CN104371000A - Method for extracting glutathione and ribonucleic acid in beer waste yeast with high efficiency - Google Patents
Method for extracting glutathione and ribonucleic acid in beer waste yeast with high efficiency Download PDFInfo
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- CN104371000A CN104371000A CN201310361878.3A CN201310361878A CN104371000A CN 104371000 A CN104371000 A CN 104371000A CN 201310361878 A CN201310361878 A CN 201310361878A CN 104371000 A CN104371000 A CN 104371000A
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Abstract
The invention provides a method for extracting glutathione and ribonucleic acid in beer waste yeast with high efficiency, which comprises the following steps: adding an amphoteric biological flocculating agent, simultaneous absorbing hydrophilic and lipophilic pigment and bitter taste substance, performing enzyme-assistant thermal lysing extraction on wet yeast, centrifuging and collecting a supernatant, employing a ceramic membrane with interception aperture of 10000 Dalton for ultrafiltration on the supernatant, wherein substance with molecular weight of greater than 10000 is nucleic acid and macromolecular hybrid protein, and substance with molecular weight of less than 10000 is glutathione and small molecular impurity, adsorbing ribonucleic acid macromolecular protein by a silicagel column, washing column by ethanol with concentration of 95%, eluting by sterile water, drying to obtain high purity nucleic acid, performing nanofiltration on the glutathione small molecular impurity, performing expanded bed affinity chromatography, concentrating, depositing by ethanol, and drying to obtain glutathione. The prepared product has the advantages of high purity and good biological activity, waste enables cyclic utilization, the method has the advantages of environmental protection and high efficiency, production cost is simultaneously reduced, and the method is adapted to large-scale industrial production.
Description
One, technical field
This project independent research beer waste yeast high-efficient extraction technology prepares gsh and Yeast Nucleic Acid technique, relate to the effective debitterize of microorganism, impurity removing technology, broken born of the same parents' technology, consistency high efficiency separation and purification technique, multienzyme compound, reliable zymolysis technique produce gsh and Yeast Nucleic Acid.
Two, technical background
Containing multiple nutritional components such as rich in protein, nucleic acid, VITAMIN, carbohydrate, lipid material, mineral substance in waste beer yeast cell.Calculate with dry weight, containing Yeast Nucleic Acid (RNA), the protein (containing 18 seed amino acids) of 50%, the gsh of 1% of 4% ~ 8% in cereuisiae fermentum, the vitamin B group of 2%.In 18 seed amino acids that beer waste yeast contains, wherein 8 seed amino acid content of needed by human and the amino acid whose proportion of composing ratio of ideal amino acid of recommending close to food and agricultural organization of Lian Tai state (FA0), therefore beer waste yeast has splendid nutritive value for the metabolism and growth of biology, and the fermention medium utilizing beer waste yeast to produce has active effect to animal and microbial growth.In addition, multiple high added value physiologically active substance can also be extracted from beer waste yeast, as materials such as gsh, Yeast Nucleic Acid, dextran.Wherein, gsh is a kind of anti-oxidant and oligosaccharides that is function of detoxification of having, multiple beneficial effects such as having elimination free radical for human body, improve immunizing power, be anti-ageing and antitumor, has been widely applied at medicine, field of food, has had important economic worth.Yeast Nucleic Acid is the important source material preparing Nucleotide, is with a wide range of applications in food, plant-growth regulator and medicine are produced.
At present, beer waste yeast obtains comprehensive utilization abroad, produces high-grade substratum and extract physiologically active ingredient to create huge economic benefit by utilizing beer waste yeast.Such as, Japanese beer industry to the utilization power of beer waste yeast is: the beer waste yeast of 50% is used to produce mixed fodder; 20% is used to extract the multiple nutrients factor, for the production of nutrient fortified food and functional foodstuff; 17 ~ 18% are used to extract various physiologically active substance, for the production of medicine and healthcare products; 12 ~ 13% are used to production enhancement feed.But at home, utilize level and availability all very low at present to beer waste yeast, the Land use systems of most of waste yeast is dried makes mixed fodder or additive of protein feed.This causes us every year all will from the fermentative production of a large amount of high-grade yeast extract of external import for high-end genetic and cell engineering product.Although existing many enterprises have started to fully utilize beer waste yeast at present, take beer waste yeast as raw material production protein extract or extraction physiologically active substance, but because adopted technology is advanced not and perfect, industrial scale is little, cause there is to the utilization of beer waste yeast the situation that state of the art is lower, resource utilization is not enough, quality product is not high, the performance of enterprises is not good.The low-level duplicate construction of industry existence and the problem of disorderly competition is fully utilized in order to solve current beer waste yeast, be necessary to tackle key problems to efficient, intensive, the comprehensive utilization technique of waste beer yeast, exploitation high-level efficiency, high-caliber beer waste yeast comprehensive utilization technique and technique, realize the efficient of beer waste yeast and complicated utilization.
