CN109837316A - A method of Pfansteihl is efficiently produced using lignocellulosic corncob residue - Google Patents
A method of Pfansteihl is efficiently produced using lignocellulosic corncob residue Download PDFInfo
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Abstract
The invention discloses a kind of methods for efficiently producing Pfansteihl using lignocellulosic corncob residue, it is related to the fermenting and producing field of Pfansteihl, using bacillus coagulans as fermentation strain, utilizes single carbon source, nitrogen source, simultaneous saccharification and fermentation produces Pfansteihl, and wherein carbon source is corncob residue.The present invention realizes the utilization of cheap carbon source and nitrogen source, produces Pfansteihl to low cost, high efficiency, high yield pulp1, provides a kind of effective alternative in the application that Pfansteihl produces for lignocellulosic.
Description
Technical field
The present invention relates to the fermenting and producing field of Pfansteihl more particularly to a kind of utilization lignocellulosic corncob residue are high
The method of effect production Pfansteihl.
Background technique
In recent years, plastic products are widely used in the production and living of people, cause certain environmental pollution.With
The enhancing of environmental consciousness, demand of the people to biodegradable plastic are gradually promoted.Polylactic acid (PLA) is a kind of good biology
Degradation material is polymerize by lactic acid and is generated.The production interest of lactic acid is added significantly to the demand of PLA in recent years.However, poly- cream
The high production cost of acid has seriously affected its application, this is mainly due to monomer lactic acid occupy polylactic acid production it is most of at
This, and common carbon source starch (glucose) occupies most of cost of production of lactic acid.Therefore, the alternative of production of lactic acid is found
Cheap carbon source has become a kind of urgent demand.
Lignocellulosic is made of cellulose, hemicellulose and lignin, be it is a kind of be easy to get extensively, reproducible biomass
Resource, mainly from agricultural and forestry waste.With the potentiality for replacing starch (glucose), lignocellulosic sugar is widely used for
Bulk chemical is produced, is especially considered as a kind of economic, attractive raw material in lactic acid production process.Corncob is
China is most abundant, one of agricultural wastes being easy to get extensively.After hemicellulose removal and dilute acid hydrolysis, corncob residue
(CCR) content of cellulose reaches 56~60%.Due to previous step --- portion has been shared in the production of xylitol and furfural
Be divided into this, therefore, bulk chemical such as: in production of lactic acid, CCR is considered as a kind of great representative and cheap wood
Matter cellulosic material.
Lignocellulosic biomass resource can be efficiently converted into lactic acid by bacillus coagulans, have output Pfansteihl optics
Purity is high, high temperature resistant, nutrition is barren, carbon efficiencies are high, by-product forms few advantage.The optimum growth temp of bacillus coagulans
It is 50 DEG C, close with the optimum temperature of cellulose hydrolyzation, this is conducive to bacterial strain and synchronizes diastatic fermentation (SSF) production cream
Acid.
Currently, producing lactic acid using lignocellulosic, still there are problems to be solved, if lignocellulosic utilizes difficulty,
Enzyme hydrolysis is at high cost, and production efficiency and yield are more low.
Therefore, those skilled in the art, which is dedicated to developing, a kind of efficiently produces L- using lignocellulosic corncob residue
The method of lactic acid produces L- lactic acid to low cost, high efficiency, high yield pulp1 to realize the utilization of cheap carbon source and nitrogen source,
A kind of effective alternative is provided in the application that Pfansteihl produces for lignocellulosic.
Summary of the invention
In view of the above drawbacks of the prior art, the technical problem to be solved by the present invention is to how inexpensive, high productions
The problem of producing Pfansteihl to efficiency and high yield pulp1.
To achieve the above object, Pfansteihl is efficiently produced using lignocellulosic corncob residue the present invention provides a kind of
Method, using bacillus coagulans as fermentation strain, using single carbon source, nitrogen source, simultaneous saccharification and fermentation produces L- lactic acid,
Wherein carbon source is corncob residue.
Further, fermentation strain is bacillus coagulans H-1(Bacillus coagulans H-1), it is preserved in China
Type Tissue Collection (Luojia Mountain, Wuhan, Hubei Province Wuhan University, China), deposit number is CCTCC No.M
2013105, preservation date is on March 26th, 2013.
Further, corncob residue is product of the corncob after hemicellulose removal and dilute acid hydrolysis.Optionally,
Purchased from Shandong Longli Biology Science and Technology Co., Ltd, fiber in corncob residue is measured by two step acidolysis according to NREL method
Cellulose content is 59.51%, hemicellulose level 5.97%, content of lignin 24.10%.
Further, nitrogen source is peanut meal.
Further, method includes the following steps:
Step 1, activation culture: by strain inoculated to solid slope culture medium, 50 DEG C are cultivated 12~24 hours；
Step 2, seed culture: by the bacterial strain after activating in step 1, it is transferred to fluid nutrient medium in gnotobasis, seals
Firmly bottleneck creates gnotobasis with bacterial strain consumption oxygen, cultivates 12~16 hours under the conditions of 50 DEG C, 200rpm, obtain seed
Liquid；
Step 3, fermentation tank culture: seed liquor in step 2 is transferred to fermentation tank culture medium according to 5~25% inoculum concentration
In, while adding cellulase 0~20U/g cellulose, 0~20U/g of beta-glucosidase cellulose and neutral proteinase 0.3g/
L controls fermentation jar temperature at 50 DEG C, speed of agitator 150rpm, cultivates 30~84 hours；
Step 4, detection: sampling after fermentation, detection Pfansteihl, cellobiose and concentration of glucose.
