CN1428415A - Aspergillus niger capable of producing acidic beta-mannase and its fermentation and method for producing manno-oligose - Google Patents
Aspergillus niger capable of producing acidic beta-mannase and its fermentation and method for producing manno-oligose Download PDFInfo
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Abstract
The present invention is aimed at providing an aspergillus niger capable of secreting acid beta-mannase. The aspergillus niger provided by said invention is the aspergillus niger AS2710 CGMCC No.0658 and its mutant or variant. The fermentation process of AS2710 includes the following steps: A. preparing fermentation strain; making the slant cultured AS2710 undergo the processes of liquid strain culture and first-stage liquid culture and at least second-stage liquid culture to obtain fermentation strain; and B. utilizing the above-obtained strain to make fermentation and production of acid beta-mannase.
Description
Technical field
The present invention relates to a strain aspergillus niger, the fermentation process of this aspergillus niger and the method for producing mannooligo saccharide with its tunning, the aspergillus niger that particularly relates to a strain producing acidic beta-mannase, the fermentation process of this aspergillus niger and the method for producing mannooligo saccharide with its tunning.The mannase of preparation and mannooligo saccharide are as feed and foodstuff additive.
Background technology
The rapid progress of biotechnology provides possibility for feed develops towards direction efficient, nuisanceless, noresidue.The application of fodder enzyme preparation and oligosaccharides is aspect most widely used in fodder industry, the most promising one of the biotechnology.As a kind of biological catalyst efficiently, enzyme itself is a protein, and the enzyme of using in fodder industry is the green product of safety non-toxic.From oligosaccharides such as the mannooligo saccharide of vegetable polysaccharides by the enzymatic conversion preparation, it is the conclusive novel prebiotic substance of effect, by optimizing intestinal microecology system, can play useful nourishing function to the host, can also discern, adhere to and get rid of pathogenic micro-organism, regulate human body immune function, improve livestock and poultry production performance.Mannooligo saccharide can substitute antibiotic multiple function, and does not have side effects such as resistance, is a kind of nuisance free feed supplement of having a bright future.
The research of 'beta '-mannase and mannooligo saccharide is had the history of decades both at home and abroad, but until after 1985, along with the exploitation of enzyme and oligosaccharides Application Areas, significantly aggravation is competed in correlative study.Current research relates to the aspects such as Production by Enzymes of generation, purifying and characteristic research, gene clone and recombinant expressed and the mannooligo saccharide of enzyme.Significant progress has: the research that Japanese physics and chemistry institute in 1987 is carried out the bacillus AM-001 that produces alkaline ' beta '-mannase, by the several different methods seed selection, enzyme activity has been brought up to 430u/ml by 36u/ml.Nineteen ninety-five, U.S. ChemGen company has developed a kind of alkaline ' beta '-mannase, and the enzymatic productivity of its bacterial classification Bacillus lentus reaches 400u/ml, and exploitation subsequently is the most salable fodder enzyme of the present U.S. as fodder additives.
Domestic 'beta '-mannase and mannooligo saccharide Study on Production Technology are in international most advanced level.The Ma Yan of Institute of Microorganism, Academia Sinica and etc. (microorganism journal, 1991,31 (6): 443-448; The microbiology circular, 1992,19 (1): 13-17) early propose to utilize alkaline ' beta '-mannase to produce the method for mannooligo saccharide.Utilizing Alkaliphilic bacillus N16-5 is raw material with the Rhizoma amorphophalli powder, the fermentative production 'beta '-mannase, and the enzyme activity of fermented liquid reaches 500u/ml, and extract yield is more than 80% behind the mannooligo saccharide.The mannooligo saccharide of preparation is as protective foods and fodder additives.
Research high temperature resistant, acid and neutral 'beta '-mannase also there are many effort both at home and abroad, as (microbiology circular, 1995,22 (3): 154-157) studied separation screening and the fermentation condition that produces the 'beta '-mannase Bacillus licheniformis such as Yang Wenbo.(microorganism journal, 1999,39 (1): 60-63) reported generation and the character of the neutral 'beta '-mannase of subtilis such as Cui Fumian.The preparation of Zhang Jun etc. (food and fermentation industries, 2001,27 (2) 5-7) research Bacillus licheniformis 'beta '-mannase.But acidity and neutral 'beta '-mannase enzyme activity that the institute strain screening produces are all lower, and high yield enzyme activity is only about 120u/ml.
