CN108251334B - Mixed microbial community for producing lactic acid through fermentation and fermentation method - Google Patents
Mixed microbial community for producing lactic acid through fermentation and fermentation method Download PDFInfo
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Abstract
The invention provides a mixed microbial flora for producing lactic acid by fermentation and a fermentation method, wherein the mixed microbial flora comprises clostridium strains, escherichia strains and enterococcus strains, the total effective viable count of the mixed microbial flora accounts for more than 94% of the effective viable count of the mixed microbial flora, and the effective viable count ratio of the bacillus strains, the escherichia strains and the enterococcus strains is 40-60: 20-40: 2-20. Compared with single-strain fermentation, the microbial flora has good fermentation stability, high biological safety and high molasses tolerance, can grow and metabolize in 500g/L untreated molasses, and has high production intensity and lactic acid conversion rate. The mixed microbial flora also has the advantages of no need of sterilization in the production process, no need of pretreatment for fermenting substrate molasses, low production cost and the like.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for producing lactic acid by fermenting molasses serving as a substrate through a microorganism mixed flora.
Background
Lactic acid (Lactic acid) is an important biochemical product and is widely used in the fields of medicine, food, beverage, chemical industry, leather, cigarette industry and the like. The most promising application of lactic acid is in the field of biomaterials represented by polylactic acid (PLA). As the most potential biodegradable high polymer material, the biodegradable high polymer material is expected to replace non-degradable plastics such as polypropylene, polyethylene, polystyrene and the like, eliminate white pollution and is a biological environment-friendly material with great application potential.
The production method of lactic acid includes chemical synthesis method, enzyme method and microbial conversion method. Compared with a chemical method and an enzymatic method, the microbial fermentation method for producing the lactic acid can obtain L-lactic acid or D-lactic acid with stereospecificity or a racemate formed by mixing two isomers in a certain proportion by selecting strains and culture conditions, and can meet the requirement of producing the polylactic acid. The fermentation method for producing lactic acid is an important method for producing lactic acid due to the advantages of wide raw material source, low production cost, high optical purity of products and the like. The traditional fermentation method for producing lactic acid mainly takes glucose or grain crops, such as rice, corn, potato and the like as raw materials. In order to further reduce the production cost, the lactic acid fermentation production is tried by taking industrial and agricultural wastes (such as cane molasses) (Flores-Albino B et al.,2012,35: 1193-1200; Xu K et al.,2014,153:23-29), lignocellulose (Ou MS,2011,38: 599-605), paper-making pulp (Budhavaram NK,2009,100:5966-5972), apple pomace (Gullon B,2008,99: 308-319), kitchen garbage (Zhang B,2008,99:855-862), Jerusalem artichoke (Wang LM,2013, 130:174-180) and the like as raw materials, so that the production cost can be further reduced, the effective utilization of renewable resources can be realized, and the problems of treatment and pollution of wastes can be solved.
Molasses is a main byproduct in the sugar production process and is also an industrial waste. Wherein, the yield of the cane molasses can reach 2.5-3% of that of the raw material cane, the raw materials for production are rich, and the price is low, so the cane molasses has important economic significance for reasonable development and utilization of the cane molasses. The main components are saccharides, which contain about 30-40% (w/v) of sucrose, about 15-20% (w/v) of other sugars (mostly reducing sugars), and the contents of nitrogen source and inorganic salt are 0.5-0.9% (w/v) and 10% (w/v), respectively (Liu YP,2008,99: 1736-. At present, cane molasses is mostly used for being processed into various fermentation products, such as alcohol, lactic acid, monosodium glutamate, citric acid, lysine, caramel pigment and the like. Although the use of inexpensive raw materials can reduce the cost of raw materials to some extent, the impurities contained therein, such as volatile organic acids, colloids, ashes, etc. contained in molasses, require purification before they can be used in a pure biological process for cultivation. If purification treatment is not carried out, the single bacterium is difficult to utilize, and the toxic effect on cell growth reduces the utilization rate of raw materials, so that the final production concentration and strength are reduced.
