CN108251334A - The microorganism mixed bacterial and fermentation process of a kind of fermenting lactic acid - Google Patents

The microorganism mixed bacterial and fermentation process of a kind of fermenting lactic acid Download PDF

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CN108251334A
CN108251334A CN201810035798.1A CN201810035798A CN108251334A CN 108251334 A CN108251334 A CN 108251334A CN 201810035798 A CN201810035798 A CN 201810035798A CN 108251334 A CN108251334 A CN 108251334A
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孙亚琴
徐振振
周瑾洁
修志龙
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Dalian University of Technology
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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Abstract

The present invention provides the microorganism mixed bacterials and fermentation process of a kind of fermenting lactic acid, the microorganism mixed bacterial includes Clostridium bacterial strain, Escherichia bacterial strain and Enterococcus strain, its total living bacteria count accounts for more than 94% of living bacteria count in microorganism mixed bacterial, and the living bacteria count ratio of the Bacillus strain, Escherichia bacterial strain and Enterococcus strain is 40 60：20‑40：2‑20.Ferment relative to single bacterium, the microorganism species fermenting stability is good, biological safety is high, molasses tolerance is high, in the untreated molasses of 500g/L also can growth metabolism, there is high production intensity and rotational rate of lactic acid.The microorganism mixed bacterial also has in production process the advantages such as need not sterilize, fermentation substrate molasses need not pre-process, production cost is low.

Description

The microorganism mixed bacterial and fermentation process of a kind of fermenting lactic acid
Technical field
It is more particularly to a kind of to be mass-sended using molasses as substrate through microorganism Mixed Microbes the invention belongs to technical field of bioengineering The method that ferment produces lactic acid.
Background technology
Lactic acid (Lactic acid) is important biochemical product, is widely used in medicine, food, beverage, chemical industry, skin The fields such as leather, cigarette industry.The application of lactic acid most prospect is the technical field of biological material for representative with polylactic acid (PLA).As Most potential biodegradable polymer is expected to replace the nondegradable modelings such as polypropylene, polyethylene, polystyrene Material is eliminated " white pollution ", is a kind of biological environmental production material of great application potential.
The production method of lactic acid includes chemical synthesis, enzyme process and microbe transformation method.It is micro- compared to chemical method and enzyme method Biological fermentation process produce lactic acid, can by the selection of strain and condition of culture and obtain the Pfansteihl with stereocpecificity or The D-ALPHA-Hydroxypropionic acid raceme that either two isomers are mixed with certain proportion can meet the needs of production polylactic acid.Fermentation method is given birth to Lactic acid producing is because the advantages that its raw material sources is extensive, production cost is low, optical purity of products is high, is into the important side for lactic acid producing of making a living Method.Traditional fermentation method production lactic acid is mainly with glucose or cereal crops, such as rice, corn, potato are raw material.In order to Further reduce production cost, attempt with industrial and agricultural waste (such as cane molasses) (Flores-Albino B et al., 2012, 35:1193-1200；Xu K et al.,2014,153:23-29), lignocellulosic (Ou MS, 2011,38: 599-605)、 Pulping (Budhavaram NK, 2009,100:5966-5972), apple pomace (Gullon B, 2008,99:308-319), kitchen Room rubbish (Zhang B, 2008,99:855-862), jerusalem artichoke (Wang LM, 2013,130:174-180) etc. breast is carried out for raw material Acid fermentation produces, and not only can further reduce production cost, realizes that renewable resource efficiently uses, can also solve waste Processing and pollution problem.
Molasses are Main By product and a kind of trade waste in sugar manufacturing process.Wherein, the production of cane molasses Amount can reach the 2.5-3% of raw material sugarcane, and raw materials for production enrich, cheap, therefore to the rational exploitation and utilization of cane molasses, With important economic implications.Its main component is carbohydrate, and containing about sucrose 30-40% (w/v), other sugars are (most of for also Originality sugar) it is about 15-20% (w/v), in addition nitrogen source and the content of inorganic salts are respectively 0.5-0.9% (w/v) and 10% (w/ v)(Liu YP,2008,99:1736-1742).Cane molasses are chiefly used in being processed into various fermented products at present, such as alcohol, breast Acid, monosodium glutamate, citric acid, lysine, caramel colorant etc..Although the utilization of cheap raw material can reduce to a certain extent raw material into This, but the impurity contained by it if the volatile organic acids that contains in molasses, colloid, ash grade, needs to be used for after purification Pure culture bioprocess.If it is difficult to be utilized using single bacterium without purification process, cell growth toxic effect is dropped The low utilization rate of raw material, leads to final production concentration and strength reduction.