'beta '-mannase reorganization progress of research is unhappy.β-sweet dew glucanase gene that Mendoza-N-S (1995) will clone from Bacillus subtilisNM-39 changes intestinal bacteria PH5a expression over to.Enzyme activity only is 4.9u/ml (Biochim.Biophys.Acta-G; 1243 (3) 552-554).Stalbrand-H (1995) utilizes β-sweet dew glucanase gene of Tricho dermareesei clone to express enzyme activity also not high (Appl.Environ.Microbiol.61 (3) 1090-1097) in yeast saccharomyces cerevisiae.Tang-C-M etc. (2001) derive from the mannase gene cel4 of Agaricus bisporus strain C54-carb8 with Pichia anomala expression, expression amount has improved 3 times, is worth further research (Appl.Environ.Microbiol.67 (5) 2298-2303).
Enzyme process decomposes the preparation mannooligo saccharide, is the key content of 'beta '-mannase applied research always.Though having gone through to do fodder additives in China, microbe-derived 'beta '-mannase and mannooligo saccharide use, but because the enzyme productive rate is low, the production cost height, so far be not applicable to the mannooligo saccharide bulk article yet, the highest transformation efficiency to mannooligo saccharide of 'beta '-mannase also has only 29%.The mannooligo saccharide that extracts is sold by U.S. Ao Teqi company from fodder yeast, oligosaccharide content 65%, and 56 yuan/kilogram of prices, cost is higher.Institute of Micro-biology of the Chinese Academy of Sciences prepares mannooligo saccharide with alkaline ' beta '-mannase enzymolysis konjaku, has reduced cost by a relatively large margin.There are 6 about microbe-derived 'beta '-mannase and mannooligo saccharide research with the relevant patent that has applied for the production aspect both at home and abroad, all about alkaline ' beta '-mannase, the product enzyme activity of external 5 patents is at 36-400u/ml, during institute in 1999 such as domestic Ma Yan and grade applies for a patent, the enzyme activity of being produced is 500u/ml, bacterial classification is the obligate Alkaliphilic bacillus, and optimal pH is 10.
Summary of the invention
The aspergillus niger that the purpose of this invention is to provide strain energy secreting acidic 'beta '-mannase.
Aspergillus niger provided by the invention is aspergillus niger (Aspergillus niger) AS2710 CGMCC № 0658 and mutant or varient.
Aspergillus niger (Aspergillus niger) AS2710 is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center December 5 calendar year 2001, and it abbreviates CGMCC as, and deposit number is CGMCC № 0658.
Aspergillus niger of the present invention separates from field mountain valley with clumps of trees and bamboo seed and obtains, and seed culture medium can be with PDA or Czapek ' s substratum, well-grown on bran mass and wheat bran-dregs of beans (1: 0.1-0.4 added water 1: 1) substratum.Optimum growth temperature is 25-28 ℃.This aspergillus niger is on wheat bran dregs of beans substratum, and early growth period 1-3 days mycelia is a white, and the back forms the black spore.
Aspergillus niger AS2710 can the secreting acidic 'beta '-mannase.The resistance toheat of 'beta '-mannase is good, and operating temperature range is 30-85 ℃, 70 ℃ of optimal reactive temperatures; Reaction pH2-9.2, optimal pH 3.0-3.8; Enzyme activity is issued to 150u/ml shaking a bottle condition, meets the fodder enzyme condition, is domestic and international highest level.
Another object of the present invention provides the method with aspergillus niger AS2710 fermentative production 'beta '-mannase.
A kind of method of producing 'beta '-mannase with the described fermentation of Aspergillus niger of claim 1 may further comprise the steps:
(1) preparation fermented bacterium: with aspergillus niger (Aspergillus niger) AS2710 of slant culture through strain cultivation with level liquid is cultivated and at least after the secondary liquid culture, obtain fermented bacterium;
(2) utilize the above-mentioned strain fermentation that obtains to produce acidic beta-mannase.
