CN105154358B - A kind of method of bacillus and its simultaneous saccharification and fermentation production Pfansteihl - Google Patents
A kind of method of bacillus and its simultaneous saccharification and fermentation production Pfansteihl Download PDFInfo
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Abstract
The invention discloses a kind of method of Bacillus and its simultaneous saccharification and fermentation production Pfansteihl, the bacterial strain is bacillus (Bacillus sp.) 13002, and deposit number is CGMCC NO.7431.Method particularly includes: (1) by the bacterial suspension inoculation of bacillus 13002 in seed culture medium, continuous activation culture is carried out, seed culture fluid is obtained；(2) seed culture fluid is inoculated in fermentation medium, supplements the mixed liquor of alpha-amylase and carbohydrase into fermentation medium with constant feed rate, fermentation culture is collected in culture to 72h~96h.The present invention synchronizes diastatic fermentation production Pfansteihl using the α-amylase of starch and the operative temperature of carbohydrase are consistent with fermentation temperature range, and the yield of Pfansteihl is 150~230g/L；And there is high-optical-purity, reach 99.8% or more；It, can be to avoid the pollution of miscellaneous bacteria due to fermentation temperature height.
Description
Technical field
The invention belongs to organic acid fermentation technical fields, are related to the production method of Pfansteihl, and in particular to a kind of gemma bar
The method of bacterium extremely simultaneous saccharification and fermentation production high-optical-purity L-lactic acid.
Background technique
Biomass refers to terrestrial plant based on lignin, cellulose, hemicellulose and other organic matters and aquatic
Plant is a kind of stable Renewable Energy Resources, and abundance, only agricultural crop straw yield in China's is about 700,000,000 tons every year.?
After last century oil crisis, people start to pay attention to the technology for preparing lactic acid, fuel alcohol for raw material by biomass.
Lactic acid (molecular formula: C2H5OCOOH), as α-hydracrylate, since alpha site of carboxyl group carbon atom is not right in its molecule
Claim atom, thus there is optical activity, can be divided into two kinds of configurations of L (+) and D (-), Pfansteihl is dextroform, and D-ALPHA-Hydroxypropionic acid is left-handed type.
Lactic acid is widely used in the industries such as food, beverage, feed, modern medicine, modern, daily-use chemical industry, papermaking and electronics.Bloom
The Pfansteihl for learning purity is a kind of important food additives and intermediate, is had extensively in industries such as food, agricultural, medicine, chemical industry
General application is environmental-friendly biomass plastics by the poly (l-lactic acid) that monomer synthesizes of Pfansteihl, can replace petroleum resources
Plastics can be relieved the increasingly depleted energy crisis of petroleum, and can slow down environmental pollution.The Pfansteihl of high-optical-purity is important
Chiral intermediate and organic synthesis raw material, also play a significant role in terms of improving polylactic acid.
The main reason for extensive use of limitation Pfansteihl, has at present: (1) high production cost of Pfansteihl, fermenting and producing
The cost of raw material it is high, purify and refine and is at high cost；(2) it does not screen suitably ferment and generates high-optical-purity L-lactic acid
Bacterial strain because general lactic acid bacteria mainly with fermentation generate racemic lactic acid based on；(3) existing known lactic acid bacteria generally cannot
Using pentose, such as xylose, ribose and arabinose are as carbon source.
Microbe fermentation method is to prepare one of main method of Pfansteihl at present, due to the physicochemical property and D- of Pfansteihl
Lactic acid is extremely similar, can use for reference in production technology, therefore the key for producing Pfansteihl is to filter out high-optical-purity L-
Lactic acid high-yield strains, and ferment using the cheap raw material for having extensive source, simplify fermentation and purifying technique, reduces cost.
The strain of main research report is as shown in table 1 at present.According to the difference of used microorganism fungus kind, L- is newborn at present
The microbial fermentation of acid is divided into root arrhizus fermentation and the big main Types of bacterial fermentation two.Root arrhizus fermentation belongs to aerobic fermentation, need compared with
High-caliber air and stirring system, it is larger to the consumption of the energy such as electricity, steam, and it is also carried out while fermentation lactic acid
The metabolism of his approach generates more heteroacid, reduces the forward rate of glucose, high production cost.So for industrial production cream
The strain of acid is mostly the bacterium of homofermentative lactic, mainly there is lactobacillus (Lactobacillus), streptococcus
(Streptoccoccus), lactococcus (Lactococcus).