The research of this project adopts both sexes biological flocculants to adsorb wetting ability and lipotropy pigment and bitter substance simultaneously, realizes carrying out cleaning to beer waste yeast before broken born of the same parents and just can effectively remove these impurity; Develop consistency high efficiency separation, the purification technique of gsh and nucleic acid; Utilize the silica column of energy specificity adsorbs nucleic acid to carry out the absorption of Yeast Nucleic Acid, realize the high purity to Yeast Nucleic Acid, high yield extracts; Adopt exclusive expanding bed affinity chromatography technology to carry out the specificity purifying of gsh, adopt the affinity chromatography that affine selectivity is higher, avidity is stronger to carry out the problem that alternative traditional ion exchange chromatography thoroughly solves impurity interference; Enzyme engineering technology is adopted to carry out protein engineering transformation to traditional proteolytic enzyme, obtain the Novel protease with better enzymolysis performance, and utilize multienzyme compound, Controlled-enzymatic Hydrolysis technology, reach the optimization of protein degradation, for industrial mass production gsh and Yeast Nucleic Acid provide reliable theoretical foundation.The present invention utilizes beer waste yeast high efficiency extraction gsh and Yeast Nucleic Acid, and its production technique is simple, and productive rate is large, is suitable for scale operation.
Three, summary of the invention
1. adopt and prepared by gsh and Yeast Nucleic Acid to beer waste yeast highly-efficient processing and utilization,
Comprise following step:
(1) both sexes biological flocculant is applied to beer waste yeast debitterize, impurity elimination process
The bitter taste produced in Process of Beer Brewing in a large number and coloring matter is there is inside and outside waste beer yeast cell.These materials not only can affect color and luster and the smell of prepared yeast extract, but also can disturb the adsorption selection of affinity chromatography medium to gsh, the abstraction and purification effect of reduction gsh.The method that tradition removes these materials is repeatedly cleaned waste beer yeast cell.This method not only can consume a large amount of water and energy, and only can remove wetting ability pigment and bitter substance, not good to the removal effect of lipotropy pigment and bitter substance.This project creatively adopts both sexes biological flocculant to adsorb wetting ability and lipotropy pigment and bitter substance simultaneously, only once need clean beer waste yeast before broken born of the same parents, just can effectively remove these impurity.Then sedimentation flocculation in refrigerator is placed on, with the yeast that must wet after drum filter continuous filtration impurity elimination.The preparation of this extraction and high-end yeast extract of not being only gsh creates favourable condition, and simplifies processing step, decreases water of productive use, reduces the follow-up load reclaiming waste water and process, has significant economy and environmental benefit.For the waste water that cleaning produces, the circulation and stress treatment unit of Cleaning Wastewater is then set, collect after Cleaning Wastewater is filtered and carry out the aerobic and anaerobic treatment of continuous print, biogas is produced as boiler oil while reduction waste water BOD, a water part after process is cycled to used in the cleaning of yeast, another part is used for the flushing of equipment, reaches wastewater zero discharge.
(2) multienzyme compound, reliable zymolysis technique are applied to beer waste yeast enzyme auxiliary heat and break born of the same parents' extracting
Gsh and nucleic acid are all positioned at yeast cell, extract gsh and nucleic acid, and effective means first will be taked to carry out brokenly born of the same parents' process to yeast cell, to realize the abundant release of gsh and nucleic acid, avoid the inactivation of gsh and nucleic acid again simultaneously.This Project-developing team has carried out protein engineering transformation by adopting enzyme engineering technology to traditional proteolytic enzyme, obtain the Novel protease with better enzymolysis performance, and utilize multienzyme compound, Controlled-enzymatic Hydrolysis technology, enzyme auxiliary heat is carried out to beer waste yeast and breaks born of the same parents' extracting, reach the optimization of protein degradation.Hot-water extraction gsh: wet yeast mixes by 1: 15 mass ratio with waste water, after being heated to 90 DEG C, is cooled fast to less than 20 DEG C, then use the rear collected by centrifugation supernatant liquor of hydrochloric acid adjusted to ph to 3.5.