The Microbial flora (Microbial consortium) is an industrial application of natural mixed bacteria fermentation, and the technology is based on the principle of ecological selection and keeps the flora capable of utilizing complex substrates and special product ranges in a reactor or a fermentation tank for continuous use. Compared with pure culture technology, the microbial flora shows great potential advantages, which are mainly reflected in that: (1) diversity of raw materials: cheaper and more complex substrates such as lignocellulose, whey, molasses, crude glycerol, potato processing wastewater, corn steep liquor and the like can be used as fermentation raw materials for producing chemicals. (2) Diversification or singulation of products: by coordinating the composition of the flora, a plurality of target products can be obtained by utilizing metabolic pathways of different microorganisms, the product range can be narrowed, the separation and purification of downstream substances are facilitated, and the cost is reduced. (3) Ease, safety and stability of operation: the microbial flora has high biological diversity, can be operated under the condition of no sterilization, and improves the anti-phage infection capacity and the biological safety. In addition, the interaction relation among the cells of the mixed bacteria system is dynamically balanced, and the mixed bacteria system has stronger adaptability and stability to environmental fluctuation. Based on the characteristics of microbial flora, the microbial flora with strong complex substrate tolerance, good fermentation stability, high production intensity and no need of sterilization in the production process is developed, and the method for efficiently converting molasses to produce lactic acid becomes a new idea for industrial development.
Disclosure of Invention
The invention aims to provide a microbial flora which has stable fermentation performance and strong tolerance and can efficiently convert molasses into lactic acid and a method for preparing lactic acid by using the microbial flora. The microbial flora of the invention consists of clostridium, escherichia and enterococcus, has stable fermentation performance and strong complex substrate tolerance, efficiently converts untreated molasses into lactic acid under anaerobic condition, does not sterilize in the fermentation process, and overcomes the limitations of low tolerance, low production strength and the like of molasses fermented by single bacterium.
The technical scheme of the invention is as follows:
the mixed microbial flora for producing lactic acid by fermenting molasses as a substrate is characterized by comprising clostridium strains, escherichia strains and enterococcus strains, wherein the total effective viable count of the clostridium strains, the escherichia strains and the enterococcus strains accounts for more than 94% of the effective viable count in the mixed microbial flora, and the effective viable count ratio of the bacillus strains, the escherichia strains and the enterococcus strains is 40-60: 20-40: 2-20. Preferably, the mixed microbial flora consists of a clostridium strain, an escherichia strain and an enterococcus strain, wherein the effective viable count ratio of the bacillus strain, the escherichia strain and the enterococcus strain is 40-60: 20-40: 2-20.
Further, the clostridium strain is clostridium perfringens, the escherichia strain is escherichia coli, and the enterococcus strain is enterococcus faecalis.
Furthermore, in the microorganism mixed flora, the ratio of the effective viable count of clostridium perfringens, escherichia coli and enterococcus faecalis is 40-60: 20-40: 2-20.
Further, in the microorganism mixed flora, the effective viable count ratio of clostridium perfringens, escherichia coli and enterococcus faecalis is 57.29:34.22: 3.17.
In the invention, the clostridium perfringens, the escherichia coli and the enterococcus faecalis are all strains which are conventionally used in the field, and in the proportional relation range among the strains provided by the application, the mixed flora consisting of the strains has stable fermentation performance, and efficiently converts untreated molasses into lactic acid under the anaerobic condition, so that the fermentation process is not sterilized, and the limitations of low tolerance, low production strength and the like of molasses fermented by single bacteria are overcome.
The invention also provides a fermentation method of lactic acid, which comprises the step of inoculating the microbial flora into a fermentation medium taking molasses as a substrate for fermentation, wherein the content of fermentation sugar in the molasses is 35-50%.
Further, in the above fermentation method, the honey is cane molasses or beet molasses. The fermentation sugar comprises glucose, fructose and sucrose. Preferably, the molasses is untreated molasses, i.e. molasses that has not been pretreated by acids etc. in sugar mills. Molasses is used as a byproduct of a sugar mill, contains fermentation sugar, can be used for microbial fermentation, but can be used for fermentation after being pretreated by acid and the like before being generally used at present, and has complex treatment process, increased cost and no contribution to industrial application. The mixed microbial flora of the invention can be directly fermented by untreated molasses. The cane molasses serving as a byproduct of the sugar production process of a sugar refinery comprises the following components in percentage by weight (w/v): 35 to 50 percent of fermentation sugar, 9 to 12 percent of non-sugar organic matter, 10 to 15 percent of sulfated ash, moisture and a small amount of pigment and impurities.