1 strain of table and its fermentation temperature
Bacterial fermentation is generally homofermentation, and impurity generates few in fermentation process, and glucose forward rate is high.These two types of bacterium
Lower generally 30~42 DEG C of common feature fermentation temperature.It is easy pollution microbes, it is therefore desirable to sterilize, cause to fermentation liquid
The loss of waste and the nutrition of energy.And chemical synthesis is due to the hydrogen cyanide of acetaldehyde to be used and severe toxicity, enzymatic production process
It is complicated.Strain in microbe fermentation method mainly is lactic acid bacteria (including Bacillus acidi lactici and streptococcus) and some filamentous fungis.
The advantages such as lactic acid bacteria has fermentation period short as production bacterial strain, and metabolite is single.90% document report is about lactic acid
Bacterium is as production bacterial strain, especially Lactococcus lactis, Lactobacillus delbrueckii,
The bacterial strains such as Lactobacillus helveticus, Lactobacillus casei are reported very much, and the defect of these bacterial strains has
Following several respects: (1) it is 30~37 DEG C that its fermentation temperature is lower, which easily contaminates miscellaneous bacteria, causes tunning Pfansteihl
Optical purity it is impure；(2) itself contains a considerable amount of D-ALPHA-Hydroxypropionic acids in metabolite；(3) lactic acid bacteria itself is in synthesis dimension life
Plain B and certain amino acid aspect are defective, the complicated nutritional ingredient of addition are required supplementation in fermentation production process, to subsequent
Lactic acid isolate and purify and to reduce production cost very unfavorable.Although the fermentation of filamentous fungi can make up the one of above-mentioned lactic acid bacteria
A little disadvantages, such as can be resistant to higher Initial sugar concentration, can be extensive with carbon source, and mycelial separation is easy to carry out etc., still
Fermenting and producing there is also following disadvantage, such as filamentous fungi is aerobic fermentation, needs to configure the air supply of higher level
And stirring system, it is larger to the consumption of electricity, steam etc., and while generating lactic acid, it is also easy to generate other heteroacid, such as
The problems such as Rhizopus oryzae ferment strength is low, and raw material availability is not high, and thalli morphology is difficult to control.
The production bacterial strain of ideal Pfansteihl should have claimed below: (1) cell can mushroom out the biology for reaching high
Amount；(2) fermentation production of L-lactic acid is carried out using cheap raw material, and the inhibition for being resistant to high concentration sugar in process of production is made
With；(3) there is high throughput rate and efficiency of pcr product, and be resistant to the inhibiting effect of high concentration substrate；(4) extractive technique
Middle by-product is as few as possible.
Summary of the invention
It is an object of the invention in view of the above shortcomings of the prior art, provide a kind of bacillus and synchronize sugar using it
Change the method for fermenting and producing high-optical-purity L-lactic acid, the yield of Pfansteihl is high in this method, and carbon source is from a wealth of sources.
To achieve the goals above, present invention employs following technical solutions:
The present invention screens to obtain one plant of high-temperature resistant strain bacillus (Bacillus that can be used for Pfansteihl fermenting and producing
Sp.) 13002, which can ferment at a high temperature of 45~65 DEG C, greatly reduce the machine of pollution microbes in fermentation process
Meeting, it might even be possible to carry out fermenting and producing in the case where culture medium is unsterilised, reduce energy consumption and production cost.The bacterial strain simultaneously
Pfansteihl optical purity be up to 99.8% or more, be easy to subsequent and isolate and purify, therefore, with Bacillus 13002 ferment give birth to
Producing Pfansteihl has good industrial production prospect.13002 bacterial strain of bacillus has the property that
(1) morphological feature: bacterium colony is rounded raised and rich in gloss, and neat in edge is non-wrinkled, and surface is smooth, in milky white
Color is slightly transparent.Strain is observed as shown in Figure 1, bacterium after Gram's staining using 16 × 100 amplifications under an optical microscope
Body is rod-shaped, single arrangement；Gram-positive bacterium can produce gemma under certain condition, be amphimicrobe, thallus be it is rod-shaped,
Single arrangement, it is about 0.5~1.0 μm wide, it is about 3.0~5.0 μm.