Further, the solid slope culture medium in step 1 is GSY solid slope culture medium, formula are as follows: 100g/L
Glucose, 10g/L yeast powder, 5g/L tryptone, 50g/L CaCO3And 2% agarose；Fluid nutrient medium in step 2 is
GSY fluid nutrient medium, formula are as follows: 100g/L glucose, 10g/L yeast powder, 5g/L tryptone, 50g/L CaCO3。
Further, the fermentation tank culture based formulas in step 3 are as follows: 60~140g/L corncob residue, 9~21g/L flower
The raw dregs of rice, 18~42g/LCaCO3。
Further, step 4 also comprises the steps of:
Step 4.1, sample treatment: sample being placed in boiling water bath and is handled 10 minutes, and 2mL sample is taken to be added 2mL 2M's
H2SO4Acidolysis 10 minutes, calcium lactate is made to be converted into lactic acid, distilled water is added to make L- lactic acid, glucose, cellobiose concentration dilution extremely
1~5g/L, 6000~10000rpm are centrifuged 10 minutes, supernatant are taken, with 0.22 μm of water phase membrane filtration in liquid phase bottle；
Step 4.2, sample detection: being detected with HPLC, testing conditions are as follows: liquid-phase chromatographic column is Bio-Rad Aminex
HPX-87H, mobile phase are the H of 5mM2SO4, flow velocity 0.5mL/min, column temperature is 55 DEG C, and sample volume is 10 μ L, using showing poor folding
Photodetector (RID) detection.
In a better embodiment of the invention, using bacillus coagulans H-1 as fermentation strain, passed through with corncob
Corncob residue after crossing hemicellulose removal and dilute acid hydrolysis is as sole carbon source, using peanut meal as only nitrogen source, uses
(formula is 100g/L corncob residue, 15g/L flower for GSY solid slope culture medium, GSY fluid nutrient medium and fermentation tank culture medium
The raw dregs of rice, 30g/L CaCO3), by step difference activated strains, culture seed liquor, fermentation tank culture simultaneous saccharification and fermentation is carried out, it is raw
Output Pfansteihl, and tunning is taken to be detected.
It is equally using bacillus coagulans H-1 as fermentation strain, with jade in another better embodiment of the invention
Corncob residue of the meter Xin after hemicellulose removal and dilute acid hydrolysis is as sole carbon source, using peanut meal as unique nitrogen
Source, during fermentation tank culture, the step of also adding feed supplement, method includes the following steps:
Step 1, activation culture: being inoculated into GSY solid slope culture medium for bacillus coagulans H-1, and 50 DEG C of cultures 12~
24 hours；
Step 2, seed culture: the bacterial strain after activating in step 1 is transferred to GSY fluid nutrient medium in gnotobasis, uses
Sealed membrane and plastic film seal bottleneck, create gnotobasis with bacterial strain consumption oxygen, cultivate under the conditions of 50 DEG C, 200rpm
12~16 hours, obtain seed liquor；
Step 3, fermentation tank culture: seed liquor in step 2 is transferred to fermentation tank culture medium according to 5~25% inoculum concentration
In, while adding cellulase 0~20U/g cellulose, 0~20U/g of beta-glucosidase cellulose and neutral proteinase 0.3g/
L controls fermentation jar temperature at 50 DEG C, speed of agitator 150rpm, ferments 72~140 hours；
Step 4, feed supplement: at logarithmic growth phase 4~7 hours, feed supplement 30~70g/L corncob residue, 9~21g/L
CaCO3, the cellulase of 2~20U/g cellulose, the beta-glucosidase of 0~20U/g cellulose；
Step 5, detection: sampling after fermentation, detection Pfansteihl, cellobiose and concentration of glucose.
Further, the formula of the GSY solid slope culture medium in step 1 are as follows: 100g/L glucose, 10g/L yeast powder,
5g/L tryptone, 50g/L CaCO3And 2% agarose；The formula of GSY fluid nutrient medium in step 2 are as follows: the Portugal 100g/L
Grape sugar, 10g/L yeast powder, 5g/L tryptone, 50g/L CaCO3；The formula of fermentation tank culture medium in step 3 are as follows: 60~
140g/L corncob residue, 13.5~31.5g/L peanut meal, 18~42g/L CaCO3。
A kind of method and existing skill preparing Pfansteihl using lignocellulosic corncob residue production proposed by the present invention
Art is compared, have it is following the utility model has the advantages that
(1) cheap carbon source is realized --- the development and utilization of lignocellulosic.Utilize cheap lignocellulosic corncob
Cellulose degradation is glucose by plus fiber element enzyme, is used as production high value added product L- as sole carbon source by residue
Lactic acid advantageously reduces cost, while solving to discard bring problem of environmental pollution due to lignocellulosic.
(2) cheap nitrogen source is realized --- the development and utilization of peanut meal.High level is produced using peanut meal as only nitrogen source
Compound L-lactic acid advantageously reduces cost, carries out industrial fermentation production, has application value.
(3) the high efficiency production of Pfansteihl is realized.Using bacillus coagulans as production bacterial strain, gemma is condensed
Bacillus H-1 has that Pfansteihl optical purity height, high temperature resistant, nutrition is barren, carbon efficiencies are high, by-product forms few advantage, can be sharp
Pfansteihl is produced with corncob residue simultaneous saccharification and fermentation, the high efficiency for being able to achieve lignocellulosic sugar to Pfansteihl converts, consumption
When it is short, save the energy.
(4) the high yield pulp1 production of Pfansteihl is realized.Bacillus coagulans H-1 utilizes corncob residue fermenting and producing L- cream
Acid, cellulose convert the conversion ratio of Pfansteihl up to 76%~85%, and compared with other producers, the yield of Pfansteihl is relatively
Height provides a kind of effective alternative in the application that Pfansteihl produces for lignocellulosic.
It is described further below with reference to technical effect of the attached drawing to design of the invention, specific method and generation, with
It is fully understood from the purpose of the present invention, feature and effect.
