CN105400699A - Pycnoporus sp. for converting lactose wastewater to synthesize lactobionic acid - Google Patents
Pycnoporus sp. for converting lactose wastewater to synthesize lactobionic acid Download PDFInfo
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- CN105400699A CN105400699A CN201510125457.XA CN201510125457A CN105400699A CN 105400699 A CN105400699 A CN 105400699A CN 201510125457 A CN201510125457 A CN 201510125457A CN 105400699 A CN105400699 A CN 105400699A
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Abstract
The present invention provides a strain of Pycnoporus sp. for converting lactose wastewater so as to prepare lactobionic acid, wherein the strain is classified and named Pycnoporus sp. SYBC-L10, is preserved in China Center for Type Culture Collection, and has a preservation number of CCTCC NO: M2015020, wherein the address is Wuhan University, China. According to the present invention, the Pycnoporus sanguineus SYBC-L10 microorganisms are adopted as bacteria, lactose wastewater is adopted as a converted substrate, a nitrogen source, an inorganic salt and an inducing agent are added to form a fermentation culture medium, and microorganism conversion is performed to prepare the lactobionic acid; during the fermentation process, a succinic acid buffer liquid is added, such that the pH value during the fermentation process maintains a constant state, the lactose wastewater conversion rate is improved, and the conversion rate under the optimized condition achieves more than or equal to 85%; the research about the lactobionic acid in China is still blank; and the lactose content in the converted lactose wastewater is about 25% according to the high performance liquid chromatography detection result.
Description
Technical field
The present invention relates to the preparation method of lactobionic acid, be specifically related to the method that a strain samguineus conversion lactose waste water prepares lactobionic acid, belong to biological technical field.
Background technology
Lactobionic acid (lactobionicacid) is third generation tartaric acid, its integrate delay senility, moisturizing, anti-oxidant and promote the multiple efficacies such as body renewal.It is tartaric acid surrogate best at present.Owing to itself being present in organism, there is not pungency problem in lactobionic acid, has excellent resistance of oxidation, cell and isolated organ can be prevented to be subject to the injury of free radical; The destruction that lactobionic acid also can avoid cytolemma to be subject to because of oxidation, having splendid water-retentivity, is the important component in makeup.Lactobionic acid is the important component in organ transplantation damping fluid, and the product after metal-chelating can reduce because the hydroxyl of ionic catalysis generation is to the damage of tissue.Lactobionic acid also can increase the solubleness of macrolide antibiotics, and the solubleness as the lactobionic acid aqueous solution of erythromycin exceeds 50-100 doubly than erythromycin; The solubleness of the calcium salt of lactobionic acid is higher, can be used to the absorption of supplementary calcium, also can be used to the supersaturated solution doing calglucon.Lactobionic acid has stronger sweet taste, can reduce tart flavour, and the additive that can be used as food uses.Lactobionic acid also can be used as the component of washing composition because of the high-affinity of itself and water.Lactobionic acid is added in makeup, can be anti-oxidant, anti-ageing, the effects such as high moisturizing.The CAS accession number of lactobionic acid: 96-82-2.
The preparation method of current lactobionic acid has chemical catalysis synthesis method and microbe transformation method.Microbe transformation method comprises again Bacterial Transformation method, fungal transformation method.Chemical catalysis synthesis method has many disadvantageous factors, such as, produce multiple by product and chemical pollutant, and the numerous by products formed make separation, and purification step becomes complicated.Microbe transformation method has very large advantage, efficiently, low cost, the advantages such as transformation efficiency is high, and by product is few, but the bacterium of some galactopoiesis saccharic acid reported is not safe bacterial strain, is difficult to the production for industries such as food.
Summary of the invention
The object of this invention is to provide the method utilizing fungi to be lactobionic acid by Lactose conversion.Particular content relates to the method utilizing samguineus conversion lactose waste water to prepare lactobionic acid.Lactose conversion can be lactobionic acid by the microbial strains that the present invention adopts, and transformation efficiency is high, by product is few, achieves unusual effect by fermentation.
Technical scheme of the present invention; One strain samguineus transforms the bacterial strain that lactose waste water prepares lactobionic acid, its Classification And Nomenclature is samguineus (Pycnoporussp.), be preserved in China typical culture collection center, address is Wuhan, China Wuhan University, deposit number CCTCCNO:M2015020.
