CN105018538B - A method of magnesium lactate is produced based on crystallisation Integrated process - Google Patents

Abstract

The invention discloses a kind of methods producing Pfansteihl magnesium based on crystallisation Integrated process.The present invention cultivates 180-220h in a manner of Batch fermentation repeatedly rhamnose lactic acid bacteria at 37-42 DEG C, passes through the lactic acid magnesium crystal in situ for detaching acquisition high-purity.Fermentation lactic acid yield is 140-150g/L, and throughput rate 2.0-2.5g/L/h, saccharic acid conversion ratio is up to 94.5%.With crystallization mode in the separation process in situ of magnesium lactate, lactic acid removal rate is up to 79.10%, and fermentation waste water reclamation rate is up to 65.57%.The more traditional feed supplement of the present invention-Batch fermentation process can save water consumption 40%, save inorganic salts dosage 41%, save yeast extract dosage 43%, and in 5 stable fermentation cycles, Pfansteihl total output reaches 3.82 times of single batch fermentation.The drawbacks of a large amount of solid waste and waste water are generated in calcium salt method production process is avoided with this technique fermenting lactic acid, and without plus seed and cooling, reheating process, simplify production stage, the production time is shortened, is ensureing that technical process is environmentally friendly, sustainable while reducing production of lactic acid cost.

Description

A method for producing magnesium lactate based on crystallization fermentation separation coupling

technical field

The invention belongs to the technical field of lactic acid fermentation, and relates to an improved method for producing magnesium lactate based on a crystallization method, fermentation, separation and coupling.

Background technique

As a natural organic acid, lactic acid is widely used in food, medicine, textile, leather and chemical industries. Lactic acid can be produced by chemical synthesis or microbial fermentation, and most industrial methods for preparing lactic acid are based on microbial fermentation. Base. It is a common feature of all fermentation methods to add alkaline earth alkali neutralizer to neutralize the acid secreted by microorganisms to maintain a constant pH during fermentation. Calcium base is the most commonly used as a conventional neutralizing agent. WO 98/22611 describes a method for fermenting lactic acid with calcium base as a neutralizing agent. After the medium containing calcium lactate is obtained by fermentation, ammonium carbonate is added to the medium to convert the calcium lactate into It is ammonium lactate, and finally lactic acid is obtained by electrodialysis and separation of salt splitting. If the target product is other lactate, after the calcium lactate medium is obtained by the conventional method, lactic acid and calcium sulfate precipitation are obtained by acidification with sulfuric acid, and after solid-liquid separation, the lactic acid is reacted with the corresponding alkaline earth base to prepare the specific lactate. CN 103952331 A describes the technology of in-situ separation of calcium lactate in the semi-continuous fermentation process. In view of the high solubility of calcium lactate at fermentation temperature, crystallization precipitation needs to cooperate with cooling and external seed crystals, which undoubtedly makes the production process cumbersome and time-consuming . At the same time, the pollution of the solid waste calcium sulfate produced in the subsequent separation process to the environment is a disadvantage that is difficult to overcome for the production of lactic acid by calcium salt fermentation.

Avoiding the production of solid waste calcium sulfate, realizing the repeated reuse of neutralizers or producing other valuable by-product salts are the hotspots in the research on the preparation of lactic acid by fermentation. Substituting magnesium base for calcium base as fermentation neutralizer makes the above ideas possible. At the same time, as an intermediate product in the production process of lactic acid, magnesium lactate can be used as an excellent and economical magnesium organic fortifier, widely used in food, beverages, dairy products, flour, nutrient solution and pharmaceuticals, etc., to supplement magnesium , to prevent various magnesium deficiency diseases, and enhance vitality has a significant effect.

In the prior art, several documents describe the production of lactic acid or lactate based on magnesium bases:

CN 101886094 A describes that after using _MgCO3 as a neutralizing agent to ferment and generate magnesium lactate, it reacts with NaOH at a pH of 9.5-10.8 to generate L-sodium lactate and Mg(OH) ₂ , and L-sodium lactate undergoes decolorization, nanofiltration and The L-sodium lactate product can be obtained after vacuum distillation, purification and concentration.

