Process for producing lactic acid by fermentation of concentrated bacterial liquid
阅读说明：本技术 一种利用浓缩菌液发酵生产乳酸的工艺 (Process for producing lactic acid by fermentation of concentrated bacterial liquid ) 是由 马光 王竞辉 杨付伟 夏顺炜 吴计划 于 2021-07-16 设计创作，主要内容包括：本发明公开了一种利用浓缩菌液发酵生产乳酸的工艺,所述工艺包括以下步骤：A、配制菌种增殖培养基,通过好氧或厌氧方式培养乳酸生产菌,当菌体浓度不再增加,浓缩收集菌液,并加入菌种活性保护剂；B、将浓缩后菌液与碳源、菌种活性增强剂相混合,通过流加中和剂和碳源进行发酵培养,生产乳酸。本发明将乳酸发酵的菌种增殖和产酸阶段分开,通过专门的菌种增殖培养基,快速获得大量乳酸生产菌,然后浓缩获得高浓度的乳酸生产菌,以高浓度的乳酸生产菌进行发酵生产,具有提高产酸速率,缩短发酵周期,节约能耗的优点。(The invention discloses a process for producing lactic acid by fermenting concentrated bacterial liquid, which comprises the following steps: A. preparing a strain multiplication culture medium, culturing lactic acid producing bacteria in an aerobic or anaerobic mode, concentrating and collecting a bacterial liquid when the concentration of the bacteria is not increased any more, and adding a strain activity protective agent; B. and mixing the concentrated bacterial liquid with a carbon source and a strain activity enhancer, and carrying out fermentation culture by feeding a neutralizing agent and the carbon source to produce lactic acid. The invention separates the strain multiplication and acid production stages of lactic acid fermentation, rapidly obtains a large amount of lactic acid producing bacteria through a special strain multiplication culture medium, then concentrates the lactic acid producing bacteria to obtain high-concentration lactic acid producing bacteria, and performs fermentation production by using the high-concentration lactic acid producing bacteria.)
1. A process for producing lactic acid by utilizing concentrated bacterial liquid fermentation is characterized by comprising the following steps:
A. preparing a strain multiplication culture medium, culturing lactic acid producing bacteria in an aerobic or anaerobic mode, concentrating and collecting a bacterial liquid after the strain multiplication culture is finished, and adding a strain activity protective agent;
B. and mixing the concentrated bacterial liquid with a carbon source and a strain activity enhancer, and carrying out fermentation culture by feeding a neutralizing agent and the carbon source to produce lactic acid.
2. The process for producing lactic acid by fermentation of concentrated bacterial liquid according to claim 1, wherein the formula of the strain multiplication medium is: 10-30g/L carbon source, 2-10g/L yeast extract, 5-20g/L tryptone, 0.2-1g/L K2HPO4·3H2O，0.2-1.1g/L KH2PO4，2-10g/L(NH4)2SO4，0.2-0.5g/L MgSO4·7H2O, 5-10mL/L of trace element stock solution, adding the balance of water when preparing 1L of culture medium according to the formula to ensure that the total amount of the culture medium is 1L, and adjusting the pH value to be 6-8;
wherein the formula of the microelement stock solution comprises: 2-6g/L FeSO4·7H2O，0.2-0.8g/L ZnSO4·7H2O，0.5-1.5g/L MnSO4·4H2O，0.05-0.1g/L CuSO4·5H2O，0.05-0.15g/L(NH4)6Mo7O24·4H2O，0.05-0.1g/L H3BO3，0.05-0.2g/L CoCl2·6H2And O, adding the balance of water when preparing 1L of stock solution according to the formula so that the total amount of the trace element stock solution is 1L.
3. The process for producing lactic acid by fermentation of concentrated bacterial liquid according to claim 1, wherein the method for concentrating and collecting bacterial liquid in step a includes: the bacterial liquid is concentrated by means of ceramic membrane, centrifugation and diatomite filtration, preferably 2-20 times.
4. The process for producing lactic acid by using concentrated bacterial liquid fermentation according to any one of claims 1 to 3, wherein the bacterial strain activity protective agent in the step A is one or more of betaine, proline, trehalose, hyaluronic acid and polyethylene glycol;
preferably, the addition ratio of the strain activity protective agent to the concentrated bacterial liquid is 0.1-10 g/kg.
5. The process for producing lactic acid by utilizing concentrated bacterial liquid fermentation according to claim 4, wherein in the step B, the strain activity enhancer is one or more of vitamin B6, biotin, nicotinic acid, magnesium sulfate, citric acid and salts thereof, and ferrous sulfate;
preferably, the addition ratio of the strain activity enhancer is 0.1-10g/kg of concentrated bacterial liquid.
6. The process for producing lactic acid by fermentation of concentrated bacterial liquid according to any one of claims 1 to 3, wherein in the step B, the concentration of the carbon source for the first mixing with the concentrated bacterial liquid is 10 to 30%, and the mixing volume ratio of the bacterial liquid to the carbon source is 1 (1 to 200);
preferably, the concentration of the carbon source fed in the step B is 10-60% so as to control the content of the carbon source in the fermentation liquid to be 10-100 g/L.
