CN104531810A - Method for preparing maltonic acid through efficient microbial conversion - Google Patents
Method for preparing maltonic acid through efficient microbial conversion Download PDFInfo
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- CN104531810A CN104531810A CN201510016938.7A CN201510016938A CN104531810A CN 104531810 A CN104531810 A CN 104531810A CN 201510016938 A CN201510016938 A CN 201510016938A CN 104531810 A CN104531810 A CN 104531810A
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- maltobionic acid
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Abstract
The invention provides a method for preparing maltonic acid through efficient microbial conversion. The method comprises the steps that (1) non-protein amino acid, namely, 5-aminolevulinic acid, is added in a traditional thallus fermentation culture process, so that the thallus culture time is greatly shortened, and an obtained thallus has the capacity of efficiently converting maltose to generate the maltonic acid; (2) centrifugal cell resuspension is carried out, obtained resting cells are used for converting the maltose, and NaCO3 or CaCO3 is added in the conversion process to keep pH constant so as to improve the conversion efficiency; (3) after conversion, centrifugal supernatant extraction is carried out, extracted supernatant is subjected to evaporation and concentration, alcohol precipitation and drying to obtain maltonic acid calcium and maltonic acid sodium, and finally a sulfuric acid replacement reaction is carried out to obtain the maltonic acid product. The maltonic acid obtained through the method has the remarkable advantages of being short in preparation cycle, high in conversion efficiency and purity, safe, reliable and the like, and can be applied to the industries of food additives, pharmaceutical intermediates, cosmetics and the like.
Description
Technical field
This belongs to technical field of microbial fermentation, relates to a kind of method that maltobionic acid is prepared in microbial transformation.
Background technology
Existing patent report maltose is as the purposes of foodstuff additive, it can be used as sweetener, strengthen the natural aroma and flavor of some foodstuff products, maltobionic acid also contributes to the antioxygenation in food, can prevent in the manufacturing processed of food and feed product or stop in oxidation reaction process wherein.The preparation of current maltobionic acid has chemical catalysis synthesis method and microbe transformation method, and due to the production of chemical synthesis often with multiple by product in oxidising process, make separation, purification step becomes complicated, therefore production cost is relatively high.Fermentable conversion method does not almost have by product generation, transformation efficiency advantages of higher.
5-ALA (5-ALA) is a kind of nonprotein amino acid is biosynthesizing vitamins B
12, protoheme, the precursor substance of the tetrapyrroles such as chlorophyll, is extensively present in microorganism, plant and animal cell.A kind of porphyrin compound of the protoheme iron ion that has been chelating is the important component of aerobic repiration and anaerobic respiration electron transmission catenin, it or the prothetic group of many induction Function protein and enzyme.Protoheme participates in the many important biochemical reactions process in biomass cells, such as electron transmission, gas transfer, the signal transduction in cell, superoxide reduction etc.
Research shows, 5-ALA has obvious promoter action to plant-growth.Its growth promoting function is the interactional result of multiple effect, as promoted the photosynthesis of plant, adjustment respiration, promoting plant tissue differentiation, active cell peroxidation and improve resistance etc.At present, 5-ALA to the promoter action of plant-growth confirm by great many of experiments but to rarely have report in the application aspect of microorganism.Innovation of the present invention is 5-ALA to be applied in microorganism pseudomonas fermentation culture, 5-ALA add the growth cycle not only substantially reducing thalline, and improve the saccharic acid conversion capability of thalline.
Summary of the invention
The present invention relates to a kind of high-effective microorganism and transform the method preparing maltobionic acid.The maltobionic acid that this method obtains have with short production cycle, transformation efficiency is high and product purity is high, the remarkable advantage such as safe and reliable.
The technical solution adopted in the present invention is: a kind of high-effective microorganism transforms the method preparing maltobionic acid, and fermentation strain used is pseudomonas, comprises the steps:
(1) fermentation culture of pseudomonas: be inoculated into by pseudomonas and with the addition of in the liquid fermentation medium of 5-ALA, cultivates 8h ~ 30h for 28 DEG C ~ 32 DEG C in shaking table.
Fermention medium component in g/L can be: carbon source 0 ~ 40, nitrogenous source 0 ~ 35, described carbon source can be in high fructose syrup (fructose of 42%), sucrose, glucose, maltose, lactose any one, described nitrogenous source can be in yeast extract paste, corn steep liquor, extractum carnis, peptone at least one or other source organic or inorganic nitrogenous source; For improving biomass growth rate, add the 5-ALA of culture volume 0.1% ~ 0.5%.
Preferred fermentation culture conditions: temperature is 28 DEG C ~ 32 DEG C, liquid amount 20 ~ 30mL/250mL, inoculum size 4% ~ 8%, and shaking speed is 150rpm ~ 220rpm, and incubation time is 8h ~ 30h.