Microorganism species (Microbial consortium) are the commercial Applications of natural mixed fungus fermentation, which is to be based on The principle of ecological choice, the flora that complex matrices, product range will be utilized special, which is retained in reactor or fermentation tank, to be held It is continuous to use.Compared with pure culture technigne, microorganism species show greatly potentiality advantage, advantage and are mainly reflected in：(1) it is former The diversity of material：More cheap, complicated matrix, as lignocellulosic, whey, molasses, crude glycerine, potato processing wastewater, Corn steep liquor etc. can become fermentation raw material and be used for production of chemicals.(2) diversification or unification of product：Pass through association Flora composition is adjusted not only the metabolic pathway of different microorganisms can be utilized to obtain multiple target products, but also can become product range It is narrow, it is isolated and purified conducive to downstream substrates, reduces cost.(3) simplification of operation, safety and stability：Microorganism species have There is very high bio-diversity, can be operated under conditions of unsterilised, antiphagin infection ability and biological safety are able to It is promoted.In addition the iuntercellular interactively dynamic equilibrium of thalline system is mixed, there is more strong adaptability and stability to environmental fluctuating.It is based on The above feature of microorganism species, develops that a kind of complex substrate tolerance is strong, and fermenting stability is good, and production intensity is high, produces The microorganism species of Cheng Wuxu sterilizings, Efficient Conversion molasses production lactic acid become the new approaches of an industrialization development.
Invention content
The purpose of the present invention is to provide a kind of fermenting property stabilization, tolerance it is strong can Efficient Conversion molasses be lactic acid Microorganism species and the method that lactic acid is prepared using the microorganism species.The microorganism species of the present invention are by clostruidium Category, Escherichia and enterococcus spp composition, fermenting property are stablized, and complex substrate tolerance is strong, high under anaerobic Effect converts untreated molasses as lactic acid, and fermentation process is unsterilised, overcomes single bacterium fermentation molasses tolerance low, and production intensity is low etc. Limitation.
Technical scheme is as follows：
A kind of microorganism mixed bacterial for being used to produce lactic acid using molasses as fermenting substrate, which is characterized in that described is micro- Biological mixed bacterial includes Clostridium bacterial strain, Escherichia bacterial strain and Enterococcus strain, the fusiform bud Total living bacteria count of spore Bacillus genus strain, Escherichia bacterial strain and Enterococcus strain, which is accounted in microorganism mixed bacterial, to be had Imitate more than 94% viable count, the Bacillus strain, Escherichia bacterial strain and Enterococcus strain it is effective Viable count ratio is 40~60:20~40:2~20.Preferably, the microorganism mixed bacterial is by Clostridium bacterium Strain, Escherichia bacterial strain and Enterococcus strain composition, wherein the Bacillus strain, Escherichia bacterium The living bacteria count ratio of strain and Enterococcus strain is 40~60:20~40:2~20.
Further, the Clostridium bacterial strain is C.perfringens, and the Escherichia bacterial strain is Escherichia coli, the Enterococcus strain are enterococcus faecalis.
Further, in the microorganism mixed bacterial, the C.perfringens, Escherichia coli and excrement intestines ball The living bacteria count ratio of bacterium is 40~60:20~40:2~20.
Further, in the microorganism mixed bacterial, the C.perfringens, Escherichia coli and excrement intestines ball The living bacteria count ratio of bacterium is 57.29:34.22:3.17.
In the present invention, the C.perfringens, Escherichia coli and enterococcus faecalis are commonly used in the art Bacterial strain, in the range of the proportionate relationship between each bacterial strain provided in the application, the mixed bacterial that is made of the bacterial strain, Fermented It can stablize, the untreated molasses of Efficient Conversion are lactic acid under anaerobic, and fermentation process is unsterilised, overcome single bacterium fermentation molasses Tolerance is low, and production intensity is low to wait limitation.