7. The process for producing lactic acid by using concentrated bacterial liquid fermentation according to claim 6, wherein the pH in the fermentation liquid in step B is controlled to be 5-10, preferably 6.5-7.5 by adding a neutralizing agent;
preferably, the neutralizing agent is one or more of alkaline sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt and ammonia water, preferably one or more of sodium hydroxide, ammonia water, calcium hydroxide and calcium carbonate.
8. The process for producing lactic acid by fermentation with concentrated bacterial liquid according to any one of claims 1 to 3, wherein in step B, the carbon source feeding is stopped when the volume of the fermentation liquid reaches 80% of the liquid loading volume of the fermentation tank, and the fermentation is finished when the carbon source content is less than 1 g/L.
9. The process for producing lactic acid by using fermentation of concentrated bacterial liquid according to any one of claims 1 to 3, wherein the lactic acid-producing bacteria are Lactobacillus, Bacillus coagulans, Pseudomonas aeruginosa, Rhizopus oryzae, Lactobacillus rhamnosus, Lactobacillus paracasei, or Escherichia coli.
10. The process for producing lactic acid by using the fermentation of the concentrated bacterial liquid according to any one of claims 1 to 3, wherein the clear liquid obtained after the concentration of the bacterial liquid in the step A is reused as a strain multiplication medium after being supplemented with nutrients.
Technical Field
The invention relates to a process for producing lactic acid, in particular to a process for producing lactic acid by utilizing concentrated bacterial liquid fermentation.
Background
Lactic acid is an important organic acid in the living body, has great application value in daily life and production activities of people, and is a main raw material for synthesizing polylactic acid. The polylactic acid is recognized in the industry as a novel bio-based material, has the characteristics of no toxicity, no irritation, good biocompatibility and the like, and has wide application in a plurality of fields of clothing, construction, agriculture, forestry, paper making, medical health and the like. With the development and utilization of new environment-friendly polylactic acid biological materials in recent years, the demand of lactic acid is increasing.
Lactic acid can be obtained by chemical synthesis or fermentation of carbohydrates, and L or D lactic acid can be obtained specifically based on a fermentation method, and thus the fermentation method is widely used for the production of lactic acid. The existing fermentation method for producing lactic acid generally comprises the steps of fermenting and metabolizing lactic acid producing bacteria such as lactobacillus, bacillus or escherichia coli under anaerobic or microaerobic conditions to produce acid. According to the difference between the growth of the thalli and the acid production process of metabolism in the fermentation process, the Chinese patent publication CN1332035A supplements a carbon source at the middle and later stages of fermentation culture to provide energy for the metabolism of the thalli and promote the generation of L-lactic acid. However, because the water content in the bacteria culture medium is usually above 80%, people always use a high-concentration carbon source (usually above 60%) and a neutralizing agent as far as possible in order to take account of the acid production rate, the volume of a fermentation tank and the concentration of a lactic acid product, while for a process for producing lactic acid by starch sugar fermentation, the concentration of a starch sugar solution as a carbon source is usually not more than 40%, and the starch sugar solution needs to be concentrated in order to obtain the high-concentration carbon source, the existing process usually uses falling film evaporation or MVR to concentrate a dilute sugar solution, and the energy consumption of each ton of concentrated sugar is 0.27t of steam or 16.39 degrees of electricity, so that the investment of lactic acid equipment and the energy consumption of production are increased.
The production cost of the existing lactic acid fermentation production method is one of the key factors restricting the application of lactic acid. Therefore, the search for a technique capable of reducing the production cost of lactic acid has been a hot point of research.
Disclosure of Invention
In order to solve the technical problems, the invention provides a process for producing lactic acid by fermenting concentrated bacterial liquid. The invention separates the strain multiplication and acid production stages of lactic acid fermentation, rapidly obtains a large amount of lactic acid producing bacteria through a special strain multiplication culture medium, then obtains the high-concentration lactic acid producing bacteria through concentration, and performs fermentation production by using the high-concentration lactic acid producing bacteria. As the thalli are concentrated, on one hand, the acid production rate is obviously improved, on the other hand, the production requirement can be met by using a carbon source with lower concentration, and for the production of lactic acid by fermentation of starch sugar, the step of sugar liquid concentration can be omitted, and the energy consumption of fermentation is reduced.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a process for producing lactic acid by fermentation of concentrated bacterial liquid comprises the following steps:
A. preparing a strain multiplication culture medium, culturing lactic acid producing bacteria in an aerobic or anaerobic mode, concentrating and collecting a bacterial liquid after the strain multiplication culture is finished, and adding a strain activity protective agent;
in the step A, the inoculation amount in the strain multiplication culture medium is 5-20%, and for aerobic lactic acid producing bacteria, the culture conditions are as follows: the temperature is 30-50 ℃, the pressure in the tank is 0.02-0.1Mpa, the ventilation volume is 0.2-5vvm, the rotation speed is 200 and 1000rpm, the dissolved oxygen is controlled to be more than 50 percent, and pure oxygen can be introduced when necessary. For anaerobic lactic acid-producing bacteria, the culture conditions were: the initial temperature is 30-50 ℃, the tank pressure is 0.02-0.05Mpa, and the rotating speed is 200-1000 rpm.
