CN105018537A - Selection method for preparing L-malic acid and rhizopus oryzae mutant strain-2 by fermenting corn stalks - Google Patents

Selection method for preparing L-malic acid and rhizopus oryzae mutant strain-2 by fermenting corn stalks Download PDF

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CN105018537A
CN105018537A CN201510474784.6A CN201510474784A CN105018537A CN 105018537 A CN105018537 A CN 105018537A CN 201510474784 A CN201510474784 A CN 201510474784A CN 105018537 A CN105018537 A CN 105018537A
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mutant strain
rhizopus oryzae
concentration
malic acid
strain
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李兴江
孙婷
杨英
王海涛
吴学凤
刘亚
李顺
姜绍通
潘丽军
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Hefei University of Technology
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Hefei University of Technology
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Abstract

The invention provides a selection method for preparing L-malic acid and rhizopus oryzae mutant strain-2 by fermenting corn stalks. A corn stalk acid hydrolysate serves as a basic material of a culture medium and also serves as a carbon source, meanwhile, a nitrogen source, inorganic salt and a neutralizing agent are added to form liquid culture mediums, the rhizopus oryzae mutant strain-2 is inoculated in a fermenting tank after sterilization is conducted, and aerobic fermentation is conducted, adsorption separation is conducted on fermentation liquid through alkaline ion exchange resin D301 after solid-liquid separation is conducted, water elution is conducted, an L-malic acid aqueous solution is obtained, the rhizopus oryzae mutant strain-2 is obtained from (Rhizopus oryzae) CICC 41160 through isotope 60 Co mutagenesis spores by screening through propenol and fluoroacetic acid slabs. According to the selection method, based on the corn stalks, the yield of L-malic acid reaches 3%, second only to glucose, compared with initial strain fermentation, the yield of the L-malic acid is increased by 51.76%, the content of the impurity such as fumaric acid is lowered by 90.12%, and the content of the impurity such as succinic acid is lowered by 81.34%.