CN 101018756 A describes a method of reacting a medium containing magnesium lactate with sodium, calcium and/or ammonium hydroxide to form sodium, calcium and/or ammonia lactate and magnesium hydroxide.

In the method published in US 1,459,395, the method for purifying lactic acid from industrial grade lactic acid is described. Magnesium oxide, magnesium carbonate or magnesium hydroxide are added to crude lactic acid, and the magnesium lactate generated is acidified with concentrated sulfuric acid in a suitable solvent. Sulfuric acid The magnesium salt can be removed by filtration, and the obtained solution containing lactic acid can be distilled to remove the solvent to obtain purified lactic acid.

In the method described in GB 173,479, after the magnesium lactate solution is acidified with sulfuric acid, lactic acid is recovered by means of acetone extraction. It is also possible to suspend the magnesium lactate salt solution in acetone, realize the acidification and extraction of lactic acid at the same time, and recover the lactic acid after removing the magnesium sulfate precipitate.

In the method for producing lactic acid by sugar fermentation described in WO 00/17378, magnesium hydroxide is used as a neutralizing agent to control the fermentation pH at 5.5-6.5. After the obtained medium containing magnesium lactate is converted into lactic acid and magnesium chloride by acidification with hydrochloric acid, the lactic acid is extracted with organic solvent isoamyl alcohol, amine or ether. The obtained magnesium chloride can be decomposed into hydrochloric acid and magnesium oxide when heated.

US 3,429,777 mentions the possibility of adding soda ash, caustic soda or sodium phosphate to a magnesium lactate solution to produce sodium lactate and magnesium carbonate or hydroxide.

WO 98/37050 describes a process for the production of lactic acid from a fermentation medium containing an alkaline earth metal salt of lactic acid, the so-called SWAP reaction, by reacting an alkaline earth metal salt of lactic acid with an alkali metal base to form an alkali metal salt of lactic acid and an alkaline earth metal base, lactic acid Alkali metal salts can be further acidified and extracted to generate lactic acid and alkali metal salts. In this way, the alkaline earth metal base can be reused in the fermentation process, and the alkali metal salt by-product can be generated at the same time.

The above literature simply verified the possibility of magnesium base as a fermentation neutralizer, or conducted research on how to realize the reuse of magnesium base or generate valuable salt products in the process of producing lactic acid from magnesium lactate. However, the improvement and optimization of the magnesium lactate fermentation method has not been mentioned in the above documents. The conventional batch fermentation mode has a low product concentration, and the necessary processes such as seed cultivation, fermentation liquid discharge, and tank cleaning prolong the production cycle and reduce the fermentation rate. The utilization rate of the tank increases the production cost. In addition, the massive discharge of fermentation wastewater during the production process will cause serious environmental pollution.

The fermentative production of magnesium lactate based on a magnesium base is also described by:

NL-A-288829 describes the possibility of continuous removal of magnesium lactate or zinc lactate salts by means of filtration under fermentation conditions maintaining a constant concentration of fermentable sugars and 45-50°C. According to this method (clearly referring to the maintenance of a constant sugar concentration during the continuous removal of magnesium lactate), the removal of magnesium lactate while inevitably entraining fermentative sugars by adsorption leads to a decrease in sugar-acid conversion. At the same time, since there is no "pH correction step" in the magnesium lactate removal process, but it is carried out at a fermentation pH of 5-6, this will affect the crystallization and precipitation of magnesium lactate and reduce the product removal rate. In addition, how to implement this process and under what conditions are not provided in detail. For example, there is an optimal value for the recycling rate of the fermentation broth. deterioration. With the addition of feed medium and neutralizer, the reuse of all the fermentation broth will increase the volume of the feed solution, which is not conducive to the balance of the reaction system, and will also reduce the removal rate of lactic acid. How to make the fermentation reaction system including multiple product on-line removal and feeding cycle to reach a steady state equilibrium is not mentioned in the literature.

Therefore, the present invention provides an improved on-line magnesium lactate removal method coupled with fermentation, which does not require additional seed crystals and cooling and reheating processes, thereby simplifying production steps and shortening production time. The high-efficiency reuse of fermentation wastewater containing bacteria is realized, and the consumption of fermentation water is reduced by 40%. At the same time, 43% of yeast extract powder consumption and 41% of inorganic salt consumption are saved, thereby reducing production cost.