Further, in the above fermentation method, the composition of the fermentation medium is: 100-700 g of molasses and 10-30 g of corn steep liquor dry powder, and adding distilled water to 1L. Wherein the concentration of molasses in the fermentation medium is preferably 200-500 g/L, and more preferably 350-500 g/L. The pH value of the fermentation medium prepared according to the components and the proportion is acidic and is about 6.3.
Further, in the above fermentation method, neither the fermentation medium nor the fermentation equipment is sterilized during the fermentation.
Further, in the fermentation method, the microbial flora is inoculated into a fermentation medium taking 100-700 g/L of molasses as a substrate for batch fermentation. The concentration of the molasses can be 200-500 g/L, and can also be 350-500 g/L. The microbial flora of the invention can grow and metabolize in 500g/L untreated molasses, and has high production intensity and lactic acid conversion rate.
Furthermore, in the fermentation method, the microbial flora is inoculated into a fermentation medium taking 200-400g/L molasses as a substrate to carry out continuous fed-batch fermentation, and the concentration of fermentation sugar is controlled to be 50-90g/L in the fermentation process.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention discloses a mixed microbial community for preparing lactic acid by fermentation, which has the advantages of good fermentation stability, strong tolerance of complex substrates and high production strength.
(2) The microbial community utilizes industrial waste molasses as a substrate to prepare the lactic acid through fermentation, the molasses is not pretreated, the production process of the lactic acid is reduced, and the production cost is reduced.
(3) The fermentation medium has simple composition, only contains two components of molasses and corn steep liquor dry powder, and reduces the production cost.
(4) In the fermentation process, the fermentation medium and the fermentation equipment do not need to be sterilized, so that the production cost is further reduced.
(5) The microorganism mixed bacteria are utilized for fermentation, a batch or batch fed-batch fermentation strategy is adopted, the operation is simple and convenient, and the production labor cost is reduced.
By adopting the mixed flora, in batch fermentation consisting of 350g/L of cane molasses serving as a substrate and 18.5 g/L of corn steep liquor dry powder serving as a culture medium, the concentration of lactic acid can reach 112.34g/L, the mass conversion rate is 81.14%, and the production intensity is 4.49 g/(L.h). In the batch fed-batch fermentation with cane molasses as a substrate and corn steep liquor dry powder as a culture medium, the concentration of lactic acid can reach 120.24g/L, the mass conversion rate is 71.95%, and the production intensity is 1.19 g/(L.h).
Drawings
FIG. 1 results of lactic acid metabolism of microbial flora under different enrichment conditions;
FIG. 2 results of the metabolism of lactic acid by the microbial colony CEE-DL15 under different operating conditions;
FIG. 3 results of the metabolism of lactic acid by the microbial colony CEE-DL15 under different medium conditions;
FIG. 4 sugar consumption, cell growth and lactate metabolism at different initial molasses concentrations for the microbial colony CEE-DL 15;
FIG. 5 shows the result of batch fermentation of the microbial community CEE-DL15 with 350g/L molasses as substrate;
FIG. 6 shows the results of batch fermentation of the microbial community CEE-DL15 using 500g/L molasses as substrate;
FIG. 7 batch fed-batch fermentation of the microbial community CEE-DL15 with molasses as substrate
FIG. 8 shows the results of batch fermentation of the microbial community CEE-DL15 using calcium hydroxide as a pH adjuster.
Detailed Description
The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. In the following examples, unless otherwise specified, the experimental methods used were all conventional methods, and the reagents used were all available from chemical or biological reagents companies.
The following describes the embodiments of the present invention in detail with reference to the technical solutions.
1. The media used in the following examples:
enrichment medium (g/L): glucose 40, Yeast extract powder 5, C4H2Na2O45, monensin 0.1, K2HPO4·3H2O 3，NaCl 1，(NH4)2SO41，CaCl20.5，MgCl2Sterilizing at 121 deg.C for 15min at 0.5.
Seed medium (g/L): 30 parts of glucose, 5 parts of peptone, 5 parts of beef extract, 2.5 parts of yeast extract powder and K2HPO4·3H2O 2，(NH4)2HC6H5O72，CH3COONa·3H2O 2，MgSO4·7H2O 0.2， MnSO4·H2Sterilizing at 121 deg.C for 15min under O0.05.