Description

A kind of maize straw fermentation is for the selection of L MALIC ACID and Rhizopus oryzae mutant strain-2
One, technical field
The present invention relates to the comprehensive utilization of agricultural crop straw, is exactly maize straw fermentation for the selection of L MALIC ACID and fermentation strain Rhizopus oryzae mutant strain-2.
Two, background technology
Oxysuccinic acid chemical name is 2-hydroxyl-1.4-succinic acid, because the carbon atom connecting hydroxyl is unsymmetrical carbon, causes in opticity, show as dextrorotation (D-), left-handed (L-) and racemize (DL-).Because the chiral structure of oxysuccinic acid, therefore Chang Yisan kind form exists, and comprises D-malic acid, L MALIC ACID, DL-oxysuccinic acid.Modal in fruit is L-type, presents white crystals or powder, has special tart flavour.
L MALIC ACID is a kind of important organic acid, is widely used in the field such as food and chemistry.Compared with citric acid, oxysuccinic acid acidity is large, heat production is low, and taste is soft, lasting and tool peat-reek, is described as " optimal acid condiment ".
Traditional L MALIC ACID fermentation be with glucose or starch based for raw material, if farm crop hydrolyzed solution can be utilized to ferment produce L MALIC ACID, will raw material sources be expanded, effectively reduce costs.In China's biomass cellulose abundant raw material, but utilization ratio is low, generally takes the mode process such as burning and burying, not only wastes resource and also causes destruction to ecotope.Relevant to utilize biomass cellulose to produce the research such as bio-ethanol, biofuel report a lot, but it is less to utilize biomass cellulose to produce the report of organic acid especially L MALIC ACID.How to utilize corn stalk hydrolysis fermentation to produce L MALIC ACID and there is profound significance.
Occurring in nature produces the bacterium of L MALIC ACID Rhizopus oryzae, flavus, Aspergillus parasiticus, zhizopchin etc., wherein Rhizopus oryzae produce L MALIC ACID and have that nutritional requirement is simple, aerobic fermentation, product purity high and be widely used.Mutagenic treatment is carried out for microorganism strains and can filter out suitable bacterial strain at short notice, accumulate a certain or several meta-bolites useful to the mankind, reduce the generation of by product simultaneously, screen the high yield, the good quality strain that obtain the most at last to apply, the mutafacient system used at present mainly comprises uv irradiating mutagenesis, ionizing rays mutagenesis, ion implantation mutagenesis, space breeding, complex mutation etc.
60co belongs to ionizing rays mutagenesis, because it has high-energy, directly or indirectly can change DNA structure by ionizing event, thus reach mutagenesis object.Utilize 60co irradiation vinylcarbinol plate screening ethanol dehydrogenase deficient strain, has blocked alcohol metabolism, reduces coproduct ethanol and generates.Utilize 60co irradiation gifblaar poison plate screening glyoxylate cycle deficient strain, reduces the output of by product fumaric acid and succsinic acid.
In current L MALIC ACID production process, mainly with glucose, starch etc. as fermenting carbon source, raw materials cost is high.With the generation of the by products such as ethanol, fumaric acid, succsinic acid in fermenting process, and adopt batch fermentation, fermentation period is long, and continuity is weak.
Three, summary of the invention
The object of the invention is to utilize maize straw to prepare L MALIC ACID, technical problem to be solved utilizes the method for biological fermentation and the high and mutant strain that by product is few Rhizopus oryzae is excellent of seed selection one strain output.