The purpose of the present invention is to provide an improved method based on crystallization, fermentation, separation and coupled production of magnesium lactate.

Another object of the present invention is to provide an improved method capable of realizing efficient reuse of fermentation wastewater and efficient removal of magnesium lactate during the preparation of magnesium lactate.

Another object of the present invention is to provide an efficient and balanced magnesium lactate fermentation separation and coupling method.

Other objects of the present invention will become apparent as the subsequent description proceeds.

Contents of the invention

Aiming at the deficiencies of the above-mentioned prior art, the present invention provides a method for producing L-lactic acid through fermentation, separation and coupling. In this method, Lactobacillus rhamnosus is used as acid-producing bacteria, and MgO is used as a neutralizing agent to carry out repeated batch fermentation including online removal of multiple products and feeding cycle. The fermented lactic acid yield is 140-150g/L, and the production rate It is 2.0-2.5g/L/h, and the sugar-acid conversion rate can reach 94.5%. During the in-situ separation of magnesium lactate by crystallization, the removal rate of lactic acid can reach 79.10%, and the reuse rate of fermentation wastewater can reach 65.57%. The invention can save 40% of water consumption, 41% of inorganic salt consumption, and 43% of yeast extract powder consumption, and in five stable fermentation cycles, the total output of L-lactic acid reaches 3.82 times of single batch fermentation. The fermentation and production of lactic acid by this process avoids the disadvantages of producing a large amount of solid waste and waste water in the production process of the calcium salt method, and does not require additional seed crystals and cooling and reheating processes, which simplifies the production steps and shortens the production time. It is environmentally friendly and sustainable while reducing the production cost of lactic acid.

In the production method coupled with lactic acid fermentation and separation provided by the present invention, the basic principle of lactic acid removal is crystallization of magnesium lactate and solid-liquid separation after crystallization. It is characterized in that, comprising the following steps:

(1) Incline culture: Lactobacillus rhamnosusCGMCC 1.2134 used in the practice of the present invention was purchased from China General Microorganism Culture Collection and Management Center.

The slant culture method is as follows: inoculate Lactobacillus rhamnosus CGMCC 1.2134 on a solid slant medium containing 15g/L agar by streaking, and cultivate at 37-42°C for 24-48h; preferably, at 40-42°C Under culture for 24-36h.

(2) Seed culture: Transfer the bacteria to the seed medium, cultivate at 37-42°C, 120-220rpm for 24-36h, then re-insert the primary seed liquid into the fresh seed medium according to the inoculation amount of 10% , cultivated at 37-42°C, 120-220rpm for 8-15h. Preferably, the primary seed liquid is cultivated at 40-42° C. and 150-180 rpm for 24-26 hours, and the secondary seed liquid is cultivated at 40-42° C. and 150-180 rpm for 10-12 hours. Add 15g/L calcium carbonate as a neutralizer to the liquid seed medium to maintain a constant pH.

(3) Fermentation culture: the secondary seed liquid obtained in the seed culture stage is inserted into the fermentation medium according to the inoculum amount of 3-10% (v/v), at a stirring speed of 180-240rpm, at a fermentation temperature of 37-42°C, flow Add MgO to control pH 5.5-6.5.

(4) In-situ product separation: The repeated batch fermentation process includes multiple online magnesium lactate removal and feeding cycles. When the fermented lactic acid concentration reaches 140-160g/L, and the glucose is exhausted, the lactic acid fermentation is suspended and MgO is added. , so that the pH of the fermentation broth in the fermenter rises and stabilizes to 6.5-7.5, preparing for the next step of crystallization and sedimentation. Pump the above supersaturated magnesium lactate fermentation liquid into the crystallization tank, realize solid-liquid separation by crystallization and precipitation at the fermentation temperature, pump the supernatant containing bacteria back to the fermentation tank for reuse, and obtain high-concentration magnesium lactate crystals at the same time .

Further, during the fermentation process, MgO is fed in to adjust the pH at 6.25±0.05, and the MgO concentration is 10wt%-20wt%. Preferably, the MgO concentration is 15 wt%.