Fermentation medium: the culture medium comprises the following MRS culture medium, S-MRS culture medium and CSLP culture medium, and the culture medium comprises the following components:
MRS medium (g/L): 100-500 parts of cane molasses, 10 parts of peptone, 10 parts of beef extract, 5 parts of yeast extract powder and K2HPO4·3H2O 2，(NH4)2HC6H5O72，CH3COONa·3H2O 2，MgSO4·7H2O 0.58，MnSO4·H2O 0.25。
100-500 g/L of S-MRS culture medium, 5g/L of cane molasses, 5g/L of peptone, 5g/L of beef extract and 2.5 g/L of yeast extract powder, and K2HPO4·3H2O 2，(NH4)2HC6H5O72，CH3COONa·3H2O 2，MgSO4·7H2O 0.2，MnSO4·H2O 0.25。
CSLP Medium (g/L): 100-500 parts of cane molasses and 18.5 parts of corn steep liquor dry powder.
2. Seed culture:
and (5) anaerobic culture. A250 mL penicillin bottle is adopted, and the liquid filling amount is 100 mL. After the seeds are filled into the seed culture medium, introducing common nitrogen into each bottle for 3min continuously to remove oxygen, and then sealing the top with a butyl rubber plug. In the experimental process, a disposable sterile needle tube is used for inoculation and sampling, the inoculation amount is 5-10% (v/v), the culture temperature is 37 ℃, the rotating speed of a shaking table is 200r/min, and the culture time is 8-12 h.
3. Fermentation culture conditions:
A5L full-automatic fermentation tank is adopted, the liquid loading amount is 2L, the product of seed culture is inoculated to a fermentation medium by the inoculation amount of 5% (v/v), the sugarcane honey is taken as a substrate, the fermentation is carried out at the temperature of 37 ℃ and the rotating speed of 250r/min, and the pH is controlled to be 6.3 by 5MNaOH in the fermentation process. Introducing 0.1vvm of common nitrogen within 4 hours before and after inoculation to build an anaerobic environment in the tank.
Cane molasses: composition (w/v): 44% fermented sugar, 10% non-sugar organic matter, 12% sulfated ash, moisture, and a small amount of pigments and impurities. The fermentation sugar comprises glucose, fructose and sucrose. The cane molasses is a byproduct of the sugar making process, and generally can be fermented after being pretreated by acid. The acid pretreatment process is as follows: 4mol/L of H2SO4Adjusting pH of the sugar cane molasses solution to 3.0, bathing in boiling water (100 deg.C) for 1h, standing overnight at room temperature, centrifuging at 12000rpm for 15min, and collecting supernatant for use. The acid pretreatment of molasses has complicated process and increases cost. The untreated cane molasses described in the examples below is cane molasses that has not been pretreated with acid or the like.
Example 1 screening of domesticated lactic acid producing microbial flora from bovine gastric content
The sample is collected from a live livestock slaughterhouse in a large-scale continuous process, and the sample of the content in the cattle stomach is divided into four processing modes: 1) suspension of bovine stomach contents, and anaerobic enrichment; 2) suspension of bovine gastric contents, aerobic enrichment; 3) solid bovine stomach contents, anaerobic enrichment; 4) solid bovine stomach contents, enriched with oxygen. Wherein the preparation operation of the bovine gastric inclusion suspension comprises the following steps: taking 0.1kg of fresh cow stomach contents in a triangular flask of 100mL of normal saline, filtering the solid substances with sterile gauze, taking 5mL of filtrate containing a large amount of bacteria, respectively inoculating the filtrate in the triangular flask and a penicillin bottle filled with 100mL of enrichment medium, and carrying out aerobic and anaerobic enrichment culture. Solid bovine stomach content preparation procedure: taking 0.1kg of fresh bovine stomach contents in a triangular flask with 100mL of normal saline, carrying out vortex oscillation for 2 min, respectively inoculating the solid-liquid mixture in the triangular flask and a penicillin bottle with 100mL of enrichment medium, and carrying out aerobic and anaerobic enrichment culture. The culture solution of the four treatment modes is subcultured every 12h with the inoculum size of 2% (v/v), and is subcultured for 20 times continuously to obtain the mixed strain with stable fermentation performance. And finally inoculating the mixed strain with stable fermentation performance to an MRS culture medium with cane molasses concentration of 100g/L (fermentation sugar concentration of 40g/L) for anaerobic fermentation. The fermentation results are shown in FIG. 1, which indicates that the flora treated by the 3 rd sample treatment mode has the highest lactic acid yield and the conversion rate is 90.38%. Selecting solid ox stomach inclusion, and carrying out subsequent fermentation culture on the microbial flora subjected to anaerobic enrichment culture.