The concentration of carbon source in the culture solution is maintained at 5g/L by feeding supplement solution, pH is controlled at 6.5-7.5 by feeding alkaline neutralizer, and the culture is stopped after 5-20h, preferably when OD600 is not increased any more.
B. And mixing the concentrated bacterial liquid with a carbon source and a strain activity enhancer, and carrying out fermentation culture by feeding a neutralizing agent and the carbon source to produce lactic acid.
The fermentation culture conditions in the step B are that the fermentation is carried out at the temperature of 30-50 ℃ and the rotation speed of 100-.
In a preferred embodiment, the formulation of the seed multiplication medium is: 10-30g/L carbon source, 2-10g/L yeast extract, 5-20g/L tryptone, 0.2-1g/L K2HPO4·3H2O，0.2-1.1g/L KH2PO4，2-10g/L(NH4)2SO4，0.2-0.5g/L MgSO4·7H2O, 5-10mL/L of trace element stock solution, adding the balance of water when preparing 1L of culture medium according to the formula to ensure that the total amount of the culture medium is 1L, and adjusting the pH value to be 6-8;
wherein the formula of the microelement stock solution comprises: 2-6g/L FeSO4·7H2O，0.2-0.8g/L ZnSO4·7H2O，0.5-1.5g/L MnSO4·4H2O，0.05-0.1g/L CuSO4·5H2O，0.05-0.15g/L(NH4)6Mo7O24·4H2O，0.05-0.1g/L H3BO3，0.05-0.2g/L CoCl2·6H2And O, adding the balance of water when preparing 1L of stock solution according to the formula so that the total amount of the trace element stock solution is 1L.
According to the invention, by adjusting the formula of the strain multiplication culture medium, the OD value of the bacterial liquid can be obviously improved, a culture liquid with higher concentration can be obtained, and the concentration cost can be favorably reduced when the next step of bacterial liquid concentration is carried out.
In a preferred embodiment, the method for concentrating and collecting the bacterial liquid in step A includes: the bacterial liquid is concentrated by means of ceramic membrane, centrifugation and diatomite filtration, preferably 2-20 times.
Further, the ceramic membrane concentration mode is as follows: and (3) feeding the fermentation liquor into a ceramic membrane system by using a ceramic membrane with the aperture of 20-100 nanometers and under the pressure of a centrifugal pump or a fermentation tank, and collecting concentrated bacterial liquid. The centrifugal concentration mode is as follows: centrifuging at 4 deg.C and 5000rpm for 10min, pouring out 50-90% supernatant, and resuspending cells; if a disc centrifuge is adopted for filtration, fermentation liquor is fed into the centrifugal pump through the pressure of the centrifugal pump or the fermentation tank, the working temperature of the centrifugal pump is 10-20 ℃, and thalli are collected.
To illustrate the advancement of the technical scheme of the invention, taking a 50L fermentation tank as an example, the concentration of lactic acid is 140g/L, the conversion rate is 90%, if the fermentation liquor is concentrated by 2 times, the concentration of the carbon source can be reduced from 70.71% to 33.1%, the concentration is basically equivalent to the concentration of the dilute sugar produced by preparing the sugar from the starch, and the production requirement can be met by directly using the dilute sugar.
TABLE 1 carbon source concentration requirement for concentrated fermentation broth
Multiple of concentration
Fermentation broth/L
25% calcium hydroxide/L
Carbon source/g
Carbon source concentration/%)
1
25
14
7777.77
70.71
2
12.5
14
7777.77
33.1
5
5
14
7777.77
25.09
10
2.5
14
7777.77
23.22
In a preferred embodiment, the bacterial species activity protective agent in step a is one or more of betaine, proline, trehalose, hyaluronic acid, polyethylene glycol; preferably, the components are compounded in a synergistic manner, so that the function of strain activity protection is enhanced;
preferably, the addition ratio of the strain activity protective agent to the concentrated bacterial liquid is 0.1-10 g/kg.
In a preferred embodiment, the strain activity enhancer in step B is one or more of vitamin B6, biotin, nicotinic acid, magnesium sulfate, citric acid and its salt, and ferrous sulfate; preferably, the components are compounded in a synergistic manner, so that the activity of the strain is improved to the maximum extent;
preferably, the addition ratio of the strain activity enhancer is 0.1-10g/kg of concentrated bacterial liquid.
In another design scheme of the invention, the strain activity protective agent and the strain activity reinforcing agent are creatively introduced and are respectively added into the strain concentration and fermentation production process steps, and the important synergistic effect is achieved on ensuring that the concentrated bacterial liquid still maintains higher fermentation activity.
In a preferred embodiment, in the step B, the concentration of the carbon source for the first mixing with the concentrated bacterial liquid is 10-30%, and the mixing volume ratio of the bacterial liquid to the carbon source is 1 (1-200);
preferably, the concentration of the carbon source fed in the step B is 10-60% so as to control the content of the carbon source in the fermentation liquid to be 10-100 g/L.