The detailed process of described (one) preparation fermented bacterium is:
1) slant strains: use the malt extract medium of total sugar degree 10-20%, add 2% agar, streak inoculation, under 30-35 ℃ of condition, shaking table was cultivated 20-24 hour, preserved in 4 ℃ of refrigerators in 3 months;
2) liquid spawn and level liquid are cultivated: use the malt extract medium of total sugar degree 10-20%, the inoculum size of seed liquor is respectively 5-20%, cultivated 20-24 hour at 30-35 ℃ of shaking table, or under 30-35 ℃ of condition, static cultivation 24-48 hour.
3) secondary liquid culture: use sugar degree 10-20% amylum hydrolysate of the sugar liquid, inoculum size is respectively 5-20%, cultivated 20-24 hour at 30-35 ℃ of shaking table, or under 30-35 ℃ of condition, static cultivation 24-48 hour.
Described (two) utilize the strain fermentation that obtains to produce acidic beta-mannase can adopt liquid fermentation process production, also can adopt solid fermentation method production.When producing with liquid processes, the fermentative medium formula of use comprises in parts by weight: konjaku powder or sesbania gum: 30-85 part, root of Cornu Cervi Pantotrichum powder 5-10 part, wheat bran 5-30 part, corn cob 0-30 part, dregs of beans 0-10 part, urea or ammonium sulfate 0-0.5 part; Raw material: water=1: 1.5-10, the inoculum size of bacterial classification is 5-30%, under 15-40 ℃ the condition, static cultivation 1-10 days; Or, make pO at 25-40 ℃ of following aerated culture
2=30%, pH=5-7; Filtering fermentating liquid obtains acidic beta-mannase; Corn cob in the described liquid fermentation medium, dregs of beans, urea or ammonium sulfate are respectively 10-25 part, 2-8 part, 0.1-0.4 part.When adopting solid fermentation method to produce, the fermentative medium formula of use comprises in parts by weight: konjaku powder or sesbania gum: 30-85 part, root of Cornu Cervi Pantotrichum powder 5-10 part, wheat bran 5-30 part, corn cob 0-30 part, dregs of beans 0-10 part, urea or ammonium sulfate 0-0.5 part; Raw material: water=1: 0.5-1.5.Be used to prepare 'beta '-mannase behind the fermenting-ripening; The solid fermentation product adds 2-6 times of water logging bubble after-filtration and obtains acidic beta-mannase; Corn cob, dregs of beans, urea or ammonium sulfate in the described solid fermentation substratum is respectively 10-25 part, 2-8 part, 0.1-0.4 part.
Another purpose of the present invention provides a kind of method of utilizing aspergillus niger of the present invention to produce mannooligo saccharide.
Utilizing aspergillus niger (Aspergillus niger) acidic beta-mannase that AS2710 produced to prepare the method for mannooligo saccharide, is with acidic beta-mannase hydrolysis mannosans.
Described mannosans is that konjaku powder is or/and sesbania gum.
The present invention utilizes high-cell density liquid fermentation technology and solid-fermented technique to prepare the novel acid 'beta '-mannase, shortened the time that fermentation period and mannosans production of enzyme peak occur, the strain enzyme-producing peak is promptly occurred at 60hr, reduced production cost, made the further widespread use in feed and foodstuffs industry of mannase and mannooligo saccharide become possibility.Because strain enzyme-producing fermentation of the present invention can be adopted plate method, production unit and raw materials cost are low, and the production of enzyme height is difficult for pollution microbes, and product cost is lower more than 50% than the best level of the research nineties.
From society and ecological benefits aspect, feedstuff raw material particularly in the grouts mannosans of anti-nutrition be that efficiency of feed utilization is low, ight soil nitrogen phosphorus excretion is big, the main reason of soil and waters eutrophication pollution.Use 'beta '-mannase and can improve livestock and poultry and fish greatly to the utilising efficiency of feed, save feed grain, improve production performance, reduce and pollute and administer the cost that pollutes, meaning is remarkable.
China has abundant mannosans plant material characteristic resources.Mannosans just like amyloid a large amount of accumulation, is the good raw material of preparation mannooligo saccharide in Tian Qing, konjaku.The present invention can promote the technical progress of a plurality of industries such as processing of farm products, light industry, chemical industry, and economic benefit, social benefit and industry influence highly significant.