(2) resting cell preparation: in fermention medium yeast culture good after, 5000 ~ 8000rpm centrifugal 10min collection thalline, it is resuspended as resting cell solution system to add stroke-physiological saline solution.It is the ability of maltobionic acid that described resting cell has conversion maltose.
(3) conversion of resting cells: add maltose in resting cell solution system, in system, resting cell concentration is OD
600=25 ~ 50, maltose mass percent concentration is 10% ~ 40%, and invert point is 28 DEG C ~ 32 DEG C, and shaking speed is 150rpm ~ 220rpm, and the reaction times is 24h ~ 72h.NaCO can be added in conversion process
3or CaCO
3pH is made to be held constant at about 6.0.
(4) extraction of maltobionic acid: transform after terminating, centrifuging and taking supernatant, obtains maltobionic acid calcium salt through steps such as evaporation concentration, alcohol settling, oven dry, finally obtains maltobionic acid product with sulfate substitution reaction.
Maltobionic acid product is interpreted as maltobionic acid and its esters, as maltobionic acid calcium, maltobionic acid sodium.
Maltobionic acid product prepared by the present invention can be applicable to the fields such as foodstuff additive, medicine intermediate, makeup.Purifying in various degree or post-treatment can be carried out again to meet the purity requirement in different application field as required to product.
Mentioned reagent and culture medium prescription, if no special instructions, be this area conventional reagent and substratum.
The method detecting maltobionic acid is as follows:
HPLC condition: Agilent1100 chromatographic column: nh 2 column (4.6 × 250mm); Moving phase is acetonitrile: water=60:40 (phosphoric acid containing 0.1%); Detector: UV-detector 210nm; Column temperature: 25 DEG C; Flow velocity: 1mL/min; Sample size: 10 μ L; Retention time is 6.033min.
Beneficial effect:
1. pseudomonas a kind ofly can transform the bacterium that maltose is maltobionic acid, and adding 5-ALA in the fermentation medium can significantly reduce the yeast culture time.
2. the conversion of resting cells obtained after thalline fermentation is prepared maltobionic acid and is improve transformation efficiency.
3. the maltobionic acid that this method obtains have with short production cycle, transformation efficiency is high and product purity is high, the remarkable advantage such as safe and reliable.
Embodiment
The inventive method is described below by specific embodiment.Unless stated otherwise, technique means used in the present invention is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention only limited by claims.To those skilled in the art, under the prerequisite not deviating from essence of the present invention and scope, the various change carry out the material component in these embodiments and consumption or change also belong to protection scope of the present invention.
Pseudomonas used herein, preferably can make the one in garden beet pseudomonas AS 1.6397, Pseudomonas fluorescens ATCC13525, the huge pseudomonas AS 1.3385 of chrysanthemum, pseudomonas syringae NBRC14078, be commercially available business bacterial classification.
Embodiment 1:
By pseudomonas streak inoculation on solid medium, cultivate in 30 DEG C of constant incubators.The single bacterium colony that picking has been grown from flat board is inoculated in the 30mL liquid nutrient medium of the 5-ALA adding 0.1%, 0.2%, 0.3%, 0.4%, 0.5% that (fermention medium component is counted with g/L: sucrose 10 respectively, peptone 10, yeast extract paste 30), in 30 DEG C, cultivate in the shaking table of 180r/min.With the time of pseudomonas liquid cultivation for X-coordinate, with the OD of bacterium liquid measured by each time point
600value is ordinate zou, draws out the growth curve of pseudomonas.Result is as shown in table 1
The impact that table 1 different concns 5-ALA ferments on thalline
| 5-ALA concentration | To time stationary phase | Stationary phase bacterium OD 600Value |
| 0 | 40h | 7.0 |
| 0.1％ | 25h | 10.2 |
| 0.2％ | 13h | 12.1 |
| 0.3％ | 8h | 15.0 |
| 0.4％ | 8h | 11.5 |
| 0.5％ | 8h | 10.4 |
Result display adds the thalli growth cycle in the fermention medium of 5-ALA and obviously reduces, and thalli growth about 8h enters stationary phase, decreases more than 30 hour than not adding 5-ALA culture cycle.And 5-ALA concentration cell concentration 0.1% ~ 0.3% time increases with 5-ALA concentration and increases, increase with 5-ALA concentration after exceeding this concentration and decrease, when 5-ALA concentration is 0.3%, cell concentration reaches the highest OD
600=15, improve more than 2 times than the substratum bacteria concentration not adding 5-ALA.