The present invention utilizes maize straw fermentation for the method for L MALIC ACID, comprise the preparation of liquid nutrient medium, sterilizing, inoculation, fermentation culture be separated, to be described liquid nutrient medium be difference with the prior art take concentration as the base material of corn stalk hydrolysis as substratum of 85-115g/L, also as carbon source, add nitrogenous source simultaneously, inorganic salt and neutralizing agent, be specially and add ammonium sulfate 3.5 ~ 4.5g/L in 1L hydrolyzed solution, magnesium sulfate 0.25 ~ 0.35g/L, ferrous sulfate 0.020 ~ 0.030g/L, potassium primary phosphate 0.45 ~ 0.55g/L, zinc sulfate 0.05 ~ 0.15g/L, calcium carbonate 70 ~ 90g/L, inoculate Rhizopus oryzae mutant strain-2 after medium sterilization, spore concentration is 10 6~ 10 8cFU/mL, inoculum size are 8 ~ 12vt% (volume percent of fermented liquid, lower same), described fermentation culture be in fermentor tank in 31 ~ 33 DEG C, stir fermentation 70 ~ 75 hours under air flow (air) 0.35 ~ 0.45L/Lmin condition, solid-liquid separation after fermentation, fermented liquid adopts deacidite D301 to carry out fractionation by adsorption, and pH is 3 ~ 4, temperature 20 ~ 30 DEG C, and water elution, obtains the L MALIC ACID aqueous solution, carries out qualitative analysis to it, and result as shown in Figure 2.
Preferred liquid substratum concentration: corn stalk hydrolysis 100g/L, ammonium sulfate 0.40g/L, magnesium sulfate 0.30g/L, ferrous sulfate 0.025g/L, potassium primary phosphate 0.50g/L, zinc sulfate 0.10g/L, calcium carbonate 80g/L.
Preferred spore concentration 10 7cFU/mL, inoculum size 10vt% (volume percent).
Described corn stalk hydrolysis is that corn straw smashing crosses 40 mesh sieves, adds water wet ball mill 1 ~ 2h in ball mill of 1 ~ 2 times amount (in mass, lower same); Then proceed in hydrolysis kettle, then add the water of 5 ~ 6 times amount, and with sulphuric acid soln adjust pH 2 ~ 3, stir hydrolysis 1 ~ 1.5 hour in 110 ~ 130 DEG C, with ammoniacal liquor adjust pH 4.5 ~ 5.5 after hydrolysis, solid-liquid separation; Filtrate is concentrated into concentration 85 ~ 115g/L, is namely corn stalk hydrolysis.
Described Rhizopus oryzae mutant strain is such seed selection:
1. utilize isotropic substance 60co carries out mutagenesis, Induced dosage 10 ~ 30Gy to starting strain Rhizopus oryzae (Rhizopus oryzae CICC41160), and irradiation time is 20min.
2. 60co mutagenesis obtains mutant strain-1 in conjunction with vinylcarbinol plate screening, and vinylcarbinol concentration starting strain when 6ml/L occurs that significance suppresses, therefore has filtered out mutant strain-1 when vinylcarbinol concentration 6ml/L, for follow-up mutagenesis.
3. walking the mutant strain-1 that mutagenesis obtains more than is parent strain, passes through 60co (10 ~ 30Gy, 20min) mutagenesis obtains mutant strain-2 (mutant strain) in conjunction with gifblaar poison plate screening.Choosing gifblaar poison concentration is 7g/L, the Auxotrophie mutant that picking grows in this concentration, and this bacterial strain is mutant strain-2, as fermentation strain of the present invention, as subsequent fermentation.(concrete mechanism is shown in the internal mechanism figure of Fig. 3 mutant strain seed selection).
4. get step 3 gained fermentation mutant bacterial strain spore suspension single culture 48h for fermentation, spore concentration 10 6~ 10 8cFU/mL.
Compare and prior art, the present invention has following beneficial effect:
The Rhizopus oryzae Rhizopus oryzae CICC41160 mutant strain adopted in the present invention passes through isotropic substance 60the ethanol dehydrogenase of Co irradiation mutagenesis and gifblaar poison plate screening dull and stereotyped in conjunction with vinylcarbinol and glyoxylate cycle deficient strain.Mutant strain-2 can utilize corn stalk hydrolysis to ferment very well and produce L MALIC ACID, and fumaric acid, succsinic acid, ethanol content have obvious decline.Mutant strain-2 is compared with initial strains, and L MALIC ACID output adds 51.76%, and fumaric acid output decreases 90.12%, and succinic acid production decreases 81.34%, and ethanol production is 0.In maize straw, the productive rate 3% of L MALIC ACID, is only second to glucose.
Four, accompanying drawing explanation
Fig. 1 is the process flow sheet that the present invention prepares L MALIC ACID.
Fig. 2 is the qualitative analysis collection of illustrative plates of L MALIC ACID, and A is standard substance collection of illustrative plates, and B is sample collection of illustrative plates.
Fig. 3 is the internal mechanism figure of mutant strain seed selection.