Detailed description of the invention
Fig. 1: influence of one embodiment of the present of invention cellulase concentration to enzymatic hydrolysis condition；
Fig. 2: influence of the beta-glucosidase enzyme concentration to enzymatic hydrolysis condition in one embodiment of the present of invention；
Fig. 3: influence of the cellulose concentration to microcrystalline cellulose simultaneous saccharification and fermentation in one embodiment of the present of invention；
Fig. 4: influence of one embodiment of the present of invention cellulase concentration to microcrystalline cellulose simultaneous saccharification and fermentation；
Fig. 5: shadow of the beta-glucosidase enzyme concentration to microcrystalline cellulose simultaneous saccharification and fermentation in one embodiment of the present of invention
It rings；
Fig. 6: influence of the nitrogen source type to microcrystalline cellulose simultaneous saccharification and fermentation in one embodiment of the present of invention；
Fig. 7: influence of the peanut meal concentration to microcrystalline cellulose simultaneous saccharification and fermentation in one embodiment of the present of invention；
Fig. 8: influence of the concentration of substrate to corncob residue simultaneous saccharification and fermentation in one embodiment of the present of invention；
Fig. 9: influence of one embodiment of the present of invention cellulase concentration to corncob residue simultaneous saccharification and fermentation；
Figure 10: shadow of the beta-glucosidase enzyme concentration to corncob residue simultaneous saccharification and fermentation in one embodiment of the present of invention
It rings；
Figure 11: bacillus coagulans H-1 is that raw material synchronizes in batches using corncob residue in one embodiment of the present of invention
Diastatic fermentation course；
Figure 12: bacillus coagulans H-1 is raw material fed-batch using corncob residue in one embodiment of the present of invention
Simultaneous saccharification and fermentation course.
Specific embodiment
Multiple preferred embodiments of the invention are introduced below with reference to Figure of description, keep its technology contents more clear and just
In understanding.The present invention can be emerged from by many various forms of embodiments, and protection scope of the present invention not only limits
The embodiment that Yu Wenzhong is mentioned.
Test method as used in the following examples is conventional method unless otherwise specified.Institute in following embodiments
Material, reagent etc., are commercially available unless otherwise specified.
The purpose of the present invention is provide a kind of effective alternative for application of the lignocellulosic in Pfansteihl production.
Using bacillus coagulans H-1 as fermentation strain, Pfansteihl is produced using corncob residue simultaneous saccharification and fermentation, high with yield,
Yield height, advantage at low cost, have certain realistic meaning and industrial application value.
Specifically, first using microcrystalline cellulose as model, the optimization of enzymatic hydrolysis condition and simultaneous saccharification and fermentation condition is carried out,
And have evaluated cheap carbon nitrogen source combination；Later using corncob residue as substrate, fermentation condition optimization is carried out, and by synchronizing in batches
Two methods of diastatic fermentation and fed-batch simultaneous saccharification and fermentation obtain Pfansteihl.
The product of corncob residue, preferably corncob after hemicellulose removal and dilute acid hydrolysis.Make in the present invention
Corncob residue is purchased from Shandong Longli Biology Science and Technology Co., Ltd, measures jade by two step acidolysis according to NREL method
Content of cellulose is 59.51% in rice core residue, hemicellulose level 5.97%, content of lignin 24.10%.
Material and equipment:
Fermentation strain is bacillus coagulans H-1, is preserved in China typical culture collection center (Wuhan City, Hubei Province Luo
Jia Shan Wuhan University, China), deposit number is CCTCC No.M 2013105；Corncob residue is purchased from Shandong dragon power biology
Science and Technology Co., Ltd.；Peanut meal, dregs of beans, Dried Corn Steep Liquor Powder are purchased from Beijing Kang Mingwei culture medium technology Co., Ltd；
Soy peptone is purchased from the extensive and profound in meaning star biotechnology in Beijing Co., Ltd；Ammonium sulfate is purchased from the limited public affairs of Chinese medicines group chemical reagent
Department；Yeast powder is purchased from France Oxoid；Tryptone is purchased from Britain Oxoid；Cellulase has purchased from Shanghai Mike woods biochemical reagents
Limit company；Beta-glucosidase is purchased from Shanghai Yuan Ye Biotechnology Co., Ltd；Neutral proteinase is purchased from the auspicious immortal object section in Shanghai
Skill Co., Ltd；Fermentor (5L) is purchased from Shanghai bolune Biotechnology Co., Ltd.
Embodiment one:
The optimization of microcrystalline cellulose enzymatic hydrolysis condition:
1. influence of the cellulase concentration to microcrystalline cellulose enzymatic hydrolysis condition
(1) in the triangle shake bottle that substrate microcrystalline cellulose concentration is 60g/L, 5~25U/g of cellulase fiber is added
Element, beta-glucosidase 20U/g cellulose, complement to 50mL using the buffer of the sodium citrate-citric acid of pH 6, are placed in 50
DEG C, in the shaking table of 200rpm, digest 40~75 hours；
(2) sampling analysis after digesting measures cellobiose, concentration of glucose by HPLC；
72 hours results are digested as shown in Figure 1, glucose yield and cellulose conversion ratio increase with the increase of cellulase
Add, when cellulase concentration is 15U/g cellulose, glucose yield and cellulose conversion ratio amplification highest.It is thus determined that micro-
It is 15U/g cellulose that crystalline cellulose, which digests best cellulase concentration,.
2. influence of the beta-glucosidase enzyme concentration to microcrystalline cellulose enzymatic hydrolysis condition
(1) in the triangle shake bottle that substrate microcrystalline cellulose concentration is 60g/L, cellulase 15U/g cellulose, β-are added
Glucuroide 0~30U/g cellulose complements to 50mL using the buffer of the sodium citrate-citric acid of pH 6, is placed in 50
DEG C, in the shaking table of 200rpm, digest 40~75 hours；
(2) sampling analysis after digesting measures cellobiose, concentration of glucose by HPLC；
Digest 72 hours results as shown in Fig. 2, glucose yield and cellulose conversion ratio with beta-glucosidase increase
And increase, when beta-glucosidase enzyme concentration is 7.5U/g cellulose, glucose yield and cellulose conversion ratio amplification highest.Cause
This determines that microcrystalline cellulose enzymatic hydrolysis best β-glucoside enzyme concentration is 7.5U/g cellulose.
The specific method of sample treatment is when sampling analysis in the present embodiment: sample being placed in boiling water bath and handles 10 points
Clock adds distilled water to make glucose, cellobiose concentration dilution between 1~5g/L, and 6000~10000rpm is centrifuged 10 minutes,
Supernatant is taken, with 0.22 μm of water phase membrane filtration in liquid phase bottle.