Embodiment 2:
By pseudomonas streak inoculation on solid medium, cultivate in 30 DEG C of constant incubators.The 30mL that the single bacterium colony that picking has been grown from flat board is inoculated in the 5-ALA adding 0.3% respectively without (fermention medium component is counted with g/L: peptone 10, yeast extract paste 30) in carbon source liquid nutrient medium in 30 DEG C, cultivate in the shaking table of 180r/min.The OD of bacterium liquid is surveyed in different time points
600value.Result shows thalli growth about 8h through entering stationary phase, now OD
600=9.2.
Embodiment 3:
By pseudomonas streak inoculation on solid medium, cultivate in 30 DEG C of constant incubators.The single bacterium colony that picking has been grown from flat board is inoculated in respectively and adds in the 30mL liquid nutrient medium of 0.3%5-amino-laevulic acid that (fermention medium component is counted with g/L: high fructose syrup 10, peptone 10, yeast extract paste 30) in 30 DEG C, cultivate in the shaking table of 180r/min, in fermention medium yeast culture good after, 8000rpm10min collected by centrifugation thalline, add stroke-physiological saline solution liquid resuspended as conversion fluid, add maltose in conversion of resting cells liquid system, in system, resting cell concentration is OD
600=45, maltose concentration 20%, adds CaCO in conversion process
3make pH keep constant, invert point is 30 DEG C, and shaking speed is 180rpm, and the reaction times is 24h ~ 72h.Result is as shown in table 2.
Add 5-ALA in table 2 fermented liquid and cultivate the resting cell of acquisition to the impact of maltobionic acid productive rate
| Transformation time | Productive rate | |
| Fermention medium does not add 5-ALA | 24h | 40％ |
| Fermention medium adds the 5-ALA of 0.3% | 24h | 95％ |
Result display adds 5-ALA fermention medium conversion of resting cells maltobionic acid productivity ratio and does not add 5-glycyl third and improve more than 2 times.
Embodiment 4:
By pseudomonas streak inoculation on solid medium, cultivate in 30 DEG C of constant incubators.The single bacterium colony that picking has been grown from flat board is inoculated in respectively and adds in the 30mL liquid nutrient medium of 0.3%5-amino-laevulic acid that (fermention medium component is counted with g/L: sucrose 10, peptone 10, yeast extract paste 30) in 30 DEG C, cultivate in the shaking table of 180r/min, in fermention medium yeast culture good after, 8000rpm10min collected by centrifugation thalline, add sterile phosphate buffer resuspended as conversion fluid, add different concns maltose (10%, 20%, 30%, 40%) in conversion of resting cells liquid system, in system, resting cell concentration is OD
600=45, in conversion process, add CaCO
3make pH keep constant, invert point is 30 DEG C, and shaking speed is 180rpm, and the reaction times is 24h ~ 72h.Result is as shown in table 3.
Table 3 different concns maltose is on the impact transformed
| Maltose concentration | Transformation time | Productive rate |
| 10％ | 20h | 95％ |
| 20％ | 24h | 95％ |
| 30％ | 47h | 95％ |
| 40％ | 70h | 95％ |
Result shows, and transformation time extends with the increase of maltose concentration, finally all reaches identical productive rate, considers transformation time and conversion yield, selects the maltose of 20% as preferred conversion of substrate concentration.
Embodiment 5:
1. the preparation of maltobionic acid calcium
(1) conversion of resting cells liquid collected after centrifugation supernatant, under the condition of 100 DEG C evaporation concentration to original volume 1/3 now solution be thick, then cool to room temperature; (2) add the ethanol of same volume 95% and constantly stir, now having a large amount of gluey maltobionic acid calcium deposit to separate out, suction filtration removing ethanol after fully stirring; (3) in gelatinous precipitate at the ethanol adding same volume 95%, fully stir postprecipitation and slowly become lenticular; (4) suction filtration obtains maltobionic acid calcium crude product to dry; (5) add water crude product rear identical volume concentrated with appeal, suction filtration after heating for dissolving, and suction filtration liquid cool to room temperature adds after same volume 95% ethanol stirred crystallization is separated out to be drained, and obtains maltobionic acid calcium fine work.
2. the preparation of maltobionic acid
The obtained maltobionic acid calcium furnishing aqueous solution, be placed in suitable reactor, the equimolar vitriol oil is slowly added under stirring, in 60 DEG C ~ 90 DEG C heating in water bath 1h ~ 3h, the calcium sulfate precipitation that elimination is separated out, the maltobionic acid hydrated barta obtained after filtrate cooling and oxalic acid are that precipitation agent carries out purifying, and remove a small amount of sulfate radical and calcium ion, too much barium and oxalic acid oxalic precipitated barium are removed; Or the exchange column of anion and cation exchange resin after filtrate cooling, is flow through with suitable flow velocity, obtain highly purified maltobionic acid.