Further, carbon sources with the concentration of 10-30% can be added in the step B at one time, or a few carbon sources can be added for the first time and supplemented at any time in the fermentation process, and the concentration of the carbon sources in the fermentation liquor is controlled to be stable by feeding the carbon sources.
In a preferred embodiment, step B is carried out by adding a neutralizing agent to control the pH in the fermentation broth to 5 to 10, preferably 6.5 to 7.5;
preferably, the neutralizing agent is one or more of alkaline sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt and ammonia water, such as NaOH and Na2CO3、NaHCO3、KOH、K2CO3、KHCO3、CaO、Ca(OH)2、CaCO3、Ca(HCO3)2、NH4OH、(NH4)2CO3And (NH)4)HCO3And the like, preferably one or more of sodium hydroxide, ammonia water, calcium hydroxide, and calcium carbonate.
In a preferred embodiment, in step B, the carbon source feeding is stopped when the volume of the fermentation liquid reaches 80% of the liquid loading of the fermentation tank, the carbon source content is less than 1g/L, and the fermentation is ended.
In a preferred embodiment, the lactic acid producing bacteria is lactobacillus, bacillus coagulans, pseudomonas aeruginosa, rhizopus oryzae, lactobacillus rhamnosus, lactobacillus paracasei or escherichia coli.
In a preferred embodiment, the concentrated clear liquid of the bacterial liquid obtained in the step A is used as a strain multiplication medium for recycling after being supplemented with nutrients.
The invention has the following beneficial effects:
compared with the existing lactic acid production process, the invention uses the multiplication culture medium to culture the thalli, the strains are rapidly multiplied, and almost no by-product is generated in the multiplication stage; after the fermentation liquor is concentrated into the thallus, the thallus is 1/2 to 1/20 of the original volume, and the low-concentration carbon source is added to meet the fermentation production requirement. After the fermentation liquor is concentrated and thalli are removed, the filtrate has almost no by-products and can be continuously reused for a proliferation culture medium, and the generation of wastewater is greatly reduced. After the thalli are concentrated, the using amount of lactic acid producing bacteria is increased, the acid production rate can be improved, and the fermentation period is shortened.
Detailed Description
The present invention is further illustrated by the following specific examples, which are intended to be illustrative of the invention and are not to be construed as limiting the scope of the invention.
The raw materials used in the invention are as follows:
solid LB medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride, 15g/L of agar powder and 7.4 of pH.
Liquid LB medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 7.4 of pH.
PDA slant agar culture medium: potato 200g/L, cane sugar 20g/L, agar 20g/L, natural pH.
A culture medium for fixing yeast: bran 1.5g, 1% ammonium sulphate solution 2.5ml, natural pH.
Starch sugar solution: from a Wanhua chemical corn starch sugar-making process.
OD 600: the optical density value was measured with the wavelength set at 600 nm.
[ example 1 ] production of lactic acid by fermentation of Lactobacillus
Lactic acid was produced according to the following steps:
(1) plate culture: inoculating lactobacillus in a solid LB culture medium to an activation plate, and culturing at 30 ℃ for 40 h;
(2) and (3) seed culture in a shaking flask: inoculating lactobacillus strain in plate culture into liquid LB culture medium, performing anaerobic culture at 38 deg.C for 20h, and rotating at 200rpm of shaking table;
(3) preparing a proliferation culture medium: the proliferation medium comprises 30g/L glucose, 10g/L yeast extract, 10g/L tryptone, 1g/L K2HPO4·3H2O，1g/L KH2PO4，8g/L(NH4)2SO4,0.5g/L MgSO4·7H2O, 5mL/L of a trace element stock solution is prepared according to the total volume of 1L of the culture medium, the balance is water, and the pH value is adjusted to 7.0; the microelement stock solution contains 5g/L FeSO4·7H2O，0.5g/L ZnSO4·7H2O，0.5g/L MnSO4·4H2O，0.1g/L CuSO4·5H2O，0.15g/L(NH4)6Mo7O24·4H2O，0.1g/L H3BO3，0.1g/L CoCl2·6H2O, blending according to the total volume of the trace element stock solution of 1L, and the balance being water;
(4) and (3) strain multiplication culture: inoculating the shake flask seeds into a proliferation culture medium according to the inoculation amount of 5%, carrying out anaerobic stirring fermentation at 40 ℃ and 200rpm, culturing for 11h until the OD600 reaches 40, and stopping culturing;
(5) concentrating and collecting bacterial liquid: collecting fermentation liquor, centrifuging at 4 deg.C and 8000rpm for 10min, and separating out 80% clear liquid for reuse; and (2) resuspending the concentrated bacterial liquid by using a vortex oscillator, adding 1g of strain activity protective agent into each kg of the concentrated bacterial liquid, wherein the strain activity protective agent comprises betaine: proline: hyaluronic acid 2:2: 1;
(6) and (3) lactic acid fermentation production: mixing the concentrated bacterial liquid with 20% starch sugar liquid according to the mass ratio of 1:9, and adding 1g of strain activity enhancer into each kg of the concentrated bacterial liquid, wherein the activity enhancer comprises vitamin b 6: biotin: ferrous sulfate 2:2: 1. Anaerobic fermentation is carried out at 40 ℃ and 200rpm, 20 percent of calcium hydroxide is fed-batch to control the pH of the fermentation liquor to be 7.0, and the fermentation is finished when the content of the carbon source is lower than 1 g/L. The yield of lactic acid is 143g/L, the conversion rate is 90%, and the fermentation period is 30 h.