HPLC testing conditions are: liquid-phase chromatographic column is Bio-Rad Aminex HPX-87H, and mobile phase is the H of 5mM2SO4,
Flow velocity is 0.5mL/min, and column temperature is 55 DEG C, and sample volume is 10 μ L, is detected using differential refraction detector (RID).
Embodiment two:
The optimization of microcrystalline cellulose simultaneous saccharification and fermentation condition:
1. influence of the concentration of substrate to microcrystalline cellulose simultaneous saccharification and fermentation
(1) with activation bacillus coagulans H-1 bacterial strain under the conditions of GSY solid slope culture medium, 50 DEG C；
(2) the bacillus coagulans H-1 bacterial strain after inoculation activation is thin with sealed membrane and plastics in GSY fluid nutrient medium
Film seals bottleneck, creates gnotobasis with bacterial strain consumption oxygen, cultivates 12~16 hours, obtain under the conditions of 50 DEG C, 200rpm
Seed liquor；
(3) seed liquor is transferred in fermentation medium by 20% inoculum concentration, while it is fine to add cellulase 15U/g
Dimension element, beta-glucosidase 7.5U/g cellulose seal bottleneck with sealed membrane and plastic film, create nothing with bacterial strain consumption oxygen
Collarium border is cultivated under the conditions of 50 DEG C, 200rpm；
(4) sampling analysis after fermenting 30~72 hours detects Pfansteihl, cellobiose and concentration of glucose by HPLC；
48 hours results of fermenting work as fiber as shown in figure 3, Pfansteihl concentration increases with the increase of substrate cellulose concentration
When plain concentration is 80g/L, Pfansteihl concentration is obviously increased.It is thus determined that 80g/L microcrystalline cellulose is that simultaneous saccharification and fermentation is best
Concentration of substrate.
Fermentative medium formula described in above-mentioned steps (3) is: 50~90g/L microcrystalline cellulose, 10g/L yeast powder,
5g/L tryptone and 25~45g/L CaCO3。
2. influence of the cellulase concentration to microcrystalline cellulose simultaneous saccharification and fermentation
(1) with activation bacillus coagulans H-1 bacterial strain under the conditions of GSY solid slope culture medium, 50 DEG C；
(2) the bacillus coagulans H-1 bacterial strain after inoculation activation is thin with sealed membrane and plastics in GSY fluid nutrient medium
Film seals bottleneck, creates gnotobasis with bacterial strain consumption oxygen, cultivates 12-16 hours, obtain under the conditions of 50 DEG C, 200rpm
Seed liquor；
(3) seed liquor is transferred in fermentation medium by 20% inoculum concentration, while adds 10~30U/g of cellulase
Cellulose, cellulase and beta-glucosidase ratio are 2:1, seal bottleneck with sealed membrane and plastic film, are consumed with bacterial strain
Oxygen creates gnotobasis, cultivates under the conditions of 50 DEG C, 200rpm；
(4) sampling analysis after fermenting 30~72 hours detects Pfansteihl, cellobiose and concentration of glucose by HPLC；
48 hours results of fermenting work as cellulose as shown in figure 4, Pfansteihl concentration increases with the increase of cellulase concentration
When enzyme concentration is 15U/g cellulose, Pfansteihl concentration is obviously increased.It is thus determined that microcrystalline cellulose simultaneous saccharification and fermentation is most preferably fine
Tieing up plain enzyme concentration is 15U/g cellulose.
Fermentative medium formula described in above-mentioned steps (3) is: 80g/L microcrystalline cellulose, 10g/L yeast powder, 5g/L pancreas
Peptone and 40g/L CaCO3。
3. influence of the beta-glucosidase enzyme concentration to microcrystalline cellulose simultaneous saccharification and fermentation
(1) with activation bacillus coagulans H-1 bacterial strain under the conditions of GSY solid slope culture medium, 50 DEG C；
(2) the bacillus coagulans H-1 bacterial strain after inoculation activation is thin with sealed membrane and plastics in GSY fluid nutrient medium
Film seals bottleneck, creates gnotobasis with bacterial strain consumption oxygen, cultivates 12~16 hours, obtain under the conditions of 50 DEG C, 200rpm
Seed liquor；
(3) seed liquor is transferred in fermentation medium by 20% inoculum concentration, while it is fine to add cellulase 15U/g
Dimension element, beta-glucosidase 0~30U/g cellulose seal bottleneck with sealed membrane and plastic film, are created with bacterial strain consumption oxygen
Gnotobasis is cultivated under the conditions of 50 DEG C, 200rpm；
(4) sampling analysis after fermenting 30~72 hours detects Pfansteihl, cellobiose and concentration of glucose by HPLC；
48 hours results ferment as shown in figure 5, Pfansteihl concentration increases with the increase of beta-glucosidase enzyme concentration, as β-
When glucoside enzyme concentration is 7.5U/g cellulose, Pfansteihl concentration increases obvious.But at this point, Pfansteihl concentration is relatively low.
Therefore consider from enzyme cost and lactic acid production angle, determine the best beta-glucosidase concentration of microcrystalline cellulose simultaneous saccharification and fermentation
For 15U/g cellulose.
Fermentative medium formula described in above-mentioned steps (3) is: 80g/L microcrystalline cellulose, 10g/L yeast powder, 5g/L pancreas
Peptone and 40g/L CaCO3。
GSY solid slope culture medium formula described in the present embodiment is: 100g/L glucose, 10g/L yeast powder, 5g/
L tryptone and 50g/L CaCO3, add 2% agarose.
Bacterial strain is bacillus coagulans H-1, is preserved in China typical culture collection center (Luojia Mountain, Wuhan, Hubei Province
Wuhan University, China), deposit number is CCTCC No.M 2013105.
GSY Liquid Culture based formulas is: 100g/L glucose, 10g/L yeast powder, 5g/L tryptone and 50g/L
CaCO3。
The specific method of sample treatment is when sampling analysis: sample being placed in boiling water bath and is handled 10 minutes, 2mL sample is taken
The H of product addition 2mL 2M2SO4Acidolysis 10 minutes, calcium lactate is made to be converted into lactic acid, distilled water is added to make Pfansteihl, glucose, fiber
Disaccharides concentration dilution is centrifuged 10 minutes to 1~5g/L, 6000~10000rpm, supernatant is taken, with 0.22 μm of water phase filter membrane mistake
It filters in liquid phase bottle.