Claims (6)
1. a method for maltobionic acid is prepared in microbial transformation, and starting strain is pseudomonas, comprises the steps:
(1) fermentation culture of pseudomonas: be inoculated into by pseudomonas and with the addition of in the liquid fermentation medium of 5-ALA, cultivates 8h ~ 30h for 28 DEG C ~ 32 DEG C in shaking table;
(2) resting cell preparation: in fermention medium yeast culture good after, 5000 ~ 8000rpm centrifugal 10min collection thalline, it is resuspended as resting cell solution system to add stroke-physiological saline solution;
(3) conversion of resting cells: add maltose in resting cell solution system, in system, resting cell concentration is OD
600=25 ~ 50, maltose mass percent concentration is 10% ~ 40%, and invert point is 28 DEG C ~ 32 DEG C, and shaking speed is 150rpm ~ 220rpm, and the reaction times is 24h ~ 72h;
(4) extraction of maltobionic acid: transform after terminating, centrifuging and taking supernatant, obtains maltobionic acid calcium, maltobionic acid sodium through steps such as evaporation concentration, alcohol settling, oven dry, finally obtains maltobionic acid product with sulfate substitution reaction.
2. the method for maltobionic acid is prepared in a kind of microbial transformation as claimed in claim 1, it is characterized in that, described in step (1), fermention medium component is counted with g/L: carbon source 0 ~ 40, nitrogenous source 0 ~ 35, described carbon source be high fructose syrup, sucrose, glucose, maltose, lactose, starch, dextrin, Fructus Hordei Germinatus leaching powder, semi-lactosi, in fructose any one, described nitrogenous source is the organic or inorganic nitrogenous source at least one or other source in yeast extract paste, corn steep liquor, extractum carnis, peptone.
3. the method for maltobionic acid is prepared in a kind of microbial transformation as claimed in claim 1, it is characterized in that, described in step (3), maltose mass percent concentration is 20%.
4. the method for maltobionic acid is prepared in a kind of microbial transformation as claimed in claim 1, it is characterized in that, described in step (1), the addition of 5-ALA is 0.1% ~ 0.5% of culture volume.
5. the method for maltobionic acid is prepared in a kind of microbial transformation as claimed in claim 4, it is characterized in that, the addition of described 5-ALA is 0.3% of culture volume.
6. the method for maltobionic acid is prepared in a kind of microbial transformation as claimed in claim 1, it is characterized in that, described pseudomonas is the one in beet pseudomonas AS 1.6397, Pseudomonas fluorescens ATCC 13525, the huge pseudomonas AS 1.3385 of chrysanthemum, pseudomonas syringae NBRC14078.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105063111A (en) * | 2015-08-26 | 2015-11-18 | 天津科技大学 | Method for preparing L-rhamnose acid through microbial conversion |
| CN108129290A (en) * | 2017-12-25 | 2018-06-08 | 武汉三江航天固德生物科技有限公司 | A kind of method of sulfate radical in removal lactic acid |
| CN112110961A (en) * | 2020-09-27 | 2020-12-22 | 山东齐都药业有限公司 | Preparation method of impurities in calcium gluconate |
| CN116590355A (en) * | 2022-12-27 | 2023-08-15 | 安徽斯拜科生物科技有限公司 | Method for synthesizing maltobionic acid by catalyzing maltose with glucose dehydrogenase |
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| EP0384534A1 (en) * | 1989-02-21 | 1990-08-29 | Coöperatieve Verkoop- en Productievereniging van Aardappelmeel en Derivaten 'AVEBE' B.A. | A process for the fermentative oxidation of reducing disaccharides |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105063111A (en) * | 2015-08-26 | 2015-11-18 | 天津科技大学 | Method for preparing L-rhamnose acid through microbial conversion |
| CN108129290A (en) * | 2017-12-25 | 2018-06-08 | 武汉三江航天固德生物科技有限公司 | A kind of method of sulfate radical in removal lactic acid |
| CN108129290B (en) * | 2017-12-25 | 2021-01-22 | 武汉三江航天固德生物科技有限公司 | Method for removing sulfate radical in lactic acid |
| CN112110961A (en) * | 2020-09-27 | 2020-12-22 | 山东齐都药业有限公司 | Preparation method of impurities in calcium gluconate |
| CN116590355A (en) * | 2022-12-27 | 2023-08-15 | 安徽斯拜科生物科技有限公司 | Method for synthesizing maltobionic acid by catalyzing maltose with glucose dehydrogenase |
| CN116590355B (en) * | 2022-12-27 | 2023-11-07 | 安徽斯拜科生物科技有限公司 | Method for synthesizing maltobionic acid by catalyzing maltose with glucose dehydrogenase |
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