(3) application of membrane separation technology is in consistency high efficiency separation, the purifying of gsh and nucleic acid
Break in born of the same parents' process at yeast, not only can discharge gsh, some the proteins and peptides molecules in born of the same parents also can discharge simultaneously.Research finds, some proteins and peptides molecule not only can disturb the isolation and purification of gsh, reduce the rate of recovery of gsh, and the degraded of nucleic acid can occur, and significantly reduces the yield of nucleic acid.Therefore, how to get rid of the interference of these materials, ensure that the rate of recovery of gsh is from beer waste yeast, prepare a key technical problem of gsh and nucleic acid.Because beer waste yeast breaks the impurity such as protein, polypeptide, oligopeptides that there is interference gsh separation and purification in cytosol, traditional Exchange Resin by Adsorption exists when separating glutathione that adsorption efficiency is low, the gsh rate of recovery and the not high deficiency of product purity.Meanwhile, in order to realize the simultaneous extraction to beer waste yeast amplifying nucleic acid and gsh, the transformation of novelty must be carried out to traditional extracting method.For this problem, this project team innovates from two Isolation and purification methods of aspect to gsh and nucleic acid, develops consistency high efficiency separation, the purification technique of gsh and nucleic acid.Gsh and Yeast Nucleic Acid belong to two kinds of diverse materials, there is larger difference in nature at molecular weight, electric charge etc.In the research fully utilized at existing beer waste yeast and report, all there is not the precedent simultaneously extracting gsh and Yeast Nucleic Acid.Therefore, the extraction process of these two materials is integrated, extract while realizing these two materials, be not only the key innovations of project, especially the key of project success.This project adopts and retains aperture is that 10000 daltonian ceramic membranes carry out ultrafiltration to broken cytosol, is separated by the material that relative molecular weight in broken cytosol is greater than 10000 by ultrafiltration with the material being less than 10000.The former comprises some nucleic acid and large protein impurities, and the latter comprises gsh and the less impurity of number molecular weight.Owing to achieving the shunting of Yeast Nucleic Acid and gsh in this step, therefore in follow-up separation and purification process, just need not worry the non-compatibility of two kinds of materials on extracting method again, only need pay close attention to respective isolation and purification respectively.
(4) separation and purification of Yeast Nucleic Acid
For the separation of Yeast Nucleic Acid, traditional method is first by removal protein impurities of saltouing, and then collects Yeast Nucleic Acid by carrying out iso-electric point low-temperature sludge in acid condition, then to wash Yeast Nucleic Acid precipitation with alcohol and remove protein further.Although the method is simple, not high to the clearance of protein impurities, and on the low side to the rate of recovery of nucleic acid, cause the total recovery of nucleic acid on the low side, product purity is not high.For this problem, this project team utilizes the silica column of energy specificity adsorbs nucleic acid to carry out the absorption of Yeast Nucleic Acid, realizes the high purity to Yeast Nucleic Acid, high yield extracts, make the yield of Yeast Nucleic Acid reach 4%.Adopt silicagel column absorption (high salt low ph value), realize adsorbing the specificity of nucleic acid, remove protein impurities, then wash post with the ethanol of 95%.60 DEG C of sterilized water wash-out Yeast Nucleic Acid, spraying dry, obtain high purity Yeast Nucleic Acid, product purity is greater than 80%.
(5) separation and purification of gsh
For the separation of gsh, first adopting and retaining aperture is that 100 daltonian rolled films carry out nanofiltration, removes the impurity that relative molecular weight is less than 100, realizes concentrating gsh simultaneously.Because gsh relative molecular weight is 307.33, therefore by ultrafiltration and the nanofiltration process of meticulously setting, effectively can not only remove the impurity of interference gsh separation and purification, and the loss of gsh less, processing cost is low.Secondly, in order to eliminate the interference of impurity to gsh and Ion Exchange Resin Phase mutual effect further, this project adopts exclusive expanding bed affinity chromatography technology to carry out the specificity purifying of gsh, adopts the affinity chromatography that affine selectivity is higher, avidity is stronger to carry out the problem that alternative traditional ion exchange chromatography thoroughly solves impurity interference.Because traditional fixed bed affinity chromatography exists the deficiency that treatment scale is little, processing efficiency is low, affinity chromatography is also coupled with expanding bed by this project, the advantage that the applied sample amount utilizing expanding bed unique is large, wash-out concentration is high, devise the novel affine adsorption chromatography technique of expanding bed, the indexs such as the efficiency that gsh is adsorbed, the rate of recovery and purifying purity obtain remarkable improvement all simultaneously, and primary sorption and wash-out can reclaim the gsh more than more than 80% from feed liquid.Elutriant is concentrated through nanofiltration, alcohol settling crystallization, after vacuum-drying gsh finished product, product purity is greater than 90%.