[ example 2 ] production of lactic acid by fermentation of Bacillus coagulans
Lactic acid was produced according to the following steps:
(1) plate culture: inoculating bacillus coagulans into a solid LB culture medium to activate a flat plate, and culturing overnight at 50 ℃;
(2) and (3) seed culture in a shaking flask: inoculating the lactic acid bacteria strain subjected to plate culture into a liquid LB culture medium, carrying out anaerobic culture at 50 ℃ for 12h, and rotating the shaking table at the speed of 200 rpm;
(3) preparing a proliferation culture medium: the proliferation medium comprises 20g/L glucose, 5g/L yeast extract, 10g/L tryptone, and 0.5g/L K2HPO4·3H2O，1.1g/L KH2PO4，10g/L(NH4)2SO4,0.4g/L MgSO4·7H2O, 7mL/L microelement stock solution according to the total volume of the culture mediumBlending 1L of the mixture, and adjusting the pH value to 7.0 by using the balance of water; the microelement stock solution contains 4g/L FeSO4·7H2O，0.8g/L ZnSO4·7H2O，1.0g/L MnSO4·4H2O，0.05g/L CuSO4·5H2O，0.10g/L(NH4)6Mo7O24·4H2O，0.05g/L H3BO3，0.2g/L CoCl2·6H2O, blending according to the total volume of the trace element stock solution of 1L, and the balance being water;
(4) and (3) strain multiplication culture: inoculating the shake flask seeds into a bacterial proliferation culture medium according to the inoculation amount of 5%, controlling the dissolved oxygen to be 20-40% at 50 ℃ and 200rpm and the ventilation volume to be 1.0vvm and 0.1Mpa, carrying out aerobic fermentation, adjusting the pH value to be 7.0 by ammonia water, culturing for 8h until the OD600 reaches 60, and stopping culturing;
(5) concentrating and collecting bacterial liquid: collecting fermentation liquor and conveying the fermentation liquor to a ceramic membrane system for bacterial liquid concentration, collecting concentrated bacterial liquid and clear liquid (clear liquid is reserved and recycled) after the bacterial liquid is concentrated by 5 times, adding 1g of strain activity protective agent into each kg of concentrated bacterial liquid, wherein the strain activity protective agent is proline: hyaluronic acid: trehalose is 2:1: 1;
(6) and (3) lactic acid fermentation production: mixing the concentrated bacterial liquid with 20% starch sugar liquid according to the mass ratio of 2:8, adding 5g of strain activity enhancer per kg of strain, wherein the activity enhancer comprises vitamin b 6: biotin: nicotinic acid 2:2: 2. Anaerobic fermentation is carried out at 50 ℃ and 200rpm, 25% of calcium hydroxide is fed-batch to control the pH value to be 7.0, when the content of a carbon source is lower than 1g/L, the fermentation is finished, the yield of lactic acid is 155g/L, the conversion rate is 95%, and the fermentation period is 15 h.
[ example 3 ] production of lactic acid by fermentation of Rhizopus oryzae
Lactic acid was produced according to the following steps:
(1) preparing slant strains: inoculating rhizopus oryzae on a PDA slant agar culture medium, and culturing at 34 deg.C for 14 h;
(2) preparing a spore liquid: inoculating slant spore in a solid culture medium, culturing at 34 deg.C for 20 hr until the spore is produced in large amount, adding sterile water, and shaking to suspend the spore.
(3) Preparing a proliferation culture medium: proliferation cultureThe base formula comprises 30g/L glucose, 10g/L yeast extract, 20g/L tryptone and 1g/L K2HPO4·3H2O，0.9g/L KH2PO4，9g/L(NH4)2SO4,0.5g/L MgSO4·7H2O, 10mL/L of trace element stock solution is prepared according to the total volume of the culture medium of 1L, the balance is water, and the pH value is adjusted to 7.0; the microelement stock solution contains 4g/L FeSO4·7H2O，0.8g/L ZnSO4·7H2O，1.0g/L MnSO4·4H2O，0.05g/L CuSO4·5H2O，0.10g/L(NH4)6Mo7O24·4H2O，0.05g/L H3BO3，0.2g/L CoCl2·6H2O, blending according to the total volume of the trace element stock solution of 1L, and the balance being water;
(3) and (3) strain multiplication culture: inoculating the spore suspension into proliferation culture medium at 5% inoculum size, controlling dissolved oxygen at 34 deg.C and 200rpm and ventilation amount of 1.0vvm and 0.1Mpa, performing aerobic fermentation, adjusting pH to 6.5, culturing for 20 hr until OD600 reaches 80, and stopping culturing;
(4) concentrating and collecting rhizopus oryzae mycelia: conveying the fermentation liquor to a disc centrifuge through a centrifugal pump, wherein the working temperature is 12 ℃, collecting separated wet thalli after the thalli are concentrated by 2 times, and adding 1g of strain activity protective agent into each kg of wet thalli, wherein the strain activity protective agent comprises the following components: betaine: proline: hyaluronic acid: trehalose is 2:1:1: 1;
(5) and (3) lactic acid fermentation production: mixing wet rhizopus oryzae thallus with 45% starch sugar solution, culturing at 42 ℃, 200rpm and ventilation volume of 0.5vvm, and adding 4g of strain activity enhancer into each kg of wet thallus, wherein the strain activity enhancer comprises vitamin b 6: biotin: nicotinic acid 1:2: 2. And (3) in the fermentation process, when the concentration of glucose is lower than 20g/L, adding 40% starch sugar solution, maintaining the concentration of a carbon source in the fermentation liquor at 30g/L, adjusting the pH value of 20% calcium carbonate solution to 6.5, culturing for 25h, and ending the fermentation when the concentration of lactic acid is not increased any more. The yield of lactic acid is 154g/L, the conversion rate is 88 percent, and the fermentation period is 25 h.