HPLC testing conditions are: liquid-phase chromatographic column is Bio-Rad Aminex HPX-87H, and mobile phase is the H of 5mM2SO4,
Flow velocity is 0.5mL/min, and column temperature is 55 DEG C, and sample volume is 10 μ L, is detected using differential refraction detector (RID).
Embodiment three:
The nitrogen source of microcrystalline cellulose simultaneous saccharification and fermentation optimizes:
1. influence of the nitrogen source type to microcrystalline cellulose simultaneous saccharification and fermentation
(1) with activation bacillus coagulans H-1 bacterial strain under the conditions of GSY solid slope culture medium, 50 DEG C；
(2) the bacillus coagulans H-1 bacterial strain after inoculation activation is thin with sealed membrane and plastics in GSY fluid nutrient medium
Film seals bottleneck, creates gnotobasis with bacterial strain consumption oxygen, cultivates 12~16 hours, obtain under the conditions of 50 DEG C, 200rpm
Seed liquor；
(3) seed liquor is transferred in fermentation medium by 20% inoculum concentration, while it is fine to add cellulase 15U/g
Dimension element, beta-glucosidase 15U/g cellulose seal bottleneck with sealed membrane and plastic film, create nothing with bacterial strain consumption oxygen
Collarium border is cultivated under the conditions of 50 DEG C, 200rpm；
(4) sampling analysis after fermenting 30~72 hours detects Pfansteihl, cellobiose and concentration of glucose by HPLC；
48 hours results of fermenting are as shown in fig. 6, when using peanut meal as nitrogen source, Pfansteihl yield highest, and peanut meal is cheap,
It is easy to get extensively.It is thus identified that peanut meal is microcrystalline cellulose simultaneous saccharification and fermentation optimum nitrogen source.
Fermentative medium formula described in above-mentioned steps (3) is: 80g/L microcrystalline cellulose, 40g/L CaCO3, nitrogen concentration
For 3.4g/L, nitrogen source type and concentration such as table 1；Wherein, nitrogen source peanut meal, addition 0.3g/L neutral proteinase is needed in dregs of beans.
Table 1 is total nitrogen concentration and nitrogen concentration in different nitrogen sources type
2. influence of the peanut meal concentration to microcrystalline cellulose simultaneous saccharification and fermentation
(1) with activation bacillus coagulans H-1 bacterial strain under the conditions of GSY solid slope culture medium, 50 DEG C；
(2) the bacillus coagulans H-1 bacterial strain after inoculation activation is thin with sealed membrane and plastics in GSY fluid nutrient medium
Film seals bottleneck, creates gnotobasis with bacterial strain consumption oxygen, cultivates 12~16 hours, obtain under the conditions of 50 DEG C, 200rpm
Seed liquor；
(3) seed liquor is transferred in fermentation medium by 20% inoculum concentration, while it is fine to add cellulase 15U/g
Dimension element, beta-glucosidase 15U/g cellulose, neutral proteinase 0.3g/L seal bottleneck with sealed membrane and plastic film, with bacterium
Strain consumption oxygen creates gnotobasis, cultivates under the conditions of 50 DEG C, 200rpm；
(4) sampling analysis after fermenting 30~72 hours detects Pfansteihl, cellobiose and concentration of glucose by HPLC；
48 hours results ferment as shown in fig. 7, Pfansteihl concentration increases with the increase of peanut meal concentration, when peanut meal is dense
When degree is greater than 20g/L, Pfansteihl concentration tends to be steady.It is thus determined that the best peanut meal concentration of microcrystalline cellulose simultaneous saccharification and fermentation
For 20g/L.
Fermentative medium formula described in above-mentioned steps (3) is: 80g/L microcrystalline cellulose, 0~30g/L peanut meal,
40g/L CaCO3。
GSY solid slope culture medium formula described in the present embodiment is: 100g/L glucose, 10g/L yeast powder, 5g/L
Tryptone and 50g/L CaCO3, add 2% agarose.
Bacterial strain is bacillus coagulans H-1, is preserved in China typical culture collection center (Luojia Mountain, Wuhan, Hubei Province
Wuhan University, China), deposit number is CCTCC No.M 2013105.
GSY Liquid Culture based formulas is: 100g/L glucose, 10g/L yeast powder, 5g/L tryptone and 50g/L
CaCO3。
The specific method of sample treatment is when sampling analysis: sample being placed in boiling water bath and is handled 10 minutes, 2mL sample is taken
The H of product addition 2mL 2M2SO4Acidolysis 10 minutes, calcium lactate is made to be converted into lactic acid, distilled water is added to make Pfansteihl, glucose, fiber
Disaccharides concentration dilution is centrifuged 10 minutes to 1~5g/L, 6000~10000rpm, supernatant is taken, with 0.22 μm of water phase filter membrane mistake
It filters in liquid phase bottle.
HPLC testing conditions are: liquid-phase chromatographic column is Bio-Rad Aminex HPX-87H, and mobile phase is the H of 5mM2SO4,
Flow velocity is 0.5mL/min, and column temperature is 55 DEG C, and sample volume is 10 μ L, is detected using differential refraction detector (RID).