(2) physiological characteristic: catalase, catalase, V-P experiment, hydrogen sulfide be all it is positive, can caseinhydrolysate, bright
Glue and starch can utilize citrate, nitrate, cannot utilize propionate, and indoles experiment is feminine gender, maximum growth temperature 65
℃；Glucose, sucrose, maltose, fructose, mannose, xylose, lactose, galactolipin can be utilized, rhamnose cannot be utilized.
(3) its DNA is extracted, its 16S rDNA is expanded and itself and Bacillus coagulans (bacillus coagulans) is sequenced
The 16S rDNA sequence of strain have the homology greater than 99%, nucleotide sequence is seen, such as table 1.
According to the information of 3 aspect such as form, physio-biochemical characteristics and DNA conserved sequence, bacillus coagulans are determined that it is.
A kind of method of simultaneous saccharification and fermentation production Pfansteihl, includes the following steps:
(1) prepared by seed culture fluid: the bacterial suspension inoculation of the bacillus 13002 in logarithmic phase growth is trained in seed
It supports in base, carries out continuous activation culture, obtain seed culture fluid；The seed culture medium are as follows: calculated according to mass ratio, bacteriology
0.2 part~1.0 parts of peptone, 0.1 part~0.5 part of yeast extract powder, 1.0 parts~2.0 parts of glucose, NaCl 0.5 part~1.5
Part, 100 parts are settled to distilled water, adjusts pH to 6.0~7.5, it is cooling after sterilizing；
(2) with the ratio of 2~10:100 of volume ratio, seed culture fluid simultaneous saccharification and fermentation Pfansteihl: is inoculated in fermentation
In culture medium, in the fermenter after starting fermentation, alpha-amylase and saccharification are supplemented into fermentation medium with constant feed rate
Fermentation culture is collected in the mixed liquor of enzyme, culture to 72h~96h.
The fermentation medium are as follows: based on parts by weight, 60~80 parts of 15~25% starch solution, glucose 1.0~
2.5 parts, 1.0~4.0 parts of nitrogen source, 0.01~0.10 part of bitter salt, 100 parts is settled to distilled water, adjusts medium pH
To 6.0~7.5,45~65 DEG C are cooled to after sterilizing.
The nitrogen source refers to: yeast powder, peptone, beef extract, soybean meal hydrolysate, yeast extract, corn pulp proteolysis
One or more of liquid is combined with arbitrary proportion.
The additive amount of the alpha-amylase is that alpha-amylase is added with every 100g dried starch for 0.02~0.08mL, with every
100g dried starch is added 0.05~0.20mL of carbohydrase and dilutes 5~10 times with sterile water after two kinds of enzyme mixing.
The flow rate of the mixed liquor of the alpha-amylase and carbohydrase is 0.5~2.0mL/min.
The starch be one or both of cornstarch, tapioca, potato starch, rice meal or coarse rice powder with
On.
Step (2) fermentation temperature is 45~65 DEG C.
Step (1) the continuous activation culture 2~3 times, fermentation temperature is 45 DEG C, and incubation time is for 24 hours.
Fermentation termination is determined using the content of chiral column high effective liquid chromatography for measuring Pfansteihl.Method fermentation of the invention
The yield of the Pfansteihl of production is 150~230g/L, and optical purity is 99.8% or more, and saccharic acid conversion ratio is up to 95%.
To further realize the object of the invention, fermentation temperature is preferably 50 DEG C, and incubation time is both preferably for 24 hours；Described
Starch solution preferred volume percentage is 20% cornstarch；The preferable amount of amylase is 0.03mL/100g starch；Carbohydrase
Preferable amount be 0.10mL/100g starch.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) bacillus 13002 of the invention has that resistance is strong, high temperature resistant, easy preservation, using starch α-amylase and
The operative temperature of carbohydrase it is consistent with fermentation temperature range can synchronous fermentation production high-optical-purity Pfansteihl.
(2) fermentation temperature of bacillus 13002 is 45 DEG C or more, can reduce the chance of living contaminants.
(3) 2~3 activation are successively carried out, activate the bacterium as far as possible, and are in synchronous growth state.
(4) strain fermentation working condition is simple, and fermentation temperature is high, greatly reduces the chance of living contaminants, improves L-
The optical purity of lactic acid.
(5) it ferments in higher temperature, fermentation broth viscosity can be reduced, be conducive to the operation of subsequent processing steps, very
The fermenting and producing that Pfansteihl can be extremely carried out under conditions of culture medium is unsterilised, reduces energy consumption and production cost.
