a kind of method of manufacturing feedstuff protein by solid state fermentation of apple dregs liquid.The apple residue of pulverizing is prepared burden into liquid state fermentation substratum by it, and the mixed strains then accessing seed culture maturation carries out liquid submerged fermentation, finally submerged fermentation liquid is carried out solid state fermentation in the solid-state fermentation culture medium being linked into apple residue.The feedstuff protein product protein content that this method is produced is high, nutritious, with low cost, is applicable to fermentation of apple pulp and produces the industrial scale production of feedstuff protein and apply.
Chinese patent 200710069718.6 discloses a kind of by the method for preparing functional dietary fiber through myrica ruba marc enzymolysis, comprise the discarded myrica ruba marc degreasing after red bayberry is squeezed the juice, add phosphate buffered saline buffer and cellulolytic enzyme enzyme liquid, after enzymolysis 1-2h, alcohol is analysed, vacuum-drying, obtains myrica ruba marc functional dietary fiber.Advantages of simple of the present invention, turns waste into wealth red bayberry, improves soluble dietary fibre content in myrica ruba marc, for red bayberry industrial chain extension provides method.Simultaneously the limited myrica ruba marc that solves is banked up a series of problem of environmental pollutions rotting to cause.
But the economic benefit that manufacture pomace being used for animal-feed and food fibre obtains is not high, so the product how Mierocrystalline cellulose in pomace and pectin substance being converted into the high added values such as the available fructose of suitability for industrialized production, wood sugar, glucose and oligose is one of direction studied from now on.Therefore obtaining a kind of enzyme lives high, and the pomace enzyme that glucose yield is high is also applied to just seem particularly important in the middle of the industry of pomace enzymolysis.
Summary of the invention
The object of this invention is to provide a kind of pomace enzyme, for the industry of pomace enzymolysis, overcome the shortcoming that in prior art, enzyme work is low and efficiency of pcr product is low.
Object of the present invention realizes by following technical proposal:
A kind of pomace enzyme, is characterized in that, form by following parts by weight: cellulase 10-15 part, polygalacturonase 5-10 part, zytase 3-8 part, glycerine 10-15 part, NaCl5-10 part, water 52-67 part.
The preparation method of described pomace enzyme is as follows:
(1) each component in described pomace enzyme is taken in proportion;
(2), after glycerine being mixed with water under 20-25 DEG C of condition, NaCl is dissolved in Glycerine-Aqueous Solution;
(3) will load weighted cellulase, polygalacturonase and zytase be placed in mixing machine and stir in advance, glycerine-NaCl-aqueous solution part is added mixing machine for 3-5 time, churning time be 5-10 minute simultaneously.
(4) preserve in normal temperature or cold house after stirring.
Described polygalacturonase and zytase, come from Ningxia Sunson Industrial Group Co., Ltd.'s product.
Described cellulase preparation method is as follows:
(1) aspergillus niger strain activation
(2) aspergillus niger liquid seeds is cultivated
Aspergillus niger strain is accessed in 500mL triangular flask, seed culture medium loading amount 100 milliliters, 30 DEG C, 150rpm shaking table cultivation 72-96h.
(3) fermentation of Aspergillus niger is cultivated
Aspergillus niger seed liquid seeds step (2) obtained is with 10%(v/v) inoculum size access be equipped with in the 10L fermentor tank of 7.5L fermented liquid, control ph is constant is 6.0 ± 0.2, culture temperature 30 ± 0.1 DEG C, stirring velocity 300rpm, ventilation (v/v) 1:0.8-1.2, incubation time 96h, dissolved oxygen 20-30%.
Described fermention medium consists of: cellulose powder 100g, ammonium sulfate 5g, magnesium sulfate 0.25g, potassium primary phosphate 1g, sodium-chlor 0.1g, tap water constant volume 1000mL, pH value 5-6,121 DEG C of sterilizing 20min.
(4) fermentation liquor is filtered, concentrated, allotment, essence filter, drying obtain cellulase.
The bacterial strain of the high activity cellulase of product provided by the invention is specially aspergillus niger (Aspergillusniger) Li-2013-03.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (be called for short CGMCC, address is: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute, postcode 100101) on July 15th, 2013, and preserving number is CGMCCNO.7927.
Preferably, pomace enzyme is by following parts by weight composition: cellulase 15 parts, polygalacturonase 8 parts, zytase 5 parts, glycerine 10 parts, NaCl10 part, 52 parts, water.