Another aspect of the present invention provides a kind of fermentation process of lactic acid, which connects including above-mentioned microorganism species Enter in using molasses as the fermentation medium of substrate and ferment, sugared content of fermenting in the molasses is 35%~50%.
Further, in above-mentioned fermentation process, the honey is cane molasses or beet molasses.The sugar fermentation packet Include glucose, fructose and sucrose.Preferential, the molasses are untreated molasses, i.e., sugar refinery is without pretreatments such as peracid Molasses.By-product of the molasses as sugar refinery containing sugar fermentation, available for microbial fermentation, but generally uses preceding use at present It could be used to ferment after the pretreatments such as acid, treatment process is cumbersome, increases cost, is unfavorable for industrial applications.It is micro- using the present invention Biological mixed bacterial can directly be fermented using untreated molasses.By-product sugarcane as sugar refinery's sugar refining technology Molasses, consisting of (w/v)：35%~50% sugar fermentation, 9%~12% non-saccharide organic matter, 10%~15% sulfated ass, Moisture and a small amount of pigment and impurity.
Further, in above-mentioned fermentation process, the composition of the fermentation medium is：100~700g of molasses, it is beautiful Rice ＆ peanut milk 10~30g of dry powder adds in distilled water to 1L.Wherein in the fermentation medium concentration of molasses be preferably 200~ 500g/L, more preferably 350~500g/L.PH value according to the good fermentation medium of the component and proportional arrangement is acidity, About 6.3.
Further, in above-mentioned fermentation process, fermentation medium and Zymolysis Equipment are all unsterilised in fermentation process.
Further, in above-mentioned fermentation process, the microorganism species are linked into the sugar with 100~700g/L Honey is carries out batch fermentation in the fermentation medium of substrate.Concentration of molasses can be 200~500g/L, or 350~ 500g/L.Microorganism species of the present invention in the untreated molasses of 500g/L also can growth metabolism, have high production intensity and breast Sour conversion ratio.
Further, in above-mentioned fermentation process, the microorganism species are accessed into the molasses with 200-400g/L To carry out the fedbatch culture fermentation of continuous feeding in the fermentation medium of substrate, in fermentation process the control of sugar fermentation concentration 50~ 90g/L。
The present invention has following advantageous effect relative to present technology：
(1) the invention discloses the microorganism mixed bacterial that a kind of fermentation prepares lactic acid, fermenting stability is good, complicated bottom Object tolerance is strong, and production intensity is high.
(2) microorganism species of the present invention prepare lactic acid using trade waste molasses for fermenting substrate, and molasses without locating in advance Reason reduces lactic acid production process, reduces production cost.
(3) fermentation medium composition of the present invention is simple, only two kinds of ingredients of molasses and Dried Corn Steep Liquor Powder, reduces and is produced into This.
(4) fermentation medium and Zymolysis Equipment all without sterilizing, further reduced and are produced into fermentation process of the present invention This.
(5) it is fermented using the microorganism Mixed Microbes of the present invention, takes the strategy of batch or fedbatch culture fermentation, operation Simplicity reduces productive manpower cost.
Mixed bacterial using the present invention is substrate and 18.5g/ Dried Corn Steep Liquor Powders as culture medium using 350g/L cane molasses In the batch fermentation of composition, for the concentration of lactic acid up to 112.34g/L, mass transitions rate is 81.14%, and production intensity is 4.49g/(L.h).It is during the fedbatch culture of substrate and Dried Corn Steep Liquor Powder as culture medium ferments using cane molasses, the concentration of lactic acid can Up to 120.24g/L, mass transitions rate is 71.95%, and production intensity is 1.19g/ (L.h).
Description of the drawings
Fig. 1 microorganism species lactic acid metabolism result under different enrichment conditions；
The Metabolic products of Fig. 2 microorganism species CEE-DL15 lactic acid under the conditions of different operation；
The Metabolic products of Fig. 3 microorganism species CEE-DL15 lactic acid under the conditions of different culture media；
Sugar consumption, cell growth and lactic acid metabolisms of Fig. 4 microorganism species CEE-DL15 under different starting concentration of molasses Situation；
Fig. 5 microorganism species CEE-DL15 is using 350g/L molasses as the batch fermentation result of substrate；
Fig. 6 microorganism species CEE-DL15 is using 500g/L molasses as the batch fermentation result of substrate；
Fedbatch cultures of Fig. 7 microorganism species CEE-DL15 using molasses as substrate ferments
Fig. 8 microorganism species CEE-DL15 is using calcium hydroxide as the batch fermentation result of pH adjusting agent.