(6) temperature of the fermentation production of L-lactic acid of bacterial strain is 45~55 DEG C, this temperature is in starch liquefacation enzyme and carbohydrase
Sphere of action in, produce (SSF) lactic acid for simultaneous saccharification and fermentation and provide the foundation condition.
(7) optical purity of strain fermentation production Pfansteihl meets the production such as polylactic acid, lactate 99.5% or more
Requirement of the product to the optical purity of Pfansteihl.
The bacillus (Bacillus sp.) 13002, was obtained by separation screening in Lychee juice, and April 8 in 2013
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center day (referred to as: CGMCC, address: Beijing's southern exposure
The institute 3 of area North Star West Road 1, Institute of Microorganism, Academia Sinica), deposit number is CGMCC NO.7431.
Detailed description of the invention
Fig. 1 is micromorphology of the bacillus 13002 after Gram's staining, and amplification factor is 16 × 1000 times.
Fig. 2 is the high-efficient liquid phase chromatogram of the chiral column Chirex 3126 of Pfansteihl standard items.
Fig. 3 is that 13002 fermenting L-lactic acid of bacillus is utilized in the embodiment of the present invention 1, with chiral column Chirex's 3126
High-efficient liquid phase technique detects the content of Pfansteihl, the high-efficient liquid phase chromatogram for the 96h that ferments.
Specific embodiment
Below with reference to embodiment, invention is further described in detail, but the scope of protection of present invention not office
It is limited to this.
Embodiment 1
(1) prepared by seed culture fluid: the bacterial suspension inoculation of the bacillus 13002 in logarithmic phase growth is trained in seed
It supports in base, carries out continuous 2~3 activation cultures, at 45 DEG C, shaking flask culture for 24 hours, obtains seed culture fluid；The seed culture
Base are as follows: it is calculated according to mass ratio, 0.5 part of bacteriological peptone, 0.2 part of yeast extract powder, 2.0 parts of glucose, NaCl1.0 parts,
100 parts are settled to distilled water, adjusts pH to 6.5, cooling is spare after sterilizing；
(2) Pfansteihl of simultaneous saccharification and fermentation high-optical-purity: with the ratio of volume ratio 5:100, seed culture fluid is connect
Kind in the fermenter after starting fermentation, is supplemented with constant feed rate into fermentation medium a certain amount of in fermentation medium
Alpha-amylase and Glucoamylase Solution mixed liquor, culture to 72h collect fermentation culture；The fermentation medium are as follows: with weight
Amount number meter, 70 parts of 20% corn starch solution, 2.0 parts of glucose, 2.0 parts of nitrogen source, 0.05 part of bitter salt, with steaming
Distilled water is settled to 100 parts, adjusts medium pH to 6.5,55 DEG C are cooled to after sterilizing.
The nitrogen source refers to: 0.5 part of yeast powder, 1.5 parts of peptone.
The additive amount of the alpha-amylase is that alpha-amylase is added with every 100g dried starch for 0.03mL, is formed sediment so that every 100g is dry
Powder is added carbohydrase 0.10mL and dilutes 8 times with sterile water after two kinds of enzyme mixing；The flow rate is 1.0mL/min.
D-ALPHA-Hydroxypropionic acid and L- cream of the art of this patent in the high-efficient liquid phase technique detection fermentation process of chiral column Chirex 3126
The content of acid, Chirex 3126 is a kind of ligand exchange column, with immobilization and metal ion (Cu2+) coordination ligand be fix
Phase, between the ligand based on ligand and stationary phase in mobile phase exchange equilibrium and stability come separating L-lactic acid and D-ALPHA-Hydroxypropionic acid,
Specific steps bibliography Liu Dongmei, Wu Hui, Yu Yigang, etc., high performance liquid chromatography is to Pfansteihl in pickles and D-ALPHA-Hydroxypropionic acid
Chiral separation and measurement, modern food science and technology, 2007,23 (8): 74-76 }.Using high performance liquid chromatograph system, it is equipped with
Waters 600 pumps multi-pass pipeline conveying system；717 autosampler of Waters；Waters 2996PDA Diode Array Detector
Device；Chromatographic column: chiral column Chirex 3126 (D)-penicillami, 4.6mm ID × 250mm L.Analysis condition: mobile phase
The aqueous isopropanol that copper-bath solvent for 0.002moL/L is 5%, using preceding through 0.45 μm of membrane filtration, ultrasound is de-
Gas；Flow rate of mobile phase is 0.7mL/min；30 DEG C of column temperature；Diode array Waters 2996, Detection wavelength 254nm；Standard
With the preceding ultrasonic degassing after 0.45 μm of membrane filtration of sample solution；Sample volume is 10 μ L；With Empower integral software product
Point, carry out peak area method outer marking quantitative.The efficient liquid of chiral column Chirex 3126 (D)-penicillami is used in this research
The optical purity and content of GC headspace analysis Pfansteihl, the results show that the retention time of Pfansteihl is 25.205min (see figure
2) optical purity for the Pfansteihl that, bacillus 13002 is fermented reaches 99.8% or more, sees Fig. 3.The amount of Pfansteihl when to 72h
Reach maximum, is 150.5g/L.