The microbial flora was identified to contain a Clostridium strain (57.29%), an Escherichia strain (34.22%) and an enterococcus strain (5.32%), wherein the percentage (%) indicates the effective viable count of each genus as a percentage of the total effective viable count.
The Clostridium strain is Clostridium perfringens (Clostridium perfringens), the Escherichia strain is Escherichia coli (Escherichia coli), and the enterococcus strain is enterococcus faecalis (enterococcus faecalis). The following examples were conducted to identify the performance of the mixed microorganism for the fermentative production of lactic acid using a mixed microorganism consisting of Clostridium perfringens, Escherichia coli and enterococcus faecalis as a mixed microbial population (designated CEE-DL 15). The ratio of the effective viable count of clostridium perfringens, escherichia coli and enterococcus faecalis in CEE-DL15 is 57.29:34.22: 3.17.
the clostridium perfringens, the escherichia coli and the enterococcus faecalis are all strains which are conventionally used in the field. Preferably, the Clostridium perfringens strain may be Clostridium perfringens ATCC 13124, Escherichia coli strain NCTC 9001, and Enterococcus faecalis strain ATCC 19433.
EXAMPLE 2 use of molasses by the microbial flora CEE-DL15 under different operating conditions
After the microbial flora CEE-DL15 obtained in example 1 was subjected to seed culture, the cultured mixture was inoculated into a penicillin bottle containing 100mL of MRS medium at a volume ratio of 5%, and the initial concentration of cane molasses was about 100g/L, and the fermentation culture was carried out using cane molasses as a substrate. The fermentation operating conditions are divided into three types, 1) sterilization and fermentation are carried out, and the substrate is cane molasses treated by acid; 2) sterilizing and fermenting, wherein a substrate is untreated cane molasses; 3) the fermentation is not sterilized, and the substrate is untreated cane molasses. After 48 hours of shake flask fermentation culture, the concentration, conversion rate and production intensity of lactic acid in the fermentation broth were measured and are shown in FIG. 2. The results show that the flora CEE-DL15 grows well under the conditions of no sterilization and the adoption of untreated molasses as a substrate, has higher molasses conversion rate and lactic acid production intensity, and the concentration, conversion rate and production intensity of the lactic acid respectively reach 87.98g/L, 73.21g/g and 1.83g/(L.h), which shows that the lactic acid is feasible to be produced by non-sterile culture and cane molasses fermentation without pretreatment. Example 3 selection and economic optimization of fermentation Medium
After the mixed bacterial population CEE-DL15 obtained in example 1 was cultured in a seed culture medium, it was inoculated at a volume ratio of 5% into penicillin bottles containing 100mL of a fermentation medium and cultured by fermentation using untreated cane molasses as a substrate at an initial untreated cane molasses concentration of 250 g/L. The fermentation medium is divided into three types: 1) MRS culture medium; 2) S-MRS culture medium; 3) CSLP medium; the fermentation results are shown in FIG. 3. The flora can utilize a simple and cheap corn steep liquor dry powder culture medium (CSLP culture medium) to ferment and produce lactic acid, the yield and the conversion rate of the lactic acid are close to those of other two culture media, the production intensity is higher than that of the culture of the former two culture media, and the cost of a nitrogen source is saved by 50-200%.