A kind of method preparing lactobionic acid with described bacterial strain conversion lactose waste water, take CCTCCNO:M2015020 as starting strain, with the lactose in lactose waste water for conversion of substrate, adopt carbon source, nitrogenous source and inorganic salt composition fermention medium, lactobionic acid is prepared in conversion, and its technique is:
(1) bacterial strain: employing CCTCCNO:M2015020 is starting strain;
(2) bacterial strain preservation: bacterial classification storage medium used is preserved, switching in 3-6 month is once; Storage medium: PDA substratum;
(3) seed culture: proceed to seed culture medium from storage medium and activate 1 time, at 30 DEG C, 200rmp shake-flask culture 2d;
The component of seed culture medium is counted with g/L: glucose 20, fresh potato 200, with distilled water preparation, and pH nature;
(4) microbial transformation: add in fermention medium by CCTCCNO:M2015020 bacterial strain, utilizes the desaturase of production by biological, laccase to be lactobionic acid by the Lactose conversion in lactose waste water;
Fermention medium component is counted with g/L: carbon source 20-50, organic nitrogen source 6-10, inorganic nitrogen-sourced 4-10, potassium primary phosphate 1, magnesium sulfate 0.3, calcium chloride 0.08, Zinc Sulphate Heptahydrate 0.005, four water manganous sulfates 0.0015, CoCL2 6H2O 0.0015
Green vitriol 0.005.
Described carbon source is lactose in lactose waste water, and add with 10%v/v, described nitrogenous source is: analysis for soybean powder and Secondary ammonium phosphate;
(5) need during the fermentation to add weak acid for adjusting pH, preferably succinate buffer.By adding acid succsinic acid, making pH4.5-4.8 in fermenting process keep constant, improving the transformation efficiency of lactose with this.
Described fungal transformation lactose waste water prepares the method for lactobionic acid, and conversion of substrate lactose concentration is in the fermentation medium preferably 3%-5%, at 30 DEG C, cultivates 7d under the condition of 200rmp.
Lactobionic acid is interpreted as lactobionic acid and its esters, as calcium lactobionate.
The method detecting lactobionic acid is as follows:
Get fermented liquid centrifugal, supernatant liquor is through filtering with microporous membrane (0.22 μm), and filtrate is analyzed through HPLC.HPLC condition: high performance liquid chromatograph Hitachi chromaster; Chromatographic column: BIO-RADAminexHPX-87H (7.8 × 300); Moving phase is 0.005MH
2sO
4; Detector: UV-detector, 210nm, column temperature: 50 DEG C, flow velocity: 0.4mL/min, sample size: 10 μ L, lactobionic acid standard specimen concentration: 1%.
Beneficial effect of the present invention: the invention provides a kind of method utilizing fungal transformation to prepare lactobionic acid, show that samguineus (Pycnoporussp.) SYBCL10 cultivates 7d transformation efficiency and can reach 60% when the lactose waste water added is 20%v/v (in lactose waste water, lactose concn is 25%), cultivate 9d when the lactose waste water added is 40%v/v, transformation efficiency reaches 45%.
Biological material specimens preservation: samguineus (Pycnoporussp.) SYBCL10 is preserved in China typical culture collection center, address is Wuhan, China Wuhan University, be called for short CCTCC, preservation date: on January 7th, 2015, deposit number CCTCCNO:M2015020.
Embodiment
By experiment, the principal element affecting microbe conversion lactobionic acid productive rate is obtained:
The impact of lactose on lactose output is added in embodiment 1. substratum process for preparation
Fermentation condition described in by specification, adds lactose waste water in substratum, the final concentration of the lactose in fermented liquid is 5%.By to the detection to lactose galactopoiesis saccharic acid, fermentation 7d (cellobiose dehydrogenase reached stationary phase at the 7th day) finds that the output of lactobionic acid is only 1.3g/L.Found the impact of lactobionic acid output by the lactose time of adding again, lactose has restraining effect to pycnoporussp.SYBCL10 fermentation galactopoiesis saccharic acid.Therefore lactose waste water should be added in the fermentation regular hour.
Embodiment 2. lactose adds the time to the impact of lactobionic acid output
Fermentation condition described in by specification, by to the detection of pycnoporussp.SYBCL10 at different fermentations time galactopoiesis saccharic acid, certain restraining effect is had because lactose produces cellobiose dehydrogenase to samguineus, the enzyme of CDH is lived after testing, find that thalline starts to produce enzyme at 4d, within the 7th day, reach stationary phase, so added lactose waste water (lactose-content is 25%) from the 3rd day, the final concentration making the lactose in fermented liquid is 5%, ferments by the 10th day and stops.
Table 1, lactose add the time to the impact transforming lactobionic acid
Embodiment 3 concentration of substrate is on the impact of transformation efficiency
Initial pH4.5, temperature 30 DEG C, 200rmp, ferments and adds lactose waste water after 5 days, and the lactose-content in fermented liquid is that 5%, pycnoporussp.SYBCL10 cultivates stopping fermentation in the 10th day, and Lactose conversion is the transformation efficiency of lactobionic acid is 71.6%.
Initial pH4.5, temperature 30 DEG C, 200rmp, ferments and adds lactose waste water after 5 days, and the lactose-content in fermented liquid is that 10%, pycnoporussp.SYBCL10 cultivates stopping fermentation in the 10th day, and Lactose conversion is the transformation efficiency of lactobionic acid is 58%.