Embodiment 2
(1) prepared by seed culture fluid: the bacterial suspension inoculation of the bacillus 13002 in logarithmic phase growth is trained in seed
It supports in base, carries out continuous 2~3 activation cultures, at 45 DEG C, shaking flask culture for 24 hours, obtains seed culture fluid；The seed culture
Base are as follows: it is calculated according to mass ratio, 1.0 parts of bacteriological peptone, 0.5 part of yeast extract powder, 1.5 parts of glucose, NaCl0.5 parts,
100 parts are settled to distilled water, adjusts pH to 6.0, cooling is spare after sterilizing；
(2) Pfansteihl of simultaneous saccharification and fermentation high-optical-purity: with the ratio of volume ratio 2:100, seed culture fluid is connect
Kind in the fermenter after starting fermentation, is supplemented with constant feed rate into fermentation medium a certain amount of in fermentation medium
Alpha-amylase and Glucoamylase Solution mixed liquor, culture to 96h collect fermentation culture；The fermentation medium are as follows: with weight
Amount number meter, 80 parts of 20% rice starch solution, 2.5 parts of glucose, 3.0 parts of nitrogen source, 0.10 part of bitter salt, with steaming
Distilled water is settled to 100 parts, and with neutralizer tune medium pH to 6.0,65 DEG C are cooled to after sterilizing.
The nitrogen source refers to: 0.5 part of yeast powder, 1.5 parts of peptone, 1.0 parts of beef extract.
The additive amount of the alpha-amylase is that alpha-amylase is added with every 100g dried starch for 0.05mL, is formed sediment so that every 100g is dry
Powder is added carbohydrase 0.20mL and dilutes 5 times with sterile water after two kinds of enzyme mixing；The flow rate is 2.0mL/min.
The neutralizer is sterilized ammonium hydroxide and sulfuric acid solution, is added portionwise by automatically controlling.
Fermentation termination is determined using the content of chiral column high effective liquid chromatography for measuring Pfansteihl.In entire fermentation process
The optical purity of Pfansteihl reaches 99.8% or more.Amount to 96h fermentation ends Pfansteihl is 230.15g/L.
Embodiment 3
(1) prepared by seed culture fluid: the bacterial suspension inoculation of the bacillus 13002 in logarithmic phase growth is trained in seed
It supports in base, carries out continuous 2~3 activation cultures, at 45 DEG C, shaking flask culture for 24 hours, obtains seed culture fluid；The seed culture
Base are as follows: it is calculated according to mass ratio, 0.2 part of bacteriological peptone, 0.1 part of yeast extract powder, 1.0 parts of glucose, NaCl1.5 parts,
100 parts are settled to distilled water, adjusts pH to 7.5, cooling is spare after sterilizing；
(2) Pfansteihl of simultaneous saccharification and fermentation high-optical-purity: with the ratio of volume ratio 10:100, seed culture fluid is connect
Kind in the fermenter after starting fermentation, is supplemented with constant feed rate into fermentation medium a certain amount of in fermentation medium
Alpha-amylase and Glucoamylase Solution mixed liquor, culture to 80h collect fermentation culture；The fermentation medium are as follows: with weight
Amount number meter, 80 parts of 25% corn starch solution, 1.0 parts of glucose, 3.0 parts of nitrogen source, 0.01 part of bitter salt, with steaming
Distilled water is settled to 100 parts, adjusts medium pH to 7.5,45 DEG C are cooled to after sterilizing.