Fig. 4 is different carbon source is that fermenting raw materials produces L MALIC ACID results contrast.
Five, embodiment
(1), the seed selection of Rhizopus oryzae mutant strain-2
1. strain mutagenesis screening: utilize isotropic substance 60co carries out mutagenesis, Induced dosage 10 ~ 30Gy to starting strain Rhizopus oryzae CICC41160, and irradiation time is 20min.Irradiation vinylcarbinol concentration is that 6ml/L plate screening obtains mutant strain-1.By mutant strain-1 again 60the flat board being inoculated in gifblaar poison mass concentration 7g/L after Co irradiation (10 ~ 30Gy, 20min) screens to obtain mutant strain-2, and this bacterial strain is mutant strain, for subsequent fermentation.
2. mutant strain activation culture: mutant strain is inoculated in 32 DEG C of cultivation 1-2d in potato dextrose agar (PDA substratum), the bacterium colony that picking colony is dense, mycelial growth is vigorous is used as subsequent fermentation.PDA substratum: potato 200g/L, glucose 20g/L, agar 20g/L, distilled water 1000mL, pH nature, 121 DEG C of sterilizing 15min.
3. mutant strain spore suspension preparation: to wash the form of spore by the bacterial strain access spore suspension culture medium culturing 48h on PDA substratum.Spore concentration 10 7cFU/mL.Spore suspension substratum: glucose 100g/L, ammonium sulfate 5g/L, potassium primary phosphate 0.5g/L, magnesium sulfate 0.3g/L, ferrous sulfate 0.025g/L, zinc sulfate 0.1g/L, natural pH, 121 DEG C of sterilizing 20min.
(2), corn stalk hydrolysis preparation:
The maize straw getting dry weight 200g is pulverized, and crosses 40 mesh sieve, adds 340g water, under the condition of ball mill, carry out wet-milling 1h process to maize straw.Get the good maize straw of wet-milling and water mixes according to 1:6, add 3mol/L sulfuric acid and make the pH value of mixture remain on 2-3, after 120 DEG C of process 60min, add in strong aqua and pH to 5.0.After solid-liquid separation, hydrolyzed solution being concentrated into concentration by Rotary Evaporators is 100g/L, as fermenting carbon source.
(3), fermentation is for L MALIC ACID
1, be that fermenting raw materials produces L MALIC ACID with corn stalk hydrolysis
(1). prepared by fermention medium: corn stalk hydrolysis 100g/L, ammonium sulfate concentrations 4g/L, magnesium sulfate 0.30g/L, ferrous sulfate 0.025g/L, potassium primary phosphate 0.5g/L, zinc sulfate 0.1g/L, neutralizing agent calcium carbonate 80g/L (independent sterilizing), 121 DEG C of sterilizing 20min.
(2). L MALIC ACID is produced in mutant strain fermentation: get 10L fermention medium (2000g maize straw) in 15L fermentor tank, inoculation 1L mutant strain spore suspension, spore concentration 10 7cFU/mL leavening temperature 32 DEG C, rotating speed 500r/min, air flow 0.4L/ (Lmin), every 12h sampling and measuring L MALIC ACID output.
High performance liquid phase measures L MALIC ACID output: chromatographic column Waters Carbohydrate (3.9mm × 300mm), chromatographic instrument Waters 2695, detector Waters 2424, sample size 20 μ L, flow velocity 0.8mL/min, moving phase 20mmol/L dipotassium hydrogen phosphate, column temperature 80 DEG C, single sample detects 20min.
Experiment repetition 3 times, as shown in Figure 4, every 12h sampling and measuring L MALIC ACID output, during its fermentation 72h, L MALIC ACID output is 60.15g/L to result.
Solid-liquid separation after fermentation ends, fermented liquid deacidite D301 carries out fractionation by adsorption, pH3 ~ 4, temperature 20 ~ 30 DEG C, flow velocity 2ml/min, and water elution, obtains the L MALIC ACID aqueous solution, carries out qualitative and quantitative analysis with high performance liquid chromatography.
2, be that fermenting raw materials produces L MALIC ACID with glucose
Using 100g/L glucose as carbon source, remaining and operate the same example 1, every 12h sampling and measuring L MALIC ACID output, during its fermentation 72h, output is 68.97g/L.
3, be that fermenting raw materials produces L MALIC ACID with wood sugar
Using 100g/L glucose as carbon source, remaining and operate the same example 1, every 12h sampling and measuring L MALIC ACID output, during its fermentation 72h, output is 40.27g/L.