This method produces Yeast Nucleic Acid and gsh technique is simple, and productive rate is large, and the product purity obtained is high, biological activity good, and achieve the comprehensive utilization of resource, improve the level of resource utilization of beer fermentation industry, reduce production cost simultaneously, be suitable for large-scale industrial production.
Claims (8)
1. beer waste yeast high-efficient extraction technology prepares a preparation method for gsh and Yeast Nucleic Acid, and its feature comprises following step:
(1) both sexes biological flocculant is applied to beer waste yeast debitterize, impurity elimination process
Waste beer yeast mud adds more than 2 times frozen water and stirs, and adds both sexes biological flocculant and adsorbs wetting ability and lipotropy pigment and bitter substance simultaneously, only once need clean beer waste yeast before broken born of the same parents, just can effectively remove these impurity.Then sedimentation flocculation in refrigerator is placed on, with the yeast that must wet after drum filter continuous filtration impurity elimination.
(2) multienzyme compound, reliable zymolysis technique are applied to beer waste yeast enzyme auxiliary heat and break born of the same parents' extracting
By adopting enzyme engineering technology, protein engineering transformation is carried out to traditional proteolytic enzyme, obtain the Novel protease with better enzymolysis performance, and utilize multienzyme compound, Controlled-enzymatic Hydrolysis technology, enzyme auxiliary heat is carried out to beer waste yeast and breaks born of the same parents' extracting, reach the optimization of protein degradation.Hot-water extraction gsh: wet yeast mixes by 1: 15 mass ratio with waste water, after being heated to 90 DEG C, is cooled fast to less than 20 DEG C, then use the rear collected by centrifugation supernatant liquor of hydrochloric acid adjusted to ph to 3.5.
(3) application of membrane separation technology is in consistency high efficiency separation, the purifying of gsh and nucleic acid
It is that 10000 daltonian ceramic membranes carry out ultrafiltration to broken cytosol that employing retains aperture, is separated by the material that relative molecular weight in broken cytosol is greater than 10000 by ultrafiltration with the material being less than 10000.The former comprises some nucleic acid and large protein impurities, and the latter comprises gsh and the less impurity of number molecular weight.Gsh and Yeast Nucleic Acid belong to two kinds of diverse materials, there is larger difference in nature at molecular weight, electric charge etc.In the research fully utilized at existing beer waste yeast and report, all there is not the precedent simultaneously extracting gsh and Yeast Nucleic Acid.And this step of this project achieves the shunting of gsh and Yeast Nucleic Acid, thus reach extraction while these two materials.Therefore in follow-up separation and purification process, just need not worry the non-compatibility of two kinds of materials on extracting method again, only need pay close attention to respective isolation and purification respectively.
(4) separation and purification of Yeast Nucleic Acid
Utilize and the silica column of specificity adsorbs nucleic acid can carry out the absorption of Yeast Nucleic Acid, realize the high purity to Yeast Nucleic Acid, high yield extracts, make the yield of Yeast Nucleic Acid reach 4%, purity is greater than 80%.Silicagel column absorption (high salt low ph value), realizes adsorbing the specificity of nucleic acid, removes protein impurities, then washes post with the ethanol of 95%.60 DEG C of sterilized water wash-out Yeast Nucleic Acid, spraying dry, obtains high purity Yeast Nucleic Acid.
(5) separation and purification of gsh
It is that 100 daltonian rolled films carry out nanofiltration that employing retains aperture, removes the impurity that relative molecular weight is less than 100, realizes concentrating gsh simultaneously.Because gsh relative molecular weight is 307.33, therefore by ultrafiltration and the nanofiltration process of meticulously setting, effectively can not only remove the impurity of interference gsh separation and purification, and the loss of gsh less, processing cost is low.Secondly, in order to eliminate the interference of impurity to gsh and Ion Exchange Resin Phase mutual effect further, this project adopts exclusive expanding bed affinity chromatography technology to carry out the specificity purifying of gsh, adopts the affinity chromatography that affine selectivity is higher, avidity is stronger to carry out the problem that alternative traditional ion exchange chromatography thoroughly solves impurity interference.Because traditional fixed bed affinity chromatography exists the deficiency that treatment scale is little, processing efficiency is low, affinity chromatography is also coupled with expanding bed by this project, the advantage that the applied sample amount utilizing expanding bed unique is large, wash-out concentration is high, devise the novel affine adsorption chromatography technique of expanding bed, the indexs such as the efficiency that gsh is adsorbed, the rate of recovery and purifying purity obtain remarkable improvement all simultaneously, and primary sorption and wash-out can reclaim the gsh more than more than 80% from feed liquid.Elutriant is concentrated through nanofiltration, alcohol settling crystallization, after vacuum-drying gsh finished product, product purity is greater than 90%.