The nitrogen source refers to: 0.3 part of yeast powder, 0.2 part of beef extract, 0.5 part of yeast extract, 2.0 parts of soybean meal hydrolysate.
The additive amount of the alpha-amylase is that alpha-amylase is added with every 100g dried starch for 0.02mL, is formed sediment so that every 100g is dry
Powder is added carbohydrase 0.15mL and dilutes 10 times with sterile water after two kinds of enzyme mixing；The flow rate is 2.0mL/min.
Fermentation termination is determined using the content of chiral column high effective liquid chromatography for measuring Pfansteihl.In entire fermentation process
The optical purity of Pfansteihl reaches 99.8% or more.Amount to 80h fermentation ends Pfansteihl is 165.03g/L.
Embodiment 4
(1) prepared by seed culture fluid: the bacterial suspension inoculation of the bacillus 13002 in logarithmic phase growth is trained in seed
It supports in base, carries out continuous 2~3 activation cultures, at 45 DEG C, shaking flask culture for 24 hours, obtains seed culture fluid；The seed culture
Base are as follows: it is calculated according to mass ratio, 0.8 part of bacteriological peptone, 0.3 part of yeast extract powder, 1.0 parts of glucose, NaCl1.2 parts,
100 parts are settled to distilled water, adjusts pH to 7.0, cooling is spare after sterilizing；
(2) Pfansteihl of simultaneous saccharification and fermentation high-optical-purity: with the ratio of volume ratio 8:100, seed culture fluid is connect
Kind in the fermenter after starting fermentation, is supplemented with constant feed rate into fermentation medium a certain amount of in fermentation medium
Alpha-amylase and Glucoamylase Solution mixed liquor, culture to 80h collect fermentation culture；The fermentation medium are as follows: with weight
Measure number meter, 25% 50 parts of corn starch solution, 20 parts of tapioca, 2.5 parts of glucose, 1.0 parts of nitrogen source, seven hydrated sulfuric acids
0.06 part of magnesium, 100 parts are settled to distilled water, adjusts medium pH to 7.0,65 DEG C is cooled to after sterilizing.
The nitrogen source refers to: 0.5 part of beef extract, 0.5 part of peptone.
The additive amount of the alpha-amylase is that alpha-amylase is added with every 100g dried starch for 0.08mL, is formed sediment so that every 100g is dry
Powder is added carbohydrase 0.20mL and dilutes 5 times with sterile water after two kinds of enzyme mixing；The flow rate is 1.5mL/min.
Fermentation termination is determined using the content of chiral column high effective liquid chromatography for measuring Pfansteihl.In entire fermentation process
The optical purity of Pfansteihl reaches 99.8% or more.Amount to 80h fermentation ends Pfansteihl is 172.10g/L.
Embodiment 5
(1) prepared by seed culture fluid: the bacterial suspension inoculation of the bacillus 13002 in logarithmic phase growth is trained in seed
It supports in base, carries out continuous 2~3 activation cultures, at 45 DEG C, shaking flask culture for 24 hours, obtains seed culture fluid；The seed culture
Base are as follows: it is calculated according to mass ratio, 0.8 part of bacteriological peptone, 0.3 part of yeast extract powder, 1.5 parts of glucose, NaCl0.5 parts,
100 parts are settled to distilled water, adjusts pH to 6.5, cooling is spare after sterilizing；
(2) Pfansteihl of simultaneous saccharification and fermentation high-optical-purity: with the ratio of volume ratio 8:100, seed culture fluid is connect
Kind in the fermenter after starting fermentation, is supplemented with constant feed rate into fermentation medium a certain amount of in fermentation medium
Alpha-amylase and Glucoamylase Solution mixed liquor, culture to 72h collect fermentation culture；The fermentation medium are as follows: with weight
Measure number meter, 15% 60 parts of potato starch solution, 10 parts of Brown Rice Starch, 1.0 parts of glucose, 2.5 parts of nitrogen source, seven hydration sulphur
0.1 part of sour magnesium is settled to 100 parts with distilled water, adjusts medium pH to 6.5,50 DEG C are cooled to after sterilizing.
The nitrogen source refers to: 0.5 part of beef extract, 2.0 parts of corn pulp protein hydrolyzate.