Specific embodiment
Following non-limiting examples can make those of ordinary skill in the art be more fully understood the present invention, but not with Any mode limits the present invention.In following embodiments, unless otherwise specified, used experimental method is conventional method, institute Reagent etc. can chemically or biological reagent company purchase.
Describe the specific embodiment of the present invention in detail below in conjunction with technical solution.
1st, the culture medium that following embodiments use：
Enriched medium (g/L)：Glucose 40, yeast extract 5, C4H2Na2O45, coban 0.1, K2HPO4· 3H2O 3, NaCl 1, (NH4)2SO41, CaCl20.5, MgCl20.5,121 DEG C of sterilizing 15min.
Seed culture medium (g/L)：Glucose 30, peptone 5, beef extract 5, yeast extract 2.5, K2HPO4·3H2O 2, (NH4)2HC6H5O72, CH3COONa·3H2O 2, MgSO4·7H2O 0.2, MnSO4·H20.05,121 DEG C of sterilizing 15min of O.
Fermentation medium：Including following MRS culture mediums, S-MRS culture mediums and CSLP culture mediums, the composition of each culture medium For：
MRS culture mediums (g/L)：Cane molasses 100~500, peptone 10, beef extract 10, yeast extract 5, K2HPO4· 3H2O 2, (NH4)2HC6H5O72, CH3COONa·3H2O 2, MgSO4·7H2O 0.58, MnSO4·H2O 0.25。
S-MRS culture mediums (g/L):Cane molasses 100~500, peptone 5, beef extract 5, yeast extract 2.5, K2HPO4·3H2O 2, (NH4)2HC6H5O72, CH3COONa·3H2O 2, MgSO4·7H2O 0.2, MnSO4·H2O 0.25。
CSLP culture mediums (g/L)：Cane molasses 100~500, Dried Corn Steep Liquor Powder 18.5.
2nd, seed culture：
Anaerobic culturel.Using 250mL cillin bottles, liquid amount 100mL.It is housed to after seed culture medium and persistently leads to 3min for every bottle General nitrogen deoxygenation, is bound later with butyl rubber plug.It is inoculated with and is sampled, inoculum concentration 5-10% with disposable sterilized needle tubing in experimentation (v/v), 37 DEG C of cultivation temperature, shaking speed 200r/min, 8~12h of incubation time.
3rd, fermentation culture conditions：
Using 5L automatic fermenters, the product of seed culture is seeded to hair by liquid amount 2L with inoculum concentration 5% (v/v) In ferment culture medium, using cane molasses as substrate, ferment under 37 DEG C of fermentation jar temperature, rotating speed 250r/min, fermentation process 5M NaOH controls pH is 6.3.0.1vvm general nitrogens are passed through before and after inoculation in 4h and build anaerobic environment in tank.
Cane molasses：It forms as (w/v)：44% sugar fermentation, 10% non-saccharide organic matter, 12% sulfated ass, moisture and A small amount of pigment and impurity.The sugar fermentation includes glucose, fructose and sucrose.The cane molasses are the by-product of sugar refining technology Object, generally requiring could ferment after low-kappa number.Low-kappa number process is as follows：The H of 4mol/L2SO4It is molten to adjust cane molasses Liquid pH is 3.0, and water-bath 1h in boiling water (100 DEG C), room temperature overnight is placed, and 12000rpm centrifugation 15min take supernatant spare.Acid It is cumbersome to pre-process molasses technique, increases cost.Untreated cane molasses described in following embodiments are without the sweet of the pretreatments such as acid Sucrose sugar.