Acidic beta-mannase of the present invention can directly be used as fodder additives on the one hand, can be used to prepare mannooligo saccharide on the one hand, can improve industrial scale and economic benefit greatly.The present invention has set up the technology platform of Production by Enzymes mannooligo saccharide, utilize efficient enzymatic conversion technology, improve transformation efficiency, with back extraction purification techniquess such as chromatographic separation, membrane sepn, improve the productive rate and the purity of enzyme and oligosaccharides, carry forward vigorously the study on the industrialization of mannooligo saccharide.
The present invention will be further described below in conjunction with specific embodiment.
Embodiment
The preparation of embodiment 1, acidic beta-mannase fermented bacterium
1) slant strains: use the malt extract medium of total sugar degree 15%, add 2% agar, streak inoculation, under 32 conditions, shaking table was cultivated 22 hours, preserved in 4 refrigerators in 3 months;
2) liquid spawn and level liquid are cultivated: use the malt extract medium 50ml of total sugar degree 15%, the inoculum size of seed liquor is 10ml, under 32 shaking table conditions, cultivated 22 hours, or under 33 conditions, static cultivation 40 hours.
3) secondary liquid culture: use the amylum hydrolysate of the sugar liquid 300ml of sugar degree 15%, inoculum size is 60ml, and shaking table was cultivated 20-24 hour under 33 conditions, or under 33 conditions, static cultivation 24-48 hour.Promptly make fermented bacterium.
Embodiment 2. utilizes liquid fermentation process to produce 'beta '-mannase
The fermentative medium formula that uses is counted with parts by weight: konjaku powder: 79.5 parts, 10 parts in root of Cornu Cervi Pantotrichum powder, 5 parts in wheat bran, 5 parts of dregs of beans, 0.5 part in urea; Raw material: water=1: 6, the inoculum size of bacterial classification are aerated culture under 30%, 35 the condition, make pO
2=30%, pH=6.5.Detected enzyme every 24 hours and live, reach termination fermentation when producing the enzyme peak, product is prepared into acidic beta-mannase after filtration.
The enzyme activity determination method is with reference to Akino (Appl.Microbiol.Biotechnol.1987, method 26:323-327) is measured: with 0.5ml, 0.5% (W/V) locust bean gum (Locust bean gam) is made end powder, use pH10,0.05mol/L glycine-NaOH damping fluid adds 0.1ml dilution enzyme liquid, 70 insulations 10 minutes, survey the reducing sugar that produces with edlefsen's reagent, 1 enzyme activity unit is defined as under the above-mentioned reaction conditions, discharged the enzyme amount that 1mmol is equivalent to the reducing sugar of D-seminose in one minute, the result is as shown in table 1.
Table 1: acidic beta-mannase output is:
| Fermentation time (hr) | ????24 | ???48 | ???72 | ????96 |
| Enzyme activity (u/ml) | ????103.5 | ???150.7 | ???97.4 | ????38.6 |
The output of 'beta '-mannase increased fast at preceding 2 days that cultivate, and reached in the 2nd day to produce the enzyme peak.Beginning in the 3rd day slowly reduces.
Embodiment 3. utilizes solid fermentation method to produce 'beta '-mannase
The fermentative medium formula that uses is counted with parts by weight: sesbania gum: 70 parts, 10 parts in root of Cornu Cervi Pantotrichum powder, 19.5 parts in wheat bran, 0.5 part in ammonium sulfate; Raw material: water=1: 1.Detected enzyme every 12 hours and live, reach termination fermentation when producing the enzyme peak, product adds 4 times of water logging bubble after-filtration and obtains acidic beta-mannase, and the result is as shown in table 2.The yield of enzyme of solid fermentating mode is lower than the high density liquid mode.
Table 2: acidic beta-mannase output is:
| Fermentation time (hr) | ???24 | ???48 | ???72 | ???96 | ???120 | ???144 |
| Enzyme activity (u/ml) | ???14.7 | ???93.4 | ???128.5 | ???132.1 | ???98.4 | ???79.9 |
The output of 'beta '-mannase increased fast in first three day of cultivating, and reduced after 5 days.