The application conditions of described pomace enzyme in the industry of pomace enzymolysis is:
Temperature: optimum temperuture 40 DEG C-50 DEG C, significant temp 20 DEG C-60 DEG C;
PH value: optimum pH 3.5-5.5, effective PH3.0-6.0;
Addition: 400-1000ml/t pomace;
Action time: 2h-6h.
Preferred application conditions is: 45 DEG C, and under pH value 4 condition, pomace per ton adds 800mL pomace enzyme, enzymolysis 4h.
Beneficial effect:
1, cellulase used in the present invention is obtained through biological fermentation by aspergillus niger (Aspergillusniger) Li-2013-03, this bacterial strain is through screening process medium optimization, growth rapidly compared with starting strain, 28 DEG C ferment 4 days, circumscribed beta-glucanase, Endo-β-glucanase, beta-glucosidase and filter paper enzyme activity in fermented liquid cellulase reach 620U/mL, 1289U/mL, 456U/mL and 732U/mL respectively, improve 9.21 times, 7.43 times, 8.15 times and 10.31 times respectively than starting strain.
2, the pomace enzyme enzyme in the present invention is lived high, and the glucose yield 45-55% of enzymolysis pomace, is greatly improved than the glucose yield (10%-30%) obtained by other modes.
Embodiment
Embodiment 1: a kind of cellulase preparation method comprises the steps:
(1) aspergillus niger strain activation
The slant strains of intact aspergillus niger is inoculated in slant medium, 30 DEG C cultivate 2d, until mycelium ripe, produce a large amount of black spore.
Described slant medium is composed as follows: 12
obrix wort l000mL, agar 2%, pH value nature, 121 DEG C of sterilizing 20min;
(2) aspergillus niger liquid seeds is cultivated
Bacterial strain Li-2013-03 is accessed in 500mL triangular flask, seed culture medium loading amount 100 milliliters, 30 DEG C, 150rpm shaking table cultivation 84h.
(3) fermentation of Aspergillus niger is cultivated
By seed liquor with 10%(v/v) inoculum size access be equipped with in the 10L fermentor tank of 7.5L fermented liquid, control ph is constant is 6.0, culture temperature 30 DEG C, stirring velocity 300rpm, ventilation (v/v) 1:1, incubation time 96h, dissolved oxygen 25%.
Described fermention medium consists of: cellulose powder 100g, ammonium sulfate 5g, magnesium sulfate 0.25g, potassium primary phosphate 1g, sodium-chlor 0.1g, tap water constant volume 1000mL, pH value 5.5,121 DEG C of sterilizing 20min.
(4) fermentation liquor filter, concentrated, allotment, essence filter, dry solid cellulase.
In cellulase in the fermented liquid that above-mentioned preparation method obtains, circumscribed beta-glucanase, Endo-β-glucanase, beta-glucosidase and filter paper enzyme activity reach 620U/mL, 1289U/mL, 456U/mL and 732U/mL respectively.
Embodiment 2: a kind of pomace enzyme and application thereof
A kind of pomace enzyme, forms by following parts by weight: cellulase 10 parts, polygalacturonase 5 parts, zytase 3 parts, glycerine 10 parts, NaCl5 part, 67 parts, water.
Described zytase and polygalacturonase are Ningxia Sunson Industrial Group Co., Ltd.'s product.
Described cellulase is obtained by method described in embodiment 1.
Make an addition in pending pomace by described pomace enzyme by the addition of pomace 600mL per ton, at 40 DEG C, enzymolysis 2h under pH value 3 condition, the glucose yield adopting DNS method to measure in enzymolysis solution is 45%
Embodiment 3: a kind of pomace enzyme and application thereof
A kind of pomace enzyme, forms by following parts by weight: cellulase 15 parts, polygalacturonase 8 parts, zytase 5 parts, glycerine 10 parts, NaCl10 part, 52 parts, water.
Described zytase and polygalacturonase are Ningxia Sunson Industrial Group Co., Ltd.'s product.
Described cellulase is obtained by method described in embodiment 1.
Make an addition in pending pomace by described pomace enzyme by the addition of pomace 800mL per ton, at 45 DEG C, enzymolysis 4h under pH value 4 condition, the glucose yield adopting DNS method to measure in enzymolysis solution is 55%
Embodiment 4: a kind of preparation method of pomace enzyme:
(1) each component in described pomace enzyme is taken in proportion: cellulase 15 parts, polygalacturonase 8 parts, zytase 5 parts, glycerine 10 parts, NaCl10 part, 52 parts, water;
(2), after glycerine being mixed with water under 25 DEG C of conditions, NaCl is dissolved in Glycerine-Aqueous Solution;
(3) will load weighted cellulase, polygalacturonase and zytase be placed in mixing machine and stir in advance, glycerine-NaCl-aqueous solution part is added mixing machine for 5 times, churning time be 10 minutes simultaneously;
(4) preserve in 4 DEG C of cold houses after stirring.
