CN103695392A - Pomace enzyme as well as preparation method and application thereof - Google Patents
Pomace enzyme as well as preparation method and application thereof Download PDFInfo
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- CN103695392A CN103695392A CN201310686847.5A CN201310686847A CN103695392A CN 103695392 A CN103695392 A CN 103695392A CN 201310686847 A CN201310686847 A CN 201310686847A CN 103695392 A CN103695392 A CN 103695392A
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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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- 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)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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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
- C12N9/14—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
- 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
- C12N9/88—Lyases (4.)
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Abstract
The invention discloses a pomace enzyme and application thereof, belonging to the field of an enzymic preparation. The pomace enzyme consists of the following materials in parts by weight: 10-15 parts of cellulase, 5-10 parts of pectinase, 3-8 parts of xylanase, 10-15 parts of glycerol, 5-10 parts of NaCl and 52-67 parts of water, wherein the adopted cellulase is prepared by fermenting aspergillus niger Li-2013-03 with preservation number of CGMCC (China General Microbiological Culture Collection Center) NO. 7927. Glucose yield of pomace enzymatically hydrolyzed by the pomace enzyme with high activity is 45-55%, and is much higher than glucose yield (10-30%) obtained from other ways; therefore, a new method for realizing conversion of cellulose and pectin matters in the pomace into industrially available high added-value products including fructose, xylitol, glucose, oligosaccharide and the like is provided.
Description
Technical field
The invention belongs to zymin field, be specifically related to a kind of pomace enzyme and preparation and application.
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 processing industry has been proposed to urgent requirement.But along with the increase of processing fruits amount, produce every year a large amount of fruit pomaces.Pomace is the residuum of fresh fruit after squeeze juice extracting, mainly pericarp, fruit stone and remaining pulp, consists of, and contains the abundant nutritive substances such as soluble sugar, VITAMIN, mineral substance and Mierocrystalline cellulose.After measured, in Fructus Mali pumilae dregs and peel, contain total reducing sugar 15.1%, crude fat 6.8%, crude protein content 6.2%, remaining most of material is robust fibre.Although pomace has very important industrial value, is not utilized effectively for a long time, cause the huge waste of resource, the while is serious environment pollution also, therefore develops the strategic task that apple pomace resource is current processing fruits industry.At present the utilization of pomace is mainly concentrated on to feed adds 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.Its apple residue by pulverizing is prepared burden into liquid state fermentation substratum, and the mixed strains that then accesses seed culture maturation carries out liquid submerged fermentation, finally submerged fermentation liquid is carried out in being linked into the solid-state fermentation culture medium of apple residue to solid state fermentation.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, has improved soluble dietary fibre content in myrica ruba marc, for red bayberry industrial chain extension provides method.Simultaneously limitedly a series of problem of environmental pollutions that myrica ruba marc is banked up and rotted to cause have been solved.
But the economic benefit that pomace is obtained for the manufacture of animal-feed and food fibre is not high, so how Mierocrystalline cellulose in pomace and pectin substance are converted into the product of the high added values such as the available fructose of suitability for industrialized production, wood sugar, glucose and oligose, be one of direction of studying from now on.Therefore obtain a kind of enzyme alive high, 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 can realize by following technical proposal:
A pomace enzyme, is characterized in that, forms: cellulase 10-15 part, polygalacturonase 5-10 part, zytase 3-8 part, glycerine 10-15 part, NaCl5-10 part, water 52-67 part by following parts by weight.
The preparation method of described pomace enzyme is as follows:
(1) take in proportion each component in described pomace enzyme;
(2) after glycerine being mixed with water under 20-25 ℃ of condition, NaCl is dissolved in Glycerine-Aqueous Solution;
(3) will be in advance load weighted cellulase, polygalacturonase and zytase be placed in mixing machine and stir, glycerine-NaCl-aqueous solution part is added to mixing machine for 3-5 time simultaneously, churning time is 5-10 minute.
(4) in normal temperature or cold house, preserve 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 to 100 milliliters of seed culture medium loading amounts, 30 ℃, 150rpm shaking table cultivation 72-96h.
(3) fermentation of Aspergillus niger is cultivated
The aspergillus niger seed liquid seeds that step (2) is obtained is with 10%(v/v) inoculum size access is equipped with in the 10L fermentor tank of 7.5L fermented liquid, controlling pH value constant is 6.0 ± 0.2,30 ± 0.1 ℃ of culture temperature, 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 ℃ of sterilizing 20min.