2. as (1) in claim 1 tell, it is characterized in that adding both sexes biological flocculant and wetting ability and lipotropy pigment and bitter substance adsorbed simultaneously.
3. as (2) in claim 1 tell, it is characterized in that adopt enzyme engineering technology obtain Novel protease.
4. as (2) in claim 1 tell, it is characterized in that adopting enzyme auxiliary heat to break born of the same parents' extracting.
5. as (3) in claim 1 tell, it is characterized in that employing retains aperture is 10000 daltonian ceramic membranes.
6. as (4) in claim 1 tell, it is characterized in that the high salt low ph value silicagel column adopting specificity adsorbs nucleic acid.
7. as (5) in claim 1 tell, it is characterized in that employing retains aperture is that 100 daltonian rolled films carry out nanofiltration.
8. as (5) in claim 1 tell, it is characterized in that adopting affinity chromatography and expanding bed coupling technique.
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Cited By (7)
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| CN104789554A (en) * | 2015-04-10 | 2015-07-22 | 中国海洋大学 | Method for preparing DNA (desoxyribonucleic acid) from waste liquid of porcine small intestinal mucosa after heparin extraction |
| CN106146609A (en) * | 2016-09-20 | 2016-11-23 | 济南大学 | A kind of utilize membrane separation technique method of separating-purifying glutathion from glutathion cuprous salt |
| CN106282280A (en) * | 2016-09-21 | 2017-01-04 | 河北美邦工程科技股份有限公司 | A kind of glutathion extracting method |
| CN109234268A (en) * | 2018-09-19 | 2019-01-18 | 南阳同凯生物技术有限公司 | A method of processing yeast rna mother liquor |
| CN110669673A (en) * | 2018-07-02 | 2020-01-10 | 王虎 | Method for extracting saccharomyces cerevisiae EST3 telomerase activation factor |
| CN111378714A (en) * | 2020-04-08 | 2020-07-07 | 平凉市华科生物技术有限公司 | Preparation method of yeast peptide and yeast peptide thereof |
| CN115381069A (en) * | 2022-08-12 | 2022-11-25 | 天叶生物科技有限公司 | Yeast extract, preparation method and application thereof, and seasoning |
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2013
- 2013-08-14 CN CN201310361878.3A patent/CN104371000A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104789554A (en) * | 2015-04-10 | 2015-07-22 | 中国海洋大学 | Method for preparing DNA (desoxyribonucleic acid) from waste liquid of porcine small intestinal mucosa after heparin extraction |
| CN104789554B (en) * | 2015-04-10 | 2017-11-28 | 中国海洋大学 | The method that DNA is prepared from waste liquid after the extraction of intestinal mucosa heparin |
| CN106146609A (en) * | 2016-09-20 | 2016-11-23 | 济南大学 | A kind of utilize membrane separation technique method of separating-purifying glutathion from glutathion cuprous salt |
| CN106282280A (en) * | 2016-09-21 | 2017-01-04 | 河北美邦工程科技股份有限公司 | A kind of glutathion extracting method |
| CN110669673A (en) * | 2018-07-02 | 2020-01-10 | 王虎 | Method for extracting saccharomyces cerevisiae EST3 telomerase activation factor |
| CN109234268A (en) * | 2018-09-19 | 2019-01-18 | 南阳同凯生物技术有限公司 | A method of processing yeast rna mother liquor |
| CN109234268B (en) * | 2018-09-19 | 2020-07-10 | 南阳同凯生物技术有限公司 | Method for treating yeast ribonucleic acid mother liquor |
| CN111378714A (en) * | 2020-04-08 | 2020-07-07 | 平凉市华科生物技术有限公司 | Preparation method of yeast peptide and yeast peptide thereof |
| CN115381069A (en) * | 2022-08-12 | 2022-11-25 | 天叶生物科技有限公司 | Yeast extract, preparation method and application thereof, and seasoning |
| CN115381069B (en) * | 2022-08-12 | 2024-01-05 | 天叶(南宁)生物科技有限公司 | Yeast extract, preparation method, application and seasoning thereof |
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