Example 4 tolerance of the microbial community CEE-DL15 to high concentrations of molasses under batch fermentation conditions
After the microbial colony CEE-DL15 obtained in example 1 was cultured in the seed medium, it was inoculated in a 5L fermentor at a volume ratio of 5% (CSLP medium, liquid content 2L), the initial untreated sugar cane molasses concentration was gradually increased to 500g/L at 250g/L, neither the fermentation medium nor the fermentation equipment was sterilized, and the fermentation results are shown in FIG. 4. The research result shows that the microbial community CEE-DL15 has stronger tolerance to the untreated molasses and can grow and metabolize in 500g/L of the untreated molasses. The 250g/L molasses fermentation result shows that the fermentation sugar including glucose, fructose and sucrose is completely consumed at 19h, the final concentration of lactic acid is 82.93 g/L, the mass conversion rate is 81%, and the production intensity is 4.36 g/(L.h). The microbial flora DL-LA15 grew slowly in 400g/L and 500g/L molasses, and after 48h, the lactic acid concentration was 48.28g/L and 38.7g/L, respectively, and 64.75g/L and 19.23g/L of fermentation sugar were consumed.
Example 5 batch fermentation of the microbial flora CEE-DL15 with 350g/L molasses as substrate
The microbial population CEE-DL15 obtained in example 1 was cultured in a seed medium and then inoculated at a volume ratio of 5% into a 5L fermentor for culture (CSLP medium, liquid content 2L), the initial untreated molasses concentration was 350g/L, neither the fermentation medium nor the fermentation equipment was sterilized, the fermentation time was 25 hours, and the fermentation results are shown in FIG. 5. As can be seen in FIG. 5, the final lactic acid concentration was 112.34g/L, the mass conversion was 81.14%, and the production strength was 4.49 g/(L.h). This indicates that the microbial flora has a strong ability to convert untreated molasses to lactic acid. In addition, as can be seen in fig. 5, glucose, fructose and sucrose are depleted at 8h, 12h and 25h, respectively, and the microbial flora has the ability to efficiently utilize complex substrates; the reaction byproducts mainly comprise succinic acid, acetic acid, formic acid and ethanol, which are less than 5g/L, 4.23g/L, 3.44g/L, 2.15g/L and 2.42g/L respectively.
EXAMPLE 6 batch fermentation of the microbial flora CEE-DL15 with 500g/L molasses as substrate
After the microbial colony CEE-DL15 obtained in example 1 was cultured in a seed medium, it was inoculated at a volume ratio of 5% into a 5L fermentor and cultured (CSLP medium, liquid content 2L), the initial untreated molasses concentration was 500g/L, neither the fermentation medium nor the fermentation equipment was sterilized, the fermentation time was 145 hours, and the fermentation results are shown in FIG. 6. The final lactic acid concentration was 130.89g/L, the mass conversion was 72.67%, and the production strength was 0.9 g/(L.h). This indicates that the microbial flora has higher tolerance to high-concentration untreated molasses and can produce lactic acid by fermentation under the condition of higher-concentration molasses. The reaction byproducts mainly comprise succinic acid, acetic acid, formic acid and ethanol, and are respectively 5.38g/L, 3.75g/L, 2.15g/L and 1.75 g/L.
Example 7 batch Fed fermentation of the microbial flora CEE-DL15 with molasses as substrate
After the microbial population CEE-DL15 obtained in example 1 was cultured in the seed medium, it was inoculated into a 5L fermentor at a volume ratio of 5% (CSLP medium, liquid content 1.5L), initial untreated molasses concentration was 250g/L, neither the fermentation medium nor the fermentation equipment was sterilized, feeding was performed for 5h, 13h, 35h, and 48h of fermentation, total reducing sugar concentration in the fermentation was maintained at 50-90g/L, and the fermentation was performed for 101h, with the results shown in FIG. 7. 437g of co-consumed sugar, 315g of lactic acid yield, 72.08% of lactic acid conversion rate, 120.24g/L of lactic acid final concentration and 1.19g/(L.h) of production intensity. The microbial community CEE-DL15 is proved to have good adaptability to untreated molasses, the fermentation medium is simple, and the production and investment costs are saved.