[ example 4 ] production of lactic acid by fermentation of Escherichia coli
Lactic acid was produced according to the following steps:
(1) plate culture: inoculating Escherichia coli in solid LB culture medium to activate plate, and culturing at 37 deg.C for 30 hr;
(2) and (3) seed culture in a shaking flask: inoculating the escherichia coli strain subjected to plate culture into a liquid LB culture medium, culturing for 11h at 37 ℃, and rotating the shaking table at the speed of 200 rpm;
(3) preparing a proliferation culture medium: the proliferation medium comprises 30g/L glucose, 10g/L yeast extract, 10g/L tryptone, 1g/L K2HPO4·3H2O，1g/L KH2PO4，8g/L(NH4)2SO4,0.5g/L MgSO4·7H2O, 5mL/L of a trace element stock solution is prepared according to the total volume of 1L of the culture medium, the balance is water, and the pH value is adjusted to 7.0; the microelement stock solution contains 5g/L FeSO4·7H2O，0.5g/L ZnSO4·7H2O，0.5g/L MnSO4·4H2O，0.1g/L CuSO4·5H2O，0.15g/L(NH4)6Mo7O24·4H2O，0.1g/L H3BO3，0.1g/L CoCl2·6H2O, blending according to the total volume of the trace element stock solution of 1L, and the balance being water;
(4) and (3) strain multiplication culture: inoculating the shake flask seeds into a proliferation culture medium according to the inoculation amount of 5%, stirring and fermenting at 37 ℃, 200rpm, the ventilation volume of 1.0vvm and the tank pressure of 0.1Mpa for 11h until the OD600 reaches 70, and stopping culturing;
(5) and (3) strain concentration and collection: collecting fermentation liquor and conveying the fermentation liquor to a ceramic membrane system, collecting concentrated bacterial liquor after the bacterial liquor is concentrated by 10 times, and adding 5g of strain activity protective agent into each kg of concentrated bacterial liquor, wherein the strain activity protective agent comprises the following components: betaine: proline: hyaluronic acid: trehalose is 2:2:1: 1;
(6) and (3) lactic acid fermentation production: mixing the concentrated bacterial liquid with 30% starch sugar liquid, and adding 5g of strain activity enhancer per kg of concentrated bacterial liquid, wherein the strain activity enhancer comprises vitamin b 6: biotin: nicotinic acid 1:2: 2. And (3) raising the culture temperature to 42 ℃, rotating at 200rpm, beginning to supplement 30% of starch sugar solution when the glucose concentration is lower than 10g/L in the fermentation process, maintaining the carbon source concentration in the fermentation broth at 30g/L, adjusting the pH to 7 by adopting 20% magnesium hydroxide, culturing for 25h, and ending the fermentation when the lactic acid concentration is not increased any more. The yield of lactic acid is 145g/L, the conversion rate is 95 percent, and the fermentation period is 20 h.