Example IV:
The optimization of corncob residue simultaneous saccharification and fermentation condition:
1. influence of the concentration of substrate to corncob residue simultaneous saccharification and fermentation
(1) with activation bacillus coagulans H-1 bacterial strain under the conditions of GSY solid slope culture medium, 50 DEG C；
(2) the bacillus coagulans H-1 bacterial strain after inoculation activation is thin with sealed membrane and plastics in GSY fluid nutrient medium
Film seals bottleneck, creates gnotobasis with bacterial strain consumption oxygen, cultivates 12~16 hours, obtain under the conditions of 50 DEG C, 200rpm
Seed liquor；
(3) seed liquor is transferred in fermentation medium by 20% inoculum concentration, while it is fine to add cellulase 15U/g
Dimension element, beta-glucosidase 15U/g cellulose, neutral proteinase 0.3g/L seal bottleneck with sealed membrane and plastic film, with bacterium
Strain consumption oxygen creates gnotobasis, cultivates under the conditions of 50 DEG C, 200rpm；
(4) sampling analysis after fermenting 30~72 hours detects Pfansteihl, cellobiose and concentration of glucose by HPLC；
48 hours results of fermenting as shown in figure 8, when corncob sludge concentration be 120g/L when, Pfansteihl yield highest, so
And the increase of Pfansteihl concentration is relatively fewer at this time.On the one hand, excessively high concentration of substrate is unfavorable for enzyme-to-substrate contact, reduces
Enzymolysis efficiency, on the other hand, excessively high concentration of substrate keep mass-and heat-transfer difficult, it is suppressed that the growth metabolism of bacterial strain.Therefore, from enzyme
Solution efficiency and lactic acid production angle comprehensively consider, and determine that 100g/L corncob residue is the best concentration of substrate of simultaneous saccharification and fermentation.
Fermentative medium formula described in above-mentioned steps (3) is: 60~140g/L corncob residue, cellulose and peanut meal
Concentration ratio is 4:1, cellulose and CaCO3Concentration ratio is 2:1.
2. influence of the cellulase concentration to corncob residue simultaneous saccharification and fermentation
(1) with activation bacillus coagulans H-1 bacterial strain under the conditions of GSY solid slope culture medium, 50 DEG C；
(2) the bacillus coagulans H-1 bacterial strain after inoculation activation is thin with sealed membrane and plastics in GSY fluid nutrient medium
Film seals bottleneck, creates gnotobasis with bacterial strain consumption oxygen, cultivates 12~16 hours, obtain under the conditions of 50 DEG C, 200rpm
Seed liquor；
(3) seed liquor is transferred in fermentation medium by 20% inoculum concentration, while adds 2~20U/g of cellulase
Cellulose, beta-glucosidase 15U/g cellulose, neutral proteinase 0.3g/L seal bottleneck with sealed membrane and plastic film, with
Bacterial strain consumes oxygen and creates gnotobasis, cultivates under the conditions of 50 DEG C, 200rpm；
(4) sampling analysis after fermenting 30~72 hours detects Pfansteihl, cellobiose and concentration of glucose by HPLC；
As shown in figure 9, when cellulase concentration is greater than 10U/g cellulose, Pfansteihl concentration becomes 48 hours results of fermenting
In steady.It is thus determined that the best cellulase concentration of corncob residue simultaneous saccharification and fermentation is 10U/g cellulose.
Fermentative medium formula described in above-mentioned steps (3) is: 100g/L corncob residue, 15g/L peanut meal concentration,
30g/L CaCO3。
3. influence of the beta-glucosidase enzyme concentration to corncob residue simultaneous saccharification and fermentation
(1) with activation bacillus coagulans H-1 bacterial strain under the conditions of GSY solid slope culture medium, 50 DEG C；
(2) the bacillus coagulans H-1 bacterial strain after inoculation activation is thin with sealed membrane and plastics in GSY fluid nutrient medium
Film seals bottleneck, creates gnotobasis with bacterial strain consumption oxygen, cultivates 12~16 hours, obtain under the conditions of 50 DEG C, 200rpm
Seed liquor；
(3) seed liquor is transferred in fermentation medium by 20% inoculum concentration, while it is fine to add cellulase 10U/g
Dimension element, beta-glucosidase 0~20U/g cellulose, neutral proteinase 0.3g/L seal bottleneck with sealed membrane and plastic film,
Gnotobasis is created with bacterial strain consumption oxygen, is cultivated under the conditions of 50 DEG C, 200rpm；
(4) sampling analysis after fermenting 30~72 hours detects Pfansteihl, cellobiose and concentration of glucose by HPLC；
The results are shown in Figure 10 within 48 hours for fermentation, when beta-glucosidase enzyme concentration is 15U/g cellulose, Pfansteihl concentration
Highest, it is thus determined that the best beta-glucosidase concentration of corncob residue simultaneous saccharification and fermentation is 15U/g cellulose.
Fermentative medium formula described in above-mentioned steps (3) is: 100g/L corncob residue, 15g/L peanut meal, 30g/L
CaCO3。
GSY solid slope culture medium formula described in the present embodiment is: 100g/L glucose, 10g/L yeast powder, 5g/
L tryptone and 50g/L CaCO3, add 2% agarose.
Bacterial strain is bacillus coagulans H-1, is preserved in China typical culture collection center (Luojia Mountain, Wuhan, Hubei Province
Wuhan University, China), deposit number is CCTCC No.M 2013105.
GSY Liquid Culture based formulas is: 100g/L glucose, 10g/L yeast powder, 5g/L tryptone and 50g/L
CaCO3。
Corncob residue in fermentative medium formula is production of the corncob after hemicellulose removal and dilute acid hydrolysis
Object is purchased from Shandong Longli Biology Science and Technology Co., Ltd, is measured in corncob residue according to NREL method by two step acidolysis
Content of cellulose is 59.51%, hemicellulose level 5.97%, content of lignin 24.10%.
The specific method of sample treatment is when sampling analysis: sample being placed in boiling water bath and is handled 10 minutes, 2mL sample is taken
The H of product addition 2mL 2M2SO4Acidolysis 10 minutes, calcium lactate is made to be converted into lactic acid, distilled water is added to make Pfansteihl, glucose, fiber
Disaccharides concentration dilution is centrifuged 10 minutes to 1~5g/L, 6000~10000rpm, supernatant is taken, with 0.22 μm of water phase filter membrane mistake
It filters in liquid phase bottle.
HPLC testing conditions are: liquid-phase chromatographic column is Bio-Rad Aminex HPX-87H, and mobile phase is the H of 5mM2SO4,
Flow velocity is 0.5mL/min, and column temperature is 55 DEG C, and sample volume is 10 μ L, is detected using differential refraction detector (RID).