Claims (5)
1. a pomace enzyme, is characterized in that, described pomace enzyme is by following parts by weight composition: cellulase 10-15 part, polygalacturonase 5-10 part, zytase 3-8 part, glycerine 10-15 part, NaCl5-10 part, water 52-67 part;
Described cellulase is obtained by aspergillus niger (Aspergillusniger) Li-2013-03 fermentation, strain number CGMCCNO.7927.
2. a kind of pomace enzyme as claimed in claim 1, is characterized in that, concrete preparation method is as follows for described cellulase:
(1) aspergillus niger strain activation;
(2) aspergillus niger liquid seeds is cultivated:
Aspergillus niger strain is accessed in 500mL triangular flask, seed culture medium loading amount 100 milliliters, 30 DEG C, 150rpm shaking table cultivation 72-96h;
(3) fermentation of Aspergillus niger is cultivated:
Aspergillus niger liquid seeds step (2) obtained is equipped with in the 10L fermentor tank of 7.5L fermented liquid with the access of volume ratio 10% inoculum size, control ph is constant is 6.0 ± 0.2, culture temperature 30 ± 0.1 DEG C, stirring velocity 300rpm, ventilation 1:0.8-1.2, incubation time 96h, dissolved oxygen 20-30%;
(4) fermentation liquor is filtered, concentrated, allotment, essence filter, drying obtain cellulase.
3. a kind of pomace enzyme as claimed in claim 2, it is characterized in that, the fermention medium that described fermentation culture uses consists of: cellulose powder 100g, ammonium sulfate 5g, magnesium sulfate 0.25g, potassium primary phosphate 1g, sodium-chlor 0.1g, tap water constant volume 1000mL, pH value 5-6,121 DEG C of sterilizing 20min.
4. a kind of pomace enzyme as claimed in claim 1, is characterized in that, form by following parts by weight: cellulase 15 parts, polygalacturonase 8 parts, zytase 5 parts, glycerine 10 parts, NaCl10 part, 52 parts, water.
5. a preparation method for pomace enzyme, is characterized in that, concrete steps are as follows:
(1) each component taken in described pomace enzyme is formed by following parts by weight: cellulase 10-15 part, polygalacturonase 5-10 part, zytase 3-8 part, glycerine 10-15 part, NaCl5-10 part, water 52-67 part; (2), after glycerine being mixed with water under 20-25 DEG C of condition, NaCl is dissolved in Glycerine-Aqueous Solution;
(3) will load weighted cellulase, polygalacturonase and zytase be placed in mixing machine and stir in advance, divided by the glycerine-NaCl-aqueous solution simultaneously and add mixing machine for 3-5 time, churning time be 5-10 minute;
(4) preserve in normal temperature or cold house after stirring;
Described cellulase preparation method is as follows:
1. aspergillus niger (Aspergillusniger) Li-2013-03 activates;
2. aspergillus niger (Aspergillusniger) Li-2013-03 liquid seeds is cultivated:
Aspergillus niger strain Li-2013-03 is accessed in 500mL triangular flask, seed culture medium loading amount 100 milliliters, 30 DEG C, 150rpm shaking table cultivation 72-96h;
3. fermentation culture:
Aspergillus niger liquid seeds step 2. obtained is equipped with in the 10L fermentor tank of 7.5L fermented liquid with the access of volume ratio 10% inoculum size, and control ph is constant is 6.0 ± 0.2, culture temperature 30 ± 0.1 DEG C, stirring velocity 300rpm, ventilation 1:0.8-1.2, incubation time 96h, dissolved oxygen 20-30%;
4. fermentation liquor is filtered, concentrated, allotment, essence filter, drying obtain cellulase.
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| CN106472598A (en) * | 2016-08-30 | 2017-03-08 | 北京山北绿色农业有限公司 | A kind of non-toxic organic pesticide and preparation method and application |
| CN109913434A (en) * | 2019-04-08 | 2019-06-21 | 宁夏夏盛实业集团有限公司 | A kind of complex enzyme and its preparation method and application |
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