Claims (4)

1. a maize straw fermentation is for the method for L MALIC ACID, comprise the preparation of liquid nutrient medium, sterilizing, inoculation, fermentation culture and be separated, it is characterized in that: described liquid nutrient medium is the base material using corn stalk hydrolysis as substratum, concentration 85-115g/L, adds ammonium sulfate 3.5 ~ 4.5g/L, magnesium sulfate 0.25 ~ 0.35g/L, ferrous sulfate 0.020 ~ 0.030g/L, potassium primary phosphate 0.45 ~ 0.55g/L, zinc sulfate 0.05 ~ 0.15g/L, calcium carbonate 70 ~ 90g/L simultaneously; Rhizopus oryzae mutant strain-2 is inoculated, spore concentration 10 after medium sterilization 6~ 10 7cFU/min, inoculum size 8 ~ 12vt%; Described fermentation culture be in fermentor tank in 31 ~ 33 DEG C, stir fermentation 70 ~ 75 hours under air flow 0.35 ~ 0.45L/L.min condition; Described corn stalk hydrolysis was water wet ball mill 1 ~ 2 hour in ball mill that the corn stalk powder of 40 mesh sieves adds 1 ~ 2 times amount, then the water adding 5 ~ 6 times amount in hydrolysis kettle is again proceeded to, and with sulphuric acid soln adjust pH 2 ~ 3, stir hydrolysis 1 ~ 1.5 hour in 110 ~ 130 DEG C; Hydrolysis terminates rear ammoniacal liquor adjust pH 4.5 ~ 5.5, is concentrated into concentration 85 ~ 115g/L after solid-liquid separation; Described Rhizopus oryzae mutant strain-2 utilizes isotropic substance 60co carries out mutagenesis, Induced dosage 10 ~ 30Gy to starting strain Rhizopus oryzae CICC41160, and irradiation time is 20min; Mutant strain-1 is obtained with vinylcarbinol concentration 6ml/L plate screening, by mutant strain-1 again after radiation 60be inoculated in after Co irradiation on flat board that gifblaar poison mass concentration is 7g/L and screen to obtain mutant strain-2.
2. method according to claim 1, is characterized in that: in liquid nutrient medium, each group concentration is corn stalk hydrolysis 100g/L, ammonium sulfate 0.40g/L, magnesium sulfate 0.30g/L, ferrous sulfate 0.025g/L, potassium primary phosphate 0.50g/L, zinc sulfate 0.10g/L, calcium carbonate 80g/L.
3. method according to claim 1, is characterized in that: spore concentration 10 7cFU/ml, inoculum size 10vt%.
4. a selection for the Rhizopus oryzae mutant strain-2 fermented in the method for claim 1, is characterized in that:
(1). utilize isotropic substance 60co carries out mutagenesis, Induced dosage 10 ~ 30Gy, irradiation time 20min to starting strain Rhizopus oryzae CICC41160, is that 6ml/L plate screening obtains mutant strain-1, by mutant strain-1 again in conjunction with vinylcarbinol concentration 60co irradiation, 10 ~ 30Gy, 20min, be inoculated on flat board that gifblaar poison mass concentration is 7g/L, screen to obtain mutant strain-2; This bacterial strain is mutant strain;
(2). mutant strain activation culture: mutant strain to be inoculated in PDA substratum 32 DEG C and to cultivate 1-2d, picking colony is dense, the vigorous bacterium colony of mycelial growth is used as subsequent fermentation; PDA substratum: potato 200g/L, glucose 20g/L, agar 20g/L, distilled water 1000mL, pH nature;
(3). prepared by mutant strain spore suspension: to wash the form of spore by the bacterial strain access spore suspension culture medium culturing 48h on PDA substratum; Spore concentration 10 6~ 10 7cFU/ml; Spore suspension substratum: glucose 100g/L, ammonium sulfate 5g/L, potassium primary phosphate 0.5g/L, magnesium sulfate 0.3g/L, ferrous sulfate 0.025g/L, zinc sulfate 0.1g/L, natural pH, 121 DEG C of sterilizing 20min.
CN201510474784.6A 2015-08-05 2015-08-05 Selection method for preparing L-malic acid and rhizopus oryzae mutant strain-2 by fermenting corn stalks Pending CN105018537A (en)

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Publication number Priority date Publication date Assignee Title
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Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101255450A (en) * 2008-01-31 2008-09-03 南京工业大学 Process for producing L-malic acid by using rhizopus oryzae fermentation
CN101724663A (en) * 2008-10-24 2010-06-09 中国农业大学 Method for producing L-lactic acid by utilizing corn cob and special rhizopus oryzae thereof
CN102618589A (en) * 2012-04-12 2012-08-01 苏州百趣食品有限公司 Method for producing L-malic acid by means of rhizopus oryzae one-step fermentation
CN103966301A (en) * 2014-05-14 2014-08-06 南京林业大学 Method for producing L-malic acid through coupling rhizopus oryzae and candida rugosa

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Application publication date: 20151104