Embodiment five:
A method of using lignocellulosic corncob residue, simultaneous saccharification and fermentation produces Pfansteihl, step in batches
Are as follows:
(1) with activation bacillus coagulans H-1 bacterial strain under the conditions of GSY solid slope culture medium, 50 DEG C；
(2) the bacillus coagulans H-1 bacterial strain after inoculation activation is thin with sealed membrane and plastics in GSY fluid nutrient medium
Film seals bottleneck, creates gnotobasis with bacterial strain consumption oxygen, cultivates 12~16 hours, obtain under the conditions of 50 DEG C, 200rpm
Seed liquor；
(3) seed liquor is transferred in fermentation tank culture medium by 20% inoculum concentration, while adds cellulase 10U/g
Cellulose, beta-glucosidase 15U/g cellulose, neutral proteinase 0.3g/L, control fermentation jar temperature is at 50 DEG C, speed of agitator
For 150rpm, ferment 30~60 hours；
(4) it samples within every 12 hours during fermenting, with HPLC detection Pfansteihl, cellobiose and concentration of glucose；
The lactic acid production of fermentation 36 hours is 68g/L, and yield is 0.85g/g cellulose；Its fermentation diagram is as shown in figure 11.
GSY solid slope culture medium formula described in the present embodiment is: 100g/L glucose, 10g/L yeast powder, 5g/
L tryptone and 50g/L CaCO3, add 2% agarose.
Bacterial strain is bacillus coagulans H-1, is preserved in China typical culture collection center (Luojia Mountain, Wuhan, Hubei Province
Wuhan University, China), deposit number is CCTCC No.M 2013105.
GSY Liquid Culture based formulas is: 100g/L glucose, 10g/L yeast powder, 5g/L tryptone and 50g/L
CaCO3。
Fermentation tank culture based formulas is: 100g/L corncob residue, 15g/L peanut meal, 30g/L CaCO3。
Corncob residue is product of the corncob after hemicellulose removal and dilute acid hydrolysis, purchased from Shandong dragon power biology
Science and Technology Co., Ltd., measuring content of cellulose in corncob residue by two step acidolysis according to NREL method is
59.51%, hemicellulose level 5.97%, content of lignin 24.10%.
The specific method of sample treatment is when sampling analysis: sample being placed in boiling water bath and is handled 10 minutes, 2mL sample is taken
The H of product addition 2mL 2M2SO4Acidolysis 10 minutes, calcium lactate is made to be converted into lactic acid, distilled water is added to make Pfansteihl, glucose, fiber
Disaccharides concentration dilution is centrifuged 10 minutes to 1~5g/L, 6000~10000rpm, supernatant is taken, with 0.22 μm of water phase filter membrane mistake
It filters in liquid phase bottle.
HPLC testing conditions are: liquid-phase chromatographic column is Bio-Rad Aminex HPX-87H, and mobile phase is the H of 5mM2SO4,
Flow velocity is 0.5mL/min, and column temperature is 55 DEG C, and sample volume is 10 μ L, is detected using differential refraction detector (RID).
Embodiment six:
A method of Pfansteihl, step are produced using lignocellulosic corncob residue fed-batch simultaneous saccharification and fermentation
Suddenly are as follows:
(1) bacillus coagulans H-1 activation culture: is inoculated into GSY solid slope culture medium, 50 DEG C of cultures 12~24
Hour activation；
(2) seed culture: the bacillus coagulans H-1 bacterial strain after inoculation activation is transferred to described in gnotobasis
GSY fluid nutrient medium seals bottleneck with sealed membrane and plastic film, with bacterial strain consumption oxygen create gnotobasis, 50 DEG C,
It is cultivated 12~16 hours under the conditions of 200rpm, obtains seed liquor；
(3) seed liquor is transferred in fermentation tank culture medium according to 20% inoculum concentration, while adds cellulase 10U/
G cellulose, beta-glucosidase 15U/g cellulose and neutral proteinase 0.3g/L control fermentation jar temperature at 50 DEG C, stir
Revolving speed is 150rpm, is fermented 72~140 hours；
(4) at logarithmic growth phase 5 hours, feed supplement 50g/L corncob residue, 15g/L CaCO3, 10U/g cellulose
Cellulase, the beta-glucosidase of 15U/g cellulose；
(5) it samples within every 12 hours during fermenting, with HPLC detection Pfansteihl, cellobiose and concentration of glucose；
The lactic acid production of fermentation 84 hours is 79.1g/L, and yield is 0.76g/g cellulose；Its fermentation diagram such as Figure 12 institute
Show.
GSY solid slope culture medium formula described in the present embodiment is: 100g/L glucose, 10g/L yeast powder, 5g/
L tryptone and 50g/L CaCO3, add 2% agarose.
Bacterial strain is bacillus coagulans H-1, is preserved in China typical culture collection center (Luojia Mountain, Wuhan, Hubei Province
Wuhan University, China), deposit number is CCTCC No.M 2013105.
GSY Liquid Culture based formulas is: 100g/L glucose, 10g/L yeast powder, 5g/L tryptone and 50g/L
CaCO3。
Fermentation tank culture based formulas is: 100g/L corncob residue, 22.5g/L peanut meal, 30g/L CaCO3。
Corncob residue is product of the corncob after hemicellulose removal and dilute acid hydrolysis, purchased from Shandong dragon power biology
Science and Technology Co., Ltd., measuring content of cellulose in corncob residue by two step acidolysis according to NREL method is
59.51%, hemicellulose level 5.97%, content of lignin 24.10%.
The specific method of sample treatment is when sampling analysis: sample being placed in boiling water bath and is handled 10 minutes, 2mL sample is taken
The H of product addition 2mL 2M2SO4Acidolysis 10 minutes, calcium lactate is made to be converted into lactic acid, distilled water is added to make Pfansteihl, glucose, fiber
Disaccharides concentration dilution is centrifuged 10 minutes to 1~5g/L, 6000~10000rpm, supernatant is taken, with 0.22 μm of water phase filter membrane mistake
It filters in liquid phase bottle.
HPLC testing conditions are: liquid-phase chromatographic column is Bio-Rad Aminex HPX-87H, and mobile phase is the H of 5mM2SO4,
Flow velocity is 0.5mL/min, and column temperature is 55 DEG C, and sample volume is 10 μ L, is detected using differential refraction detector (RID).