[ example 5 ] production of lactic acid by fermentation of Escherichia coli
Lactic acid was produced according to the following steps:
(1) plate culture: inoculating Escherichia coli in solid LB culture medium to activate plate, and culturing at 37 deg.C for 30 hr;
(2) and (3) seed culture in a shaking flask: inoculating the escherichia coli strain subjected to plate culture into a liquid LB culture medium, culturing for 11h at 37 ℃, and rotating the shaking table at the speed of 200 rpm;
(3) preparing a proliferation culture medium: the proliferation medium comprises 30g/L glucose, 10g/L yeast extract, 10g/L tryptone, 1g/L K2HPO4·3H2O，1g/L KH2PO4，8g/L(NH4)2SO4,0.5g/L MgSO4·7H2O, 5mL/L of a trace element stock solution is prepared according to the total volume of 1L of the culture medium, the balance is water, and the pH value is adjusted to 7.0; the microelement stock solution contains 5g/L FeSO4·7H2O，0.5g/L ZnSO4·7H2O，0.5g/L MnSO4·4H2O，0.1g/L CuSO4·5H2O，0.15g/L(NH4)6Mo7O24·4H2O，0.1g/L H3BO3，0.1g/L CoCl2·6H2O, blending according to the total volume of the trace element stock solution of 1L, and the balance being water;
(4) and (3) strain multiplication culture: inoculating the shake flask seeds into a proliferation culture medium according to the inoculation amount of 5%, stirring and fermenting at 37 ℃, 200rpm, the ventilation volume of 1.0vvm and the tank pressure of 0.1Mpa for 11h until the OD600 reaches 70, and stopping culturing;
(5) and (3) strain concentration and collection: collecting fermentation liquor and conveying the fermentation liquor to a ceramic membrane system, collecting concentrated bacterial liquor after the bacterial liquor is concentrated by 10 times, and adding 5g of strain activity protective agent into each kg of concentrated bacterial liquor, wherein the strain activity protective agent is betaine;
(6) and (3) lactic acid fermentation production: mixing the concentrated bacterial liquid with 30% starch sugar liquid, and adding 5g of strain activity enhancer per kg of concentrated bacterial liquid, wherein the strain activity enhancer comprises vitamin b 6: biotin: nicotinic acid 1:2: 2. And (3) raising the culture temperature to 42 ℃, fermenting at the rotating speed of 200rpm, beginning to supplement 30% of starch sugar solution when the glucose concentration is lower than 10g/L in the fermentation process, maintaining the carbon source concentration in the fermentation broth at 30g/L, adjusting the pH to be 7 by adopting 20% magnesium hydroxide, and ending the fermentation when the lactic acid concentration is not increased any more. The yield of lactic acid is 130g/L, the conversion rate is 90 percent, and the fermentation period is 23 h.
[ example 6 ] production of lactic acid by fermentation of Escherichia coli
Lactic acid was produced according to the following steps:
(1) plate culture: inoculating Escherichia coli in solid LB culture medium to activate plate, and culturing at 37 deg.C for 30 hr;
(2) and (3) seed culture in a shaking flask: inoculating the escherichia coli strain subjected to plate culture into a liquid LB culture medium, culturing for 11h at 37 ℃, and rotating the shaking table at the speed of 200 rpm;
(3) preparing a proliferation culture medium: the proliferation medium comprises 30g/L glucose, 10g/L yeast extract, 10g/L tryptone, 1g/L K2HPO4·3H2O，1g/L KH2PO4，8g/L(NH4)2SO4,0.5g/L MgSO4·7H2O, 5mL/L of a trace element stock solution is prepared according to the total volume of 1L of the culture medium, the balance is water, and the pH value is adjusted to 7.0; the microelement stock solution contains 5g/L FeSO4·7H2O，0.5g/L ZnSO4·7H2O，0.5g/L MnSO4·4H2O，0.1g/L CuSO4·5H2O，0.15g/L(NH4)6Mo7O24·4H2O，0.1g/L H3BO3，0.1g/L CoCl2·6H2O, blending according to the total volume of the trace element stock solution of 1L, and the balance being water;
(4) and (3) strain multiplication culture: inoculating the shake flask seeds into a proliferation culture medium according to the inoculation amount of 5%, stirring and fermenting at 37 ℃, 200rpm, the ventilation volume of 1.0vvm and the tank pressure of 0.1Mpa for 11h until the OD600 reaches 70, and stopping culturing;
(5) and (3) strain concentration and collection: collecting fermentation liquor and conveying the fermentation liquor to a ceramic membrane system, collecting concentrated bacterial liquor after the bacterial liquor is concentrated by 10 times, and adding 5g of strain activity protective agent into each kg of concentrated bacterial liquor, wherein the strain activity protective agent comprises the following components: betaine: proline: hyaluronic acid: trehalose is 2:2:1: 1;
(6) and (3) lactic acid fermentation production: mixing the concentrated bacterial liquid with 30% starch sugar liquid, and adding 5g of strain activity enhancer per kg of concentrated bacterial liquid, wherein the strain activity enhancer is nicotinic acid. And (3) raising the culture temperature to 42 ℃, fermenting at the rotating speed of 200rpm, beginning to supplement 30% starch sugar solution when the glucose concentration is lower than 10g/L in the fermentation process, maintaining the carbon source concentration in the fermentation broth at 30g/L, adjusting the pH to be 7 by adopting 20% magnesium hydroxide, and ending the fermentation when the lactic acid concentration is not increased any more. The yield of lactic acid is 130g/L, the conversion rate is 92 percent, and the fermentation period is 23 h.