The additive amount of the alpha-amylase is that alpha-amylase is added with every 100g dried starch for 0.06mL, is formed sediment so that every 100g is dry
Powder is added carbohydrase 0.15mL and dilutes 10 times with sterile water after two kinds of enzyme mixing；The flow rate is 2.0mL/min.
Fermentation termination is determined using the content of chiral column high effective liquid chromatography for measuring Pfansteihl.In entire fermentation process
The optical purity of Pfansteihl reaches 99.8% or more.Amount to 72h fermentation ends Pfansteihl is 180.32g/L.
Embodiment 6
(1) prepared by seed culture fluid: the bacterial suspension inoculation of the bacillus 13002 in logarithmic phase growth is trained in seed
It supports in base, carries out continuous 2~3 activation cultures, at 45 DEG C, shaking flask culture for 24 hours, obtains seed culture fluid；The seed culture
Base are as follows: it is calculated according to mass ratio, 0.8 part of bacteriological peptone, 0.4 part of yeast extract powder, 1.2 parts of glucose, NaCl1.2 parts,
100 parts are settled to distilled water, adjusts pH to 7.0, cooling is spare after sterilizing；
(2) Pfansteihl of simultaneous saccharification and fermentation high-optical-purity: with the ratio of volume ratio 8:100, seed culture fluid is connect
Kind in the fermenter after starting fermentation, is supplemented with constant feed rate into fermentation medium a certain amount of in fermentation medium
Alpha-amylase and Glucoamylase Solution mixed liquor, culture to 96h collect fermentation culture；The fermentation medium are as follows: with weight
Amount number meter, 80 parts of 23% corn starch solution, 1.5 parts of glucose, 3.5 parts of nitrogen source, 0.08 part of bitter salt, with steaming
Distilled water is settled to 100 parts, adjusts medium pH to 7.0,45 DEG C are cooled to after sterilizing.
The nitrogen source refers to: 0.5 part of beef extract, 0.5 part of peptone, 2.5 parts of soybean meal hydrolysate.
The additive amount of the alpha-amylase is that alpha-amylase is added with every 100g dried starch for 0.06mL, is formed sediment so that every 100g is dry
Powder is added carbohydrase 0.10mL and dilutes 10 times with sterile water after two kinds of enzyme mixing；The flow rate is 2.0mL/min.
Fermentation termination is determined using the content of chiral column high effective liquid chromatography for measuring Pfansteihl.In entire fermentation process
The optical purity of Pfansteihl reaches 99.8% or more.Amount to 96h fermentation ends Pfansteihl is 220.40g/L.
Claims (10)
1. a kind of bacillus, which is characterized in that the bacterial strain be bacillus (Bacillus sp.) 13002, in 2013 4
It is preserved within 8th China Committee for Culture Collection of Microorganisms's common micro-organisms center the moon, deposit number is CGMCC NO.
7431。
2. a kind of method of simultaneous saccharification and fermentation production Pfansteihl, which comprises the steps of:
(1) prepared by seed culture fluid: the bacteria suspension of bacillus 13002 described in the claim 1 in logarithmic phase growth is connect
Kind carries out continuous activation culture, obtains seed culture fluid in seed culture medium；The seed culture medium are as follows: according to mass ratio
It calculates, 0.2 part ~ 1.0 parts of bacteriological peptone, 0.1 part ~ 0.5 part of yeast extract powder, 1.0 parts ~ 2.0 parts of glucose, NaCl 0.5
Part ~ 1.5 parts, 100 parts are settled to distilled water, adjusts pH to 6.0 ~ 7.5, it is cooling after sterilizing；
(2) with the ratio of 2 ~ 10:100 of volume ratio, seed culture fluid simultaneous saccharification and fermentation Pfansteihl: is inoculated in fermentation medium
In, in the fermenter after starting fermentation, supplement mixing for alpha-amylase and carbohydrase into fermentation medium with constant feed rate
Liquid is closed, fermentation culture is collected in culture to the h of 72 h ~ 96.
3. according to the method described in claim 2, it is characterized in that, the fermentation medium are as follows: based on parts by weight, 15 ~ 25%
60 ~ 80 parts of starch solution, 1.0 ~ 2.5 parts of glucose, 1.0 ~ 4.0 parts of nitrogen source, 0.01 ~ 0.10 part of bitter salt, with steaming
Distilled water is settled to 100 parts, adjusts medium pH to 6.0 ~ 7.5,45 ~ 65 °C are cooled to after sterilizing.