The preparation of embodiment 4. acidic beta-mannases
The fermented liquid direct filtration of liquid fermenting, solid fermentation product add 2-6 times of water logging bubble after-filtration.Adopt filter press, filtrate adds diatomite as flocculating aids, and filter pressure is at 0-5kg/cm
2Between.Filtrate adds ammonium sulfate to saturated, and 4 left standstill 24 hours, the thick enzyme preparation of centrifugal preparation.High density liquid fermenting enzyme output is 0.2-1.0% (w/v), and the solid fermentation production of enzyme is 0.1-0.5%.The acidic beta-mannase that obtains can be used for making feeding, medical and industrial enzymes.
Embodiment 5. enzymolysis prepare mannooligo saccharide
Konjaku powder adds 4 times in water, and heated and boiled is fully dissolved powder, is cooled to 40, adds 8% the thick enzyme preparation of acidic beta-mannase, stirs, and reacts 10-48 hour, and after the colloid complete digestion became liquid, sampling detected sugar and forms.Oligosaccharides account for total reducing sugar more than 80% the back termination reaction.Drying is made mannooligo saccharide.Made oligosaccharides is as feed and foodstuff additive, agricultural chemicals etc.
Embodiment 6. enzymolysis prepare mannooligo saccharide
Sesbania gum adds 8 times in water, and heated and boiled is fully dissolved glue, is cooled to 60, adds the thick enzyme preparation of acidic beta-mannase of 2% (0.5-10%), stirs, and reacts 10-48 hour, and after the colloid complete digestion became liquid, sampling detected sugar and forms.Oligosaccharides account for total reducing sugar more than 80% the back termination reaction.Drying is made mannooligo saccharide.Made oligosaccharides is as feed and foodstuff additive, agricultural chemicals etc.
Claims (10)
1, aspergillus niger (Aspergillus niger) AS2710 CGMCC № 0658 and mutant or varient.
2, a kind of method of producing 'beta '-mannase with the described fermentation of Aspergillus niger of claim 1 may further comprise the steps:
(1) preparation fermented bacterium: with aspergillus niger (Aspergillus niger) AS2710 of slant culture through strain cultivation with level liquid is cultivated and at least after the secondary liquid culture, obtain fermented bacterium;
(2) utilize the above-mentioned strain fermentation that obtains to produce acidic beta-mannase.
3, zymotechnique according to claim 2 is characterized in that: the detailed process of described (one) preparation fermented bacterium is:
1) slant strains: use the malt extract medium of total sugar degree 10-20%, add 2% agar, streak inoculation, under 30-35 ℃ of condition, shaking table was cultivated 20-24 hour, preserved in 4 ℃ of refrigerators in 3 months;
2) liquid spawn and level liquid are cultivated: use the malt extract medium of total sugar degree 10-20%, the inoculum size of seed liquor is respectively 5-20%, cultivated 20-24 hour at 30-35 ℃ of shaking table, or under 30-35 ℃ of condition, static cultivation 24-48 hour.
3) secondary liquid culture: use sugar degree 10-20% amylum hydrolysate of the sugar liquid, inoculum size is respectively 5-20%, cultivated 20-24 hour at 30-35 ℃ of shaking table, or under 30-35 ℃ of condition, static cultivation 24-48 hour.
4, zymotechnique according to claim 2, it is characterized in that: described (two) utilize the strain fermentation that obtains to produce acidic beta-mannase is to adopt liquid fermentation process production, and the fermentative medium formula of use comprises in parts by weight: konjaku powder or sesbania gum: 30-85 part, root of Cornu Cervi Pantotrichum powder 5-10 part, wheat bran 5-30 part, corn cob 0-30 part, dregs of beans 0-10 part, urea or ammonium sulfate 0-0.5 part; Raw material: water=1: 1.5-10, the inoculum size of bacterial classification is 5-30%, under 15-40 ℃ the condition, static cultivation 1-10 days; Or, make pO at 25-40 ℃ of following aerated culture
2=30%, pH=5-7; Filtering fermentating liquid obtains acidic beta-mannase.
5, zymotechnique according to claim 4 is characterized in that: the corn cob in the described liquid fermentation medium, dregs of beans, urea or ammonium sulfate are respectively 10-25 part, 2-8 part, 0.1-0.4 part.