The preferred embodiment of the present invention has been described in detail above.It should be appreciated that the ordinary skill of this field is without wound
The property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art
Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Scheme, all should be within the scope of protection determined by the claims.
Claims (10)
1. a kind of method for efficiently producing Pfansteihl using lignocellulosic corncob residue, which is characterized in that condense gemma
Bacillus is fermentation strain, and using single carbon source, nitrogen source, simultaneous saccharification and fermentation produces Pfansteihl, wherein the carbon source is corn
Core residue.
2. the method for efficiently producing Pfansteihl using lignocellulosic corncob residue as described in claim 1, feature exist
In the fermentation strain is bacillus coagulans H-1, is preserved in China typical culture collection center (Wuhan City, Hubei Province Luo
Jia Shan Wuhan University, China), deposit number is CCTCC No.M 2013105.
3. the method for efficiently producing Pfansteihl using lignocellulosic corncob residue as described in claim 1, feature exist
In the corncob residue is product of the corncob after hemicellulose removal and dilute acid hydrolysis.
4. the method for efficiently producing Pfansteihl using lignocellulosic corncob residue as described in claim 1, feature exist
In the nitrogen source is peanut meal.
5. the method for efficiently producing Pfansteihl using lignocellulosic corncob residue as described in claim 1, feature exist
In the described method comprises the following steps:
Step 1, activation culture: the fermentation strain is inoculated into solid slope culture medium, 50 DEG C are cultivated 12~24 hours；
Step 2, seed culture: by the fermentation strain after activating in step 1, Liquid Culture is transferred in gnotobasis
Base seals bottleneck, creates gnotobasis with fermentation strain consumption oxygen, 12~16 are cultivated under the conditions of 50 DEG C, 200rpm
Hour, obtain seed liquor；
Step 3, fermentation tank culture: seed liquor described in step 2 is transferred to fermentation tank culture medium according to 5~25% inoculum concentration
In, while adding cellulase 0~20U/g cellulose, 0~20U/g of beta-glucosidase cellulose and neutral proteinase 0.3g/
L controls fermentation jar temperature at 50 DEG C, speed of agitator 150rpm, cultivates 30~84 hours；
Step 4, detection: sampling after fermentation, detection Pfansteihl, cellobiose and concentration of glucose.
6. method as claimed in claim 5, which is characterized in that the solid slope culture medium in the step 1 is solid for GSY
Body slant medium, formula are as follows: 100g/L glucose, 10g/L yeast powder, 5g/L tryptone, 50g/L CaCO3And 2%
Agarose；The fluid nutrient medium in the step 2 is GSY fluid nutrient medium, formula are as follows: 100g/L glucose, 10g/L
Yeast powder, 5g/L tryptone, 50g/L CaCO3。
7. method as claimed in claim 5, which is characterized in that the fermentation tank culture based formulas in the step 3 are as follows: 60
~140g/L corncob residue, 9~21g/L peanut meal, 18~42g/L CaCO3。
8. method as claimed in claim 5, which is characterized in that the step 4 also comprises the steps of:
Step 4.1, sample treatment: sample being placed in boiling water bath and is handled 10 minutes, and sample described in 2mL is taken to be added 2mL 2M's
H2SO4Acidolysis 10 minutes, calcium lactate is made to be converted into lactic acid, distilled water is added to make Pfansteihl, glucose, cellobiose concentration dilution to 1
~5g/L, 6000~10000rpm are centrifuged 10 minutes, supernatant are taken, with 0.22 μm of water phase membrane filtration in liquid phase bottle；
Step 4.2, sample detection: being detected with HPLC, testing conditions are as follows: and liquid-phase chromatographic column is Bio-RadAminex HPX-87H,
Mobile phase is the H of 5mM2SO4, flow velocity 0.5mL/min, column temperature is 55 DEG C, and sample volume is 10 μ L, uses differential refraction detector
(RID) it detects.
9. the method for efficiently producing Pfansteihl using lignocellulosic corncob residue as claimed in claim 2, feature exist
In the described method comprises the following steps:
Step 1, activation culture: being inoculated into GSY solid slope culture medium for the bacillus coagulans H-1, and 50 DEG C of cultures 12~
24 hours；
Step 2, seed culture: the bacillus coagulans H-1 after activating in step 1 is transferred to GSY liquid in gnotobasis
Body culture medium seals bottleneck with sealed membrane and plastic film, creates asepsis ring with bacillus coagulans H-1 consumption oxygen
Border is cultivated 12~16 hours under the conditions of 50 DEG C, 200rpm, obtains seed liquor；
Step 3, fermentation tank culture: seed liquor described in step 2 is transferred to fermentation tank culture medium according to 5~25% inoculum concentration
In, while adding cellulase 0~20U/g cellulose, 0~20U/g of beta-glucosidase cellulose and neutral proteinase 0.3g/
L controls fermentation jar temperature at 50 DEG C, speed of agitator 150rpm, ferments 72~140 hours；
Step 4, feed supplement: at logarithmic growth phase 4~7 hours, corncob residue described in 30~70g/L of feed supplement, 9~21g/L
CaCO3, the cellulase of 2~20U/g cellulose, the beta-glucosidase of 0~20U/g cellulose；
Step 5, detection: sampling after fermentation, detection Pfansteihl, cellobiose and concentration of glucose.
10. the method for efficiently producing Pfansteihl using lignocellulosic corncob residue as claimed in claim 9, feature exist
In the formula of the GSY solid slope culture medium in the step 1 are as follows: 100g/L glucose, 10g/L yeast powder, 5g/L pancreas
Peptone, 50g/L CaCO3And 2% agarose；The formula of the GSY fluid nutrient medium in the step 2 are as follows: 100g/L
Glucose, 10g/L yeast powder, 5g/L tryptone, 50g/L CaCO3；The fermentation tank culture medium in the step 3 is matched
Side are as follows: 60~140g/L corncob residue, 13.5~31.5g/L peanut meal, 18~42g/L CaCO3。
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