4. according to the method described in claim 3, it is characterized in that, the nitrogen source refers to: yeast powder, peptone, beef extract, beans
One or more of dregs of rice hydrolyzate, yeast extract, corn pulp protein hydrolyzate are combined with arbitrary proportion.
5. according to the method described in claim 3, it is characterized in that, the additive amount of the alpha-amylase is formed sediment so that every 100 g is dry
It is 0.02 ~ 0.08 mL that alpha-amylase, which is added, in powder, and 0.05 ~ 0.20 mL of carbohydrase, two kinds of enzyme mixing are added with every 100 g dried starch
Afterwards, 5 ~ 10 times are diluted with sterile water.
6. according to the method described in claim 5, it is characterized in that, the dosage of the alpha-amylase is dry for 0.03 mL/100 g
Starch；The dosage of carbohydrase is 0.10 mL/100 g dried starch.
7. according to method described in claim 2 ~ 6 any one, which is characterized in that the mixing of the alpha-amylase and carbohydrase
The flow rate of liquid is 0.5 ~ 2.0 mL/min.
8. according to method described in claim 3 ~ 6 any one, which is characterized in that the starch is cornstarch, para arrowroot
One or more of powder, potato starch, rice meal or coarse rice powder.
9. according to method described in claim 2 ~ 6 any one, which is characterized in that step (2) fermentation temperature is 45 ~ 65
°C。
10. according to method described in claim 2 ~ 6 any one, which is characterized in that step (1) the continuous activation culture 2 ~
3 times, fermentation temperature is 45 °C, and incubation time is 24 h.
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| CN107857606A (en) * | 2017-11-08 | 2018-03-30 | 贵州省五谷惠生态农业科技有限公司 | A kind of ureaformaldehyde modifying agent and its preparation method and application |
| CN108264399A (en) * | 2018-02-05 | 2018-07-10 | 砀山县绿源生态肥料有限公司 | A kind of preparation method of agricultural amino acid fertilizer |
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| CN110403011B (en) * | 2019-07-17 | 2022-12-16 | 华南理工大学 | Bacillus coagulans fermented milk and preparation method thereof |
| CN110358798B (en) * | 2019-08-21 | 2020-09-11 | 浙江省桐庐汇丰生物科技有限公司 | Method for producing validamycin by fermentation |
| CN110734873B (en) * | 2019-10-10 | 2022-04-22 | 华南理工大学 | Method for increasing number of bacillus coagulans spores and application |
| CN110656136B (en) * | 2019-10-25 | 2021-08-17 | 江南大学 | Method for producing menadione-7 by using starch |
| CN112501219B (en) * | 2020-12-30 | 2022-12-13 | 天津科技大学 | Method for producing lactic acid monomer by fermenting sucrose as raw material |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002074934A1 (en) * | 2001-03-16 | 2002-09-26 | University Of Tartu | Thermophilic microorganism bacillus coagulans strain sim-t dsm 14043 for the production of l(+)-lactate from fermentable sugars and their mixtures |
| CN101173242A (en) * | 2007-10-18 | 2008-05-07 | 中国科学院微生物研究所 | Method for producing L-lactic acid and coagulate bacillus cereus special for the same |
-
2015
- 2015-08-19 CN CN201510510895.8A patent/CN105154358B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002074934A1 (en) * | 2001-03-16 | 2002-09-26 | University Of Tartu | Thermophilic microorganism bacillus coagulans strain sim-t dsm 14043 for the production of l(+)-lactate from fermentable sugars and their mixtures |
| CN101173242A (en) * | 2007-10-18 | 2008-05-07 | 中国科学院微生物研究所 | Method for producing L-lactic acid and coagulate bacillus cereus special for the same |
Non-Patent Citations (3)
| Title |
|---|
| Production of D-lactic acid from defatted rice bran by simultaneous saccharification and fermentation;Takaaki Tanaka等;《Bioresource Technology》;20050413;第97卷;第211-217页 * |
| 直接乳酸发酵工艺的研究进展;赵国振等;《食品工业科技》;20091231;第30卷(第12期);第410-413页，尤其是第410页摘要、右栏第1段、第411页左栏第3段和图1 * |
| 马铃薯淀粉同步糖化发酵制备L-乳酸条件的统计学优化;赵国振等;《生物加工过程》;20100731;第8卷(第4期);第6-11页 * |
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