6, zymotechnique according to claim 2, it is characterized in that: described (two) utilize the strain fermentation that obtains to produce acidic beta-mannase is to adopt solid fermentation method production, and the fermentative medium formula of use comprises in parts by weight: konjaku powder or sesbania gum: 30-85 part, root of Cornu Cervi Pantotrichum powder 5-10 part, wheat bran 5-30 part, corn cob 0-30 part, dregs of beans 0-10 part, urea or ammonium sulfate 0-0.5 part, its material; Raw material: water=1: 0.5-1.5.Be used to prepare 'beta '-mannase behind the fermenting-ripening; The solid fermentation product adds 2-6 times of water logging bubble after-filtration and obtains acidic beta-mannase.
7, zymotechnique according to claim 6 is characterized in that: corn cob, dregs of beans, urea or the ammonium sulfate in the described solid fermentation substratum is respectively 10-25 part, 2-8 part, 0.1-0.4 part.
8, the 'beta '-mannase that utilizes the described fermentation of Aspergillus niger of claim 1 to obtain prepares the method for mannooligo saccharide, is with acidic beta-mannase hydrolysis mannosans.
9, method according to claim 8 is characterized in that: described mannosans is that konjaku powder is or/and sesbania gum.
10, 'beta '-mannase that obtains with the method for claim 2 and the application of mannooligo saccharide in feed and foodstuff additive that obtain with the method for claim 8.
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| CN100410370C (en) * | 2005-11-03 | 2008-08-13 | 暨南大学 | Process for producing bata-mannanase fermented by pseudo honey agaric and application thereof |
| CN102002465A (en) * | 2010-11-05 | 2011-04-06 | 广东溢多利生物科技股份有限公司 | Acidic belta-mannase VMAN, gene thereof and application |
| CN102268468A (en) * | 2011-07-20 | 2011-12-07 | 上海海洋大学 | Method for preparing mannan-oligosaccharide by using waste beer yeast |
| CN101392241B (en) * | 2007-09-18 | 2012-01-04 | 中国科学院微生物研究所 | Beta-mannanase, gene, preparation method, vector and host cell |
| CN102586209A (en) * | 2012-03-15 | 2012-07-18 | 济南诺能生物工程有限公司 | Preparation method for liquid high temperature resistant acidic mannase |
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| CN102002465A (en) * | 2010-11-05 | 2011-04-06 | 广东溢多利生物科技股份有限公司 | Acidic belta-mannase VMAN, gene thereof and application |
| CN102268468A (en) * | 2011-07-20 | 2011-12-07 | 上海海洋大学 | Method for preparing mannan-oligosaccharide by using waste beer yeast |
| CN102586209B (en) * | 2012-03-15 | 2013-08-07 | 济南诺能生物工程有限公司 | Preparation method for liquid high temperature resistant acidic mannase |
| CN102586209A (en) * | 2012-03-15 | 2012-07-18 | 济南诺能生物工程有限公司 | Preparation method for liquid high temperature resistant acidic mannase |
| CN103173427A (en) * | 2012-07-12 | 2013-06-26 | 北京伟嘉人生物技术有限公司 | Method for producing beta-mannase by utilizing Aspergillus niger fermentation |
| CN105087717A (en) * | 2015-07-31 | 2015-11-25 | 丽江玉元食品有限公司 | Method of utilizing acidic mannase to hydrolyze refined konjac powder to prepare konjac oligosaccharide |
| CN107805635A (en) * | 2017-12-26 | 2018-03-16 | 马鞍山市五谷禽业专业合作社 | A kind of method that β mannases are prepared using pomace solid state fermentation |
| CN109679936A (en) * | 2019-01-31 | 2019-04-26 | 北京华美源生物科技有限公司 | Functional mannase preparation and its production technology |
| CN113718002A (en) * | 2021-08-31 | 2021-11-30 | 江苏菁康生物科技有限公司 | Method for preparing micromolecular galactomannan and galactomannan-oligosaccharide by using alkali extraction residues of corncobs |
| CN113718002B (en) * | 2021-08-31 | 2024-04-12 | 南京林业大学 | Method for preparing micromolecular galactomannan and galactomannan-oligosaccharide by using corncob alkali extraction residues |
| CN114984664A (en) * | 2022-06-09 | 2022-09-02 | 成都长力元生物科技有限公司 | Method for filtering viscous konjac flour enzymatic hydrolysate |
| CN114984664B (en) * | 2022-06-09 | 2023-12-15 | 成都长力元生物科技有限公司 | Method for filtering viscous konjaku flour enzymatic hydrolysate |
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