[ COMPARATIVE EXAMPLE 1 ] production of lactic acid by fermentation of Escherichia coli
Lactic acid was produced according to the following steps:
(1) plate culture: inoculating Escherichia coli in solid LB culture medium to activate plate, and culturing at 37 deg.C for 30 hr;
(2) and (3) seed culture in a shaking flask: inoculating the escherichia coli strain subjected to plate culture into a liquid LB culture medium, culturing for 11h at 37 ℃, and rotating the shaking table at the speed of 200 rpm;
(3) preparing a fermentation medium: the proliferation medium formula is 30g/L glucose, 1g/L K2HPO4·3H2O，1g/L KH2PO4，8g/L(NH4)2SO4,0.5g/L MgSO4·7H2O, 0.2g/L NaCl and 5mL/L of trace element stock solution are mixed according to the total volume of the culture medium of 1L, the balance is water, and the pH value is adjusted to 7.0; the microelement stock solution contains 5g/L FeSO4·7H2O，0.5g/L ZnSO4·7H2O，0.5g/L MnSO4·4H2O，0.1g/L CuSO4·5H2O，0.15g/L(NH4)6Mo7O24·4H2O，0.1g/L H3BO3，0.1g/L CoCl2·6H2O, blending according to the total volume of the trace element stock solution of 1L, and the balance being water;
(4) lactic acid fermentation aerobic proliferation: inoculating the shake flask seeds into a proliferation culture medium according to the inoculation amount of 5%, stirring and fermenting at 37 ℃ and 200rpm under the condition that the aeration quantity is 1.0vvm and the tank pressure is 0.1Mpa, adjusting the pH value to 7.0 by ammonia water, culturing for 7h until the OD600 reaches 30, and stopping culturing;
(6) and (3) lactic acid fermentation production: after the aerobic culture of the lactic acid producing bacteria is finished, stopping ventilation, raising the culture temperature to 42 ℃, rotating at the speed of 200rpm, adding 60% starch sugar solution in batches, adjusting the pH to 7.0 by using 20% calcium hydroxide when the sugar concentration in the fermentation broth reaches 50g/L, adding the starch sugar solution again when the glucose concentration is reduced to 5g/L, and ending the fermentation when the liquid filling amount of the fermentation tank reaches 80%, the sugar solution is not added any more, and the lactic acid concentration is not increased any more. The yield of lactic acid is 135g/L, the conversion rate is 95 percent, and the fermentation period is 30 h.
Comparative example 2 production of lactic acid by fermentation of Bacillus coagulans
Lactic acid was produced according to the following steps:
(1) plate culture: inoculating bacillus coagulans into a solid LB culture medium to activate a flat plate, and culturing overnight at 50 ℃;
(2) and (3) seed culture in a shaking flask: inoculating the lactic acid bacteria strain subjected to plate culture into a liquid LB culture medium, carrying out anaerobic culture at 50 ℃ for 12h, and rotating the shaking table at the speed of 200 rpm;
(3) preparing a fermentation medium: the proliferation medium comprises 20g/L glucose, 1g/L yeast extract, 2g/L tryptone, and 0.5g/L K2HPO4·3H2O，5g/L CH3COONa，50g/L NaCl，10g/L(NH4)2SO4,0.4g/L MgSO4·7H2O, 7mL/L of a microelement stock solution, blending according to the total volume of the culture medium of 1L, and adjusting the pH value to 7.0 by using the balance of water; the microelement stock solution contains 4g/L FeSO4·7H2O，0.8g/L ZnSO4·7H2O，1.0g/L MnSO4·4H2O，0.05g/L CuSO4·5H2O，0.10g/L(NH4)6Mo7O24·4H2O，0.05g/L H3BO3，0.2g/L CoCl2·6H2O, blending according to the total volume of the trace element stock solution of 1L, and the balance being water;
(4) lactic acid fermentation aerobic proliferation: inoculating the shake flask seeds into a proliferation culture medium according to the inoculation amount of 5%, stirring and fermenting at 50 ℃, 200rpm, the ventilation volume of 1.0vvm and the tank pressure of 0.1Mpa, controlling the dissolved oxygen to be 20-40%, adjusting the pH value of ammonia water to be 7.0, culturing for 8h until the OD600 reaches 30, and stopping culturing;
(5) and (3) lactic acid fermentation production: after the aerobic culture of the lactic acid producing bacteria is finished, stopping ventilation, raising the culture temperature to 50 ℃, carrying out anaerobic fermentation under the condition of 200rpm, adding 60% starch sugar solution (the source is the same as that of comparative example 1) in batches, adjusting the pH to 7.0 by using 20% calcium hydroxide, adding the starch sugar solution again when the glucose concentration is reduced to 5g/L, and stopping adding the sugar solution when the liquid filling amount of the fermentation tank reaches 80%, and finishing the fermentation when the lactic acid concentration is not increased any more. The yield of lactic acid is 135g/l, the conversion rate is 95 percent, and the fermentation period is 35 h.
Comparative example 3
Lactic acid was produced by fermentation of E.coli using substantially the same conditions as in example 4 except that no strain activity protector was added at the time of strain concentration collection in step 5. The yield of lactic acid is 135g/L, the conversion rate is 90 percent, and the fermentation period is 25 hours.
Comparative example 4
Lactic acid was produced by fermentation of E.coli using substantially the same conditions as in example 4 except that no strain activity enhancer was added during the lactic acid fermentation production in step 6. The yield of lactic acid is 135g/L, the conversion rate is 90 percent, and the fermentation period is 25 hours.
Comparative example 5
Lactic acid was produced by fermentation of E.coli under substantially the same conditions as in example 4, except that no strain activity protector was added during the concentration and collection of the strain in step 5, and no strain activity enhancer was added during the fermentation production of lactic acid in step 6. The yield of lactic acid is 125g/L, the conversion rate is 85 percent, and the fermentation period is 33 h.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.
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