(4) fermented liquid after filtration, concentrated, allotment, essence filter, the dry cellulase that obtains.
The bacterial strain of the high activity cellulase of product provided by the invention is specially aspergillus niger (Aspergillus niger) Li-2013-03.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 15th, 2013 and (is called for short CGMCC, address is: No. 3, No. 1, North Star West Road, Chaoyang District, BeiJing, China city institute, postcode 100101), preserving number is CGMCC NO.7927.
Preferably, pomace enzyme forms by following parts by weight: 52 parts of 15 parts of cellulases, 8 parts of polygalacturonases, 5 parts of zytases, 10 parts of glycerine, NaCl10 part, water.
The application conditions of described pomace enzyme in the industry of pomace enzymolysis is:
Temperature: 40 ℃-50 ℃ of optimum temperutures, 20 ℃-60 ℃ of significant temps;
PH value: optimum pH 3.5-5.5, effectively PH3.0-6.0;
Addition: 400-1000ml/t pomace;
Action time: 2h-6h.
Preferred application conditions is: 45 ℃, under pH value 4 conditions, pomace per ton is added 800mL pomace enzyme, enzymolysis 4h.
Beneficial effect:
1, cellulase used in the present invention is obtained through biological fermentation by aspergillus niger (Aspergillus niger) Li-2013-03, this bacterial strain is through screening process medium optimization, compare growth with starting strain rapidly, 28 ℃ ferment 4 days, circumscribed beta-glucanase, Endo-β-glucanase, beta-glucosidase and filter paper enzyme activity in fermented liquid cellulase reach respectively 620U/mL, 1289U/mL, 456U/mL and 732U/mL, have improved 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 alive high, and the glucose yield 45-55% of enzymolysis pomace, is greatly improved than the glucose yield (10%-30%) obtaining 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 to slant medium, cultivates 2d for 30 ℃, until mycelium ripe, produce a large amount of black spores.
Described slant medium is composed as follows: 12
obrix wort l000mL, agar 2%, pH value nature, 121 ℃ of sterilizing 20min;
(2) aspergillus niger liquid seeds is cultivated
By in bacterial strain Li-2013-03 access 500mL triangular flask, 100 milliliters of seed culture medium loading amounts, 30 ℃, 150rpm shaking table cultivation 84h.
(3) fermentation of Aspergillus niger is cultivated
By seed liquor with 10%(v/v) inoculum size access is equipped with in the 10L fermentor tank of 7.5L fermented liquid, controlling pH value constant is 6.0,30 ℃ of culture temperature, 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, 5.5,121 ℃ of sterilizing 20min of pH value.
(4) fermented liquid after filtration, concentrated, allotment, essence filter, the dry solid cellulase that to obtain.
In cellulase in the fermented liquid obtaining through above-mentioned preparation method, circumscribed beta-glucanase, Endo-β-glucanase, beta-glucosidase and filter paper enzyme activity reach respectively 620U/mL, 1289U/mL, 456U/mL and 732U/mL.
Embodiment 2: a kind of pomace enzyme and application thereof
A pomace enzyme, forms by following parts by weight: 67 parts of 10 parts of cellulases, 5 parts of polygalacturonases, 3 parts of zytases, 10 parts of glycerine, NaCl5 part, water.
Described zytase and polygalacturonase are Ningxia Sunson Industrial Group Co., Ltd.'s product.
Described cellulase is obtained by method described in embodiment 1.
Described pomace enzyme is made an addition in pending pomace by the addition of pomace 600mL per ton, at 40 ℃, enzymolysis 2h under pH value 3 conditions, the glucose yield that adopts DNS method to measure in enzymolysis solution is 45%
Embodiment 3: a kind of pomace enzyme and application thereof
A pomace enzyme, forms by following parts by weight: 52 parts of 15 parts of cellulases, 8 parts of polygalacturonases, 5 parts of zytases, 10 parts of glycerine, NaCl10 part, water.
Described zytase and polygalacturonase are Ningxia Sunson Industrial Group Co., Ltd.'s product.
Described cellulase is obtained by method described in embodiment 1.