Embodiment 1 screens the microorganism species of domestication lactic acid producing from ox stomach content
Sample collection is derived from Dalian work poultry slaughterhouse, ox stomach content four kinds of processing modes of sample point：1) ox stomach includes Object suspension, anaerobism enrichment；2) ox stomach content suspension, there is oxygen coalescence；3) solid ox stomach content, anaerobism enrichment；4) solid ox Stomach content, there is oxygen coalescence.Wherein ox stomach content suspension preparation manipulation：The fresh content of 0.1kg ox stomaches is taken in 100mL physiology In the triangular flask of brine, solid matter therein is filtered with sterile gauze, filtrates of the 5mL containing a large amount of thalline is taken to be inoculated in dress respectively There are the triangular flask and cillin bottle of 100mL enriched mediums, carry out aerobic and anaerobism enrichment culture.Solid ox stomach content prepares behaviour Make：The fresh content of 0.1kg ox stomaches is taken in the triangular flask of 100mL physiological saline, 2 min of vortex oscillation, solidliquid mixture point It is not inoculated in triangular flask and cillin bottle equipped with 100mL enriched mediums, carries out aerobic and anaerobism enrichment culture.Four kinds of processing sides The culture solution of formula is passed on every 12h with the inoculum concentration of 2% (v/v), continuous passage 20 times, obtains the mixed bacterium of fermenting property stabilization. The MRS that the mixed bacterium that fermenting property is stablized finally is inoculated in a concentration of 100g/L of cane molasses (a concentration of 40g/L of sugar fermentation) is trained It supports base and carries out anaerobic fermentation.Fermentation results as shown in Figure 1, show flora lactic acid production highest obtained by the 3rd kind of sample handling characteristics, Conversion ratio is 90.38%.Solid ox stomach content is chosen, the microorganism species of anaerobism enrichment culture are subsequent fermentation culture.
Identified, which contains Clostridium bacterial strain (57.29%), Escherichia bacterial strain (34.22%) and it is total effective to represent that the living bacteria count of each Pseudomonas accounts for for Enterococcus strain (5.32%), wherein percentage (%) The percentage of viable count.
The Clostridium bacterial strain be C.perfringens (Clostridium perfringens), angstrom Xi Shi Bacillus genus strain is Escherichia coli (Escherichia coli), and Enterococcus strain is enterococcus faecalis (Enterococcus faecalis).Following embodiments using by the Mixed Microbes that C.perfringens, Escherichia coli and enterococcus faecalis form as microorganism Mixed bacterial (is named as CEE-DL15), identifies the performance of mixed fermentation production lactic acid.Aerogenesis pod described in CEE-DL15 Film clostridium, Escherichia coli and enterococcus faecalis living bacteria count ratio are 57.29：34.22：3.17.
The C.perfringens, Escherichia coli and enterococcus faecalis are bacterial strain commonly used in the art.Preferably, institute It states C.perfringens and Clostridium perfringens ATCC 13124 can be used, Escherichia coli can be used Enterococcus faecalis ATCC 19433 can be used in Escherichia coli NCTC 9001, enterococcus faecalis.
Microorganism species CEE-DL15 utilizes the situation of molasses under the conditions of 2 different operation of embodiment
By the microorganism species CEE-DL15 obtained in embodiment 1 after seed culture, dress is seeded to by 5% volume ratio In the cillin bottle for having 100mL MRS culture mediums, using cane molasses as fermenting substrate culture, the concentration of initial cane molasses is about 100g/L.Three kinds of fermentation operation condition point, 1) sterilization fermentation, substrate is acid treated cane molasses；2) sterilization fermentation, bottom Object is untreated cane molasses；3) unsterilised fermentation, substrate are untreated cane molasses.After shake flask fermentation culture 48 hours, survey Concentration, conversion ratio and the production intensity for determining the lactic acid in zymotic fluid are as shown in Figure 2.The result shows that flora CEE-DL15 is not going out Bacterium, use untreated molasses as substrate under conditions of well-grown, have higher molasses conversion ratio and production of lactic acid intensity, breast Concentration, conversion ratio and the production intensity of acid respectively reach 87.98g/L, 73.21g/g and 1.83g/ (L.h), show unsterilised training It is feasible to support and produce lactic acid without pretreated fermentation with cane molasses.The selection of 3 fermentation medium of embodiment and economy are excellent Change
By the mixed bacterial CEE-DL15 obtained in embodiment 1 after seed culture medium culture, it is inoculated with by 5% volume ratio It is cultivated into the cillin bottle equipped with 100mL fermentation mediums, it is initial untreated using untreated cane molasses as fermenting substrate culture A concentration of 250g/L of cane molasses.Three kinds of fermentation medium point：1) MRS culture mediums；2) S-MRS culture mediums；3) CSLP is cultivated Base；Fermentation results are as shown in Figure 3.The flora can utilize simple cheap Dried Corn Steep Liquor Powder culture medium (CSLP culture mediums) to ferment Lactic acid is produced, lactic acid production and conversion ratio are close with other two kinds of culture mediums, and production intensity is higher than first two medium culture, Nitrogen source cost savings 50%~200%.