Initial pH4.5, temperature 30 DEG C, 200rmp, ferments and adds lactose waste water after 5 days, and the lactose-content in fermented liquid is that 15%, pycnoporussp.SYBCL10 cultivates stopping fermentation in the 10th day, and Lactose conversion is the transformation efficiency of lactobionic acid is 42%.
Initial pH4.5, temperature 30 DEG C, 200rmp, ferments and adds lactose waste water after 5 days, and the lactose-content in fermented liquid is that 20%, pycnoporussp.SYBCL10 cultivates stopping fermentation in the 10th day, and Lactose conversion is the transformation efficiency of lactobionic acid is 30%.
Table 2 concentration of substrate is on the impact of transformation efficiency
Claims (2)
1. a strain transforms samguineus and the method for transformation of lactose waste water synthesis lactobionic acid, it is characterized in that utilizing Classification And Nomenclature for samguineus (Pycnoporussp.) SYBC-L10, the samguineus of deposit number CCTCCNO:M2015020 transforms lactose waste water and prepares lactobionic acid, and technique is:
(1) bacterial classification: employing CCTCCNO:M2015020 is starting strain;
(2) culture presevation: bacterial strain uses therefor is preservation on storage medium, switching in 3-6 month is once;
Storage medium: PDA substratum;
(3) seed culture: proceed to seed culture medium from storage medium and need activate 1 time, cultivates 4d at each 30 DEG C;
(4) microbial transformation: add in fermention medium by CCTCCNO:M2015020 bacterial strain, utilizes the cellobiose dehydrogenase of production by biological and laccase to be lactobionic acid by Lactose conversion;
The component of fermention medium is counted with g/L: carbon source 20-50, organic nitrogen source 6-10, inorganic nitrogen-sourced 4-10, potassium primary phosphate 1, magnesium sulfate 0.3, calcium chloride 0.08, Zinc Sulphate Heptahydrate 0.005, four water manganous sulfates 0.0015, CoCL2 6H2O 0.0015
Green vitriol 0.005;
Described carbon source is lactose in lactose waste water, and described nitrogenous source is organic nitrogen source analysis for soybean powder, and inorganic nitrogen-sourced is Secondary ammonium phosphate;
Conversion of substrate lactose concentration is in the fermentation medium 3%-5%W/V, seed liquor with 10% inoculum size, access add different nitrogen sources and inorganic salt composition fermention medium in, 250mL triangular flask liquid amount is 50mL; Under the condition of 28-30 DEG C, 200-220rmp, cultivate 7d, obtain the fermented liquid containing lactobionic acid;
(5) during the fermentation by adding succinate buffer, making pH4.5-5.0 in fermenting process keep constant, improving the transformation efficiency of lactose waste water with this.
2. a strain transforms samguineus and the method for transformation of lactose waste water synthesis lactobionic acid according to claim 1, it is characterized in that conversion of substrate lactose concentration is in the fermentation medium 3%-5%W/V, under the condition of 28-30 DEG C, 200-220rpm, cultivates 7d.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110526418A (en) * | 2019-08-19 | 2019-12-03 | 江南大学 | A method of utilizing antibiotic in immobilization samguineus degrading cultivation waste water |
| CN111218486A (en) * | 2020-03-23 | 2020-06-02 | 杭州巴洛特生物科技有限公司 | Process for synthesizing lactobionic acid by biological method |
| CN113466391A (en) * | 2021-08-12 | 2021-10-01 | 深圳波顿香料有限公司 | Method for simultaneously measuring contents of lactobionic acid and sodium gluconate in fermentation liquor |
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| CN101988046A (en) * | 2010-10-09 | 2011-03-23 | 江南大学 | Method for preparing lactobionic acid by microbial transformation |
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Patent Citations (1)
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| CN101988046A (en) * | 2010-10-09 | 2011-03-23 | 江南大学 | Method for preparing lactobionic acid by microbial transformation |
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| GUPTESHWAR GUPTA ET AL.: "Production of Cellobiose Dehydrogenase from a Newly Isolated White Rot Fungus Termitomyces sp. OE147", 《APPL BIOCHEM BIOTECHNOL》 * |
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| 冯圆圆: "血红密孔菌产纤维二糖脱氢酶及其在乳糖酸合成中的初步研究", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110526418A (en) * | 2019-08-19 | 2019-12-03 | 江南大学 | A method of utilizing antibiotic in immobilization samguineus degrading cultivation waste water |
| CN111218486A (en) * | 2020-03-23 | 2020-06-02 | 杭州巴洛特生物科技有限公司 | Process for synthesizing lactobionic acid by biological method |
| CN111218486B (en) * | 2020-03-23 | 2023-06-16 | 杭州巴洛特生物科技有限公司 | Process for synthesizing lactobionic acid by biological method |
| CN113466391A (en) * | 2021-08-12 | 2021-10-01 | 深圳波顿香料有限公司 | Method for simultaneously measuring contents of lactobionic acid and sodium gluconate in fermentation liquor |
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Application publication date: 20160316 |