Example 8 batch fermentation of the microbial flora CEE-DL15 with calcium hydroxide as pH regulator
The mixed bacterial strain CEE-DL15 obtained in example 1 was inoculated into a 5L fermentor for culture (CSLP medium, liquid content 2.0L) at a volume ratio of 5% after passing through a seed medium, initial sugar cane molasses concentration was 350g/L, and pH adjusting agent was 5M Ca (OH)2Instead of 5M NaOH, neither the fermentation medium nor the fermentation apparatus was sterilized and the fermentation was carried out for 25 hours, the results of which are shown in FIG. 8. The final lactic acid concentration was 114.87g/L, the mass conversion was 82.22%, and the production strength was 4.79 g/(L.h). This shows that calcium hydroxide is slightly superior to sodium hydroxide as pH regulator, and can convert lactic acid into calcium lactate precipitate, eliminate the inhibition of high concentration lactic acid on cell growth and facilitate the subsequent separation of lactic acid.
Claims (7)
1. A fermentation method of lactic acid is characterized in that the fermentation method comprises the steps of inoculating a microorganism mixed flora into a fermentation medium taking untreated molasses as a substrate for fermentation, wherein the content of fermentation sugar in the molasses is 35-50%;
the microorganism mixed flora comprises clostridium strains, escherichia strains and enterococcus strains, the total effective viable count of the clostridium strains, the escherichia strains and the enterococcus strains accounts for more than 94% of the effective viable count in the microorganism mixed flora, and the effective viable count ratio of the bacillus strains, the escherichia strains and the enterococcus strains is 40-60: 20-40: 2-20;
the clostridium strain is clostridium perfringens, the escherichia strain is escherichia coli, and the enterococcus strain is enterococcus faecalis.
2. The method for fermenting lactic acid according to claim 1, wherein the effective viable count ratio of clostridium perfringens, escherichia coli and enterococcus faecalis in the mixed microbial population is 57.29:34.22: 3.17.
3. The method of claim 1, wherein the molasses is cane molasses or beet molasses.
4. The method of claim 1, wherein the fermentation medium comprises: 100-700 g of molasses and 10-30 g of corn steep liquor dry powder, and adding distilled water to 1L.
5. The method of claim 1, wherein neither the fermentation medium nor the fermentation equipment is sterilized during the fermentation.
6. The method for fermenting lactic acid according to claim 1, wherein the mixed microbial population is inoculated into a fermentation medium containing 100 to 700g/L of molasses as a substrate to perform batch fermentation.
7. The lactic acid fermentation method according to claim 1, wherein the mixed microbial population is inoculated into a fermentation medium containing 200-400g/L molasses as a substrate to perform continuous fed-batch fermentation, and the concentration of fermentation sugar is controlled to be 50-90g/L during the fermentation process.
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| CN108893497A (en) * | 2018-07-18 | 2018-11-27 | 大连理工大学 | A kind of many cells system and fermentation process changing aeration condition regulation product |
| CN114181872B (en) * | 2022-01-07 | 2023-11-24 | 北京大北农科技集团股份有限公司 | Enterococcus faecium high-density fermentation culture method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7039131B2 (en) * | 2002-08-02 | 2006-05-02 | Agere Systems Inc. | Carrier frequency offset estimation in a wireless communication system |
| AU2008212826A1 (en) * | 2007-02-09 | 2008-08-14 | Zeachem Inc. | Energy efficient methods to produce products |
| KR20150061448A (en) * | 2013-11-27 | 2015-06-04 | 롯데케미칼 주식회사 | Method for Producing Lactic acid Using Microalgae as a Nitrogen Source |
| CN106167785A (en) * | 2016-08-29 | 2016-11-30 | 内蒙古工业大学 | A kind of E. Faecium strains and application thereof |
-
2018
- 2018-01-15 CN CN201810035798.1A patent/CN108251334B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7039131B2 (en) * | 2002-08-02 | 2006-05-02 | Agere Systems Inc. | Carrier frequency offset estimation in a wireless communication system |
| AU2008212826A1 (en) * | 2007-02-09 | 2008-08-14 | Zeachem Inc. | Energy efficient methods to produce products |
| KR20150061448A (en) * | 2013-11-27 | 2015-06-04 | 롯데케미칼 주식회사 | Method for Producing Lactic acid Using Microalgae as a Nitrogen Source |
| CN106167785A (en) * | 2016-08-29 | 2016-11-30 | 内蒙古工业大学 | A kind of E. Faecium strains and application thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110241043B (en) * | 2019-06-10 | 2021-12-21 | 大连理工大学 | Strain for producing lactic acid through high-temperature fermentation and method for producing lactic acid |
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