Under the conditions of 4 batch fermentation of embodiment, microorganism species CEE-DL15 is to the tolerance of high concentration molasses
By the microorganism species CEE-DL15 obtained in embodiment 1 after seed culture medium culture, connect by 5% volume ratio (CSLP culture mediums, liquid amount 2L) is cultivated in kind to 5L fermentation tanks, initial untreated a concentration of 250g/L of cane molasses is gradually carried 500g/L is raised to, fermentation medium and Zymolysis Equipment are all unsterilised, and fermentation results are as shown in Figure 4.Result of study shows microorganism Flora CEE-DL15 has stronger tolerance to untreated molasses, can in the untreated molasses of 500g/L growth metabolism.250g/L It is molasses fermented the result shows that, during 19h, the sugar fermentation including glucose, fructose and sucrose is depleted, and the end of lactic acid is dense It spends for 82.93 g/L, mass transitions rate is 81%, and production intensity is 4.36g/ (L.h).Microorganism species DL-LA15 exists Slow-growing in 400g/L and 500g/L molasses, after 48h, the concentration of lactic acid is respectively 48.28g/L and 38.7g/L, is disappeared respectively 64.75g/L and 19.23g/L sugar fermentations are consumed.
5 microorganism species CEE-DL15 of embodiment is using 350g/L molasses as the batch fermentation of substrate
By the microorganism species CEE-DL15 obtained in embodiment 1 after seed culture medium culture, connect by 5% volume ratio Kind (CSLP culture mediums, liquid amount 2L) is cultivated in 5L fermentation tanks, initial untreated concentration of molasses is 350g/L, fermentation medium All unsterilised with Zymolysis Equipment, fermentation time 25h, fermentation results are as shown in Figure 5.As it can be seen that final lactic acid concn is in Fig. 5 112.34g/L, mass transitions rate are 81.14%, and production intensity is 4.49g/ (L.h).It is relatively strong that this shows that microorganism species have The untreated molasses of conversion be lactic acid ability.In addition, in Fig. 5 as it can be seen that glucose, fructose and sucrose respectively in 8h, 12h and 25 hours depleted, and microorganism species have the ability for efficiently utilizing complex substrate；Byproduct of reaction is mainly with succinic acid, second Based on acid, formic acid and ethyl alcohol, it is below 5g/L, respectively 4.23g/L, 3.44g/L, 2.15g/L and 2.42g/L.
6 microorganism species CEE-DL15 of embodiment is using 500g/L molasses as the batch fermentation of substrate
By the microorganism species CEE-DL15 obtained in embodiment 1 after seed culture medium culture, connect by 5% volume ratio Kind (CSLP culture mediums, liquid amount 2L) is cultivated in 5L fermentation tanks, initial untreated concentration of molasses is 500g/L, fermentation medium All unsterilised with Zymolysis Equipment, fermentation time 145h, fermentation results are as shown in Figure 6.Final lactic acid concn is 130.89g/L, Mass transitions rate is 72.67%, and production intensity is 0.9g/ (L.h).This shows that untreated molasses have microorganism species to high concentration Higher tolerance, can under higher concentration molasses fermenting lactic acid.Byproduct of reaction is mainly with succinic acid, acetic acid, first Based on acid and ethyl alcohol, respectively 5.38g/L, 3.75g/L, 2.15g/L and 1.75g/L.