Described pomace enzyme is made an addition in pending pomace by the addition of pomace 800mL per ton, at 45 ℃, enzymolysis 4h under pH value 4 conditions, the glucose yield that adopts DNS method to measure in enzymolysis solution is 55%
Embodiment 4: a kind of preparation method of pomace enzyme:
(1) take in proportion each component in described pomace enzyme: 52 parts of 15 parts of cellulases, 8 parts of polygalacturonases, 5 parts of zytases, 10 parts of glycerine, NaCl10 part, water;
(2) after glycerine being mixed with water under 25 ℃ of conditions, NaCl is dissolved in Glycerine-Aqueous Solution;
(3) will be in advance load weighted cellulase, polygalacturonase and zytase be placed in mixing machine and stir, glycerine-NaCl-aqueous solution part is added to mixing machine for 5 times simultaneously, churning time is 10 minutes;
(4) in 4 ℃ of cold houses, preserve after stirring.
Claims (7)
1. a pomace enzyme, is characterized in that, described pomace enzyme forms 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.
Described cellulase is made by aspergillus niger (Aspergillus niger) Li-2013-03 fermentation, strain number CGMCC NO.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 to 100 milliliters of seed culture medium loading amounts, 30 ℃, 150rpm shaking table cultivation 72-96h.
(3) fermentation of Aspergillus niger is cultivated
The aspergillus niger seed liquid seeds that step (2) is obtained is with 10%(v/v) inoculum size access is equipped with in the 10L fermentor tank of 7.5L fermented liquid, controlling pH value constant is 6.0 ± 0.2,30 ± 0.1 ℃ of culture temperature, stirring velocity 300rpm, ventilation (v/v) 1:0.8-1.2, incubation time 96h, dissolved oxygen 20-30%.
(4) fermented liquid after filtration, concentrated, allotment, essence filter, the dry cellulase that obtains.
3. a kind of pomace enzyme as claimed in claim 1 or 2, it is characterized in that, 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 ℃ of sterilizing 20min.
4. a kind of pomace enzyme as claimed in claim 1, is characterized in that, by following parts by weight, forms: 52 parts of 15 parts of cellulases, 8 parts of polygalacturonases, 5 parts of zytases, 10 parts of glycerine, NaCl10 part, water.
5. a kind of pomace enzyme as claimed in claim 1, is characterized in that, the application conditions of described pomace enzyme in the industry of pomace enzymolysis is:
(1) temperature: 40 ℃-50 ℃ of optimum temperutures, 20 ℃-60 ℃ of significant temps;
(2) pH value: optimum pH 3.5-5.5, effectively PH3.0-6.0;
(3) addition: 400-1000ml/t pomace;
(4) action time: 2h-6h.
6. a kind of pomace enzyme as claimed in claim 1, is characterized in that, the application conditions of described pomace enzyme in the industry of pomace enzymolysis is: 45 ℃, under pH value 4 conditions, pomace per ton is added 800mL pomace enzyme, enzymolysis 4h.
7. a preparation method for pomace enzyme, is characterized in that, concrete steps are as follows:
(1) by following parts by weight, form and take each component in described pomace enzyme: 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 ℃ of condition, NaCl is dissolved in Glycerine-Aqueous Solution;
(3) will be in advance load weighted cellulase, polygalacturonase and zytase be placed in mixing machine and stir, glycerine-NaCl-aqueous solution part is added to mixing machine for 3-5 time simultaneously, churning time is 5-10 minute.
(4) in normal temperature or cold house, preserve 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 (Aspergillus niger) Li-2013-03 activation;
2. aspergillus niger (Aspergillus niger) Li-2013-03 liquid seeds is cultivated
By in Aspergillus niger strain Li-2013-03 access 500mL triangular flask, 100 milliliters of seed culture medium loading amounts, 30 ℃, 150rpm shaking table cultivation 72-96h;
3. fermentation culture
The aspergillus niger seed liquid seeds that 2. step is obtained is with 10%(v/v) inoculum size access is equipped with in the 10L fermentor tank of 7.5L fermented liquid; controlling pH value constant is 6.0 ± 0.2; 30 ± 0.1 ℃ of culture temperature; stirring velocity 300rpm; ventilation (v/v) 1:0.8-1.2; incubation time 96h, dissolved oxygen 20-30%;
4. fermented liquid after filtration, concentrated, allotment, essence filter, the dry cellulase that obtains.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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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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| 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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| CN103695392B (en) | 2016-04-20 |
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