Fedbatch cultures of the 7 microorganism species CEE-DL15 of embodiment using molasses as substrate ferments
By the microorganism species CEE-DL15 obtained in embodiment 1 after seed culture medium culture, connect by 5% volume ratio Kind (CSLP culture mediums, liquid amount 1.5L) is cultivated in 5L fermentation tanks, initial untreated concentration of molasses is 250g/L, fermented and cultured Base and Zymolysis Equipment are all unsterilised, carry out feed supplement in 5h, 13h, 35h, 48h of fermentation, reduced sugar total concentration is in maintenance fermentation 50-90g/L, fermentation carry out 101h, and fermentation results are as shown in Figure 7.Consumption sugar 437g altogether, lactic acid production 315g, rotational rate of lactic acid 72.08%, lactic acid final concentration 120.24g/L, production intensity 1.19g/ (L.h).Show microorganism species CEE-DL15 to not locating The molasses of reason have good adaptability, and fermentation medium is simple, save production and cost of investment.
8 microorganism species CEE-DL15 of embodiment is using calcium hydroxide as the batch fermentation of pH adjusting agent
By the mixed bacterial CEE-DL15 obtained in embodiment 1 after seed culture medium, 5L is seeded to by 5% volume ratio (CSLP culture mediums, liquid amount 2.0L) is cultivated in fermentation tank, initial a concentration of 350g/L of cane molasses, pH adjusting agent is by 5M Ca (OH)2Instead of 5M NaOH, fermentation medium and Zymolysis Equipment are all unsterilised, and ferment 25h, and fermentation results are as shown in Figure 8.Finally Lactic acid concn is 114.87g/L, and mass transitions rate is 82.22%, and production intensity is 4.79g/ (L.h).This shows calcium hydroxide As pH adjusting agent slightly better than sodium hydroxide, product lactic acid can be converted into calcium lactate precipitate by calcium hydroxide, can be to a certain degree It is upper to release high concentration lactic acid to the inhibiting effect of cell growth and be conducive to the separation of follow-up lactic acid.

Claims (10)

  1. A kind of 1. microorganism mixed bacterial for fermenting lactic acid, which is characterized in that the microorganism mixed bacterial packet Include Clostridium bacterial strain, Escherichia bacterial strain and Enterococcus strain, the Clostridium bacterial strain, angstrom Total living bacteria count of Xi Shi Bacillus genus strains and Enterococcus strain accounts for 94% of living bacteria count in microorganism mixed bacterial More than, the living bacteria count ratio of the Bacillus strain, Escherichia bacterial strain and Enterococcus strain is 40 ~60:20~40:2~20.
  2. 2. microorganism mixed bacterial according to claim 1, which is characterized in that the Clostridium bacterial strain is production Gas capsular clostridium, the Escherichia bacterial strain are Escherichia coli, and the Enterococcus strain is enterococcus faecalis.
  3. 3. microorganism mixed bacterial according to claim 2, which is characterized in that described in the mixed bacterial The living bacteria count ratio of C.perfringens, Escherichia coli and enterococcus faecalis is 40~60:20~40:2~20.
  4. 4. microorganism mixed bacterial according to claim 3, which is characterized in that described in the mixed bacterial The living bacteria count ratio of C.perfringens, Escherichia coli and enterococcus faecalis is 57.29:34.22:3.17.
  5. 5. a kind of fermentation process of lactic acid, which is characterized in that the fermentation process includes will be described in any one of Claims 1 to 4 Microorganism species be linked into using molasses as the fermentation medium of substrate in ferment, in the molasses ferment sugared content be 35%~50%.
  6. 6. fermentation process according to claim 5, which is characterized in that the honey is cane molasses or beet molasses.
  7. 7. fermentation process according to claim 5, which is characterized in that the composition of the fermentation medium is：Molasses 100 ~700g, 10~30g of Dried Corn Steep Liquor Powder add in distilled water to 1L.
  8. 8. fermentation process according to claim 5, which is characterized in that fermentation medium and Zymolysis Equipment be all in fermentation process It is unsterilised.
  9. 9. fermentation process according to claim 5, which is characterized in that the microorganism species are linked into 100~ The molasses of 700g/L is carry out batch fermentation in the fermentation medium of substrate.
  10. 10. fermentation process according to claim 5, which is characterized in that access to the microorganism species with 200~ The molasses of 400g/L ferment to carry out the fedbatch culture of continuous feeding in the fermentation medium of substrate, and sugar fermentation is dense in fermentation process Degree control is in 50~90g/L.
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