Further, during the fed-batch fermentation process, when the glucose is exhausted, the feeding medium is added until the glucose content is 60-80 g/L. The final concentration of lactic acid after fed-batch fermentation is 110-160g/L.

Further, the initial glucose content of the fermentation medium is 100-160g/L, and the yeast extract powder content is 10-30g/L.

Further, the glucose content of the feed medium is 300-500g/L, and the yeast extract powder content is 10-30g/L.

Further, the term "repeated batch fermentation" in the present invention refers to repeated cycles of a plurality of "fed-batch fermentation" processes. From the second fed-batch fermentation, the medium for feeding is added Until the glucose content is 60-80g/L, the preferred content is 70g/L, and when the glucose is exhausted for the first time, the feeding medium is added to maintain the glucose lower than 30g/L. The preferred concentration of lactic acid after each fermentation cycle is 140g/L.

Further, after the end of each fermentation cycle, MgO is added to increase and stabilize the pH to 6.5-7.5, preferably 7.0. The MgO concentration is 10wt%-20wt%. Preferably, the MgO concentration is 15 wt%.

Further, 80-85% of the total volume of the fermentation broth is pumped into the crystallization tank, the magnesium lactate crystallization temperature is 42°C, and the crystallization time is 120min. The reuse rate of fermentation wastewater is 60-70%, of which the wastewater reuse rate and lactic acid removal rate are calculated as follows:

Among them, the determination method of L-lactic acid and glucose in the fermentation broth is that the fermentation broth is diluted to the required concentration range with 0.006mol/L hydrochloric acid, filtered through a 0.22μm microporous membrane, and then biosensing analyzer SBA- 40C (Shandong Academy of Sciences) determination. The glucose content in the culture medium was diluted with deionized water to the required concentration range and measured by the above-mentioned equipment. Under the requirement of high-precision measurement, L-lactic acid in the fermentation broth was determined by high-performance liquid chromatography (HPLC). The detection used Bio-RadAminex HPX-87H chromatographic column (300×7.8mm), and the mobile phase was 5mM H ₂ SO _4. The flow rate is 0.5ml/min, the detection wavelength and column temperature are 205nm and 55°C, respectively.

Description of drawings

Fig. 1 is based on the process diagram of magnesium lactate crystallization fermentation separation coupling production L-lactic acid;

Fig. 2 is the fed-batch fermentation of the 5L tank level of neutralizing agent with MgO;

Figure 3 5L tank level fermentation separation coupling process based on magnesium lactate crystallization;

The comparison of Fig. 4 fed-batch fermentation and repeated batch fermentation in L-lactic acid production capacity;

Table 1 Changes of lactic acid concentration in the supernatant after crystallization of magnesium lactate in the fermentation broth with temperature (initial lactic acid concentration 140g/L, crystallization time 120min)

Table 2 Changes of lactic acid concentration in crystallization supernatant with different initial lactic acid concentrations (crystallization time 120 min, crystallization temperature 42°C)

Table 3 Changes of lactic acid concentration in crystallization supernatant with different crystallization time (crystallization temperature 42°C, initial lactic acid concentration 140g/L)

Table 4 Separation coupling process of magnesium lactate fermentation at 5L tank level

Table 5 Comparison of fermentation separation coupling and fed-batch fermentation for lactic acid production

Detailed ways

The present invention will be described in detail below in conjunction with the embodiments and drawings, which are only for illustration and not limiting the scope of application of the present invention. The materials in each embodiment are analytical test supplies.

Example 1

The medium used in this embodiment is as follows:

The composition of solid slant medium is (g/L): glucose 20, yeast extract 5, soybean peptone 10, beef extract 10, sodium chloride 10, sodium acetate 5, ammonium citrate 2, magnesium sulfate heptahydrate 0.2, sulfuric acid heptahydrate Manganese 0.05, agar 15. The pH is 6.50.

The composition of the liquid seed medium is (g/L): glucose 40, peptone 10, yeast extract powder 10, sodium chloride 0.01, sodium acetate 0.5, ammonium citrate 0.2, potassium dihydrogen phosphate 0.4, magnesium sulfate heptahydrate 0.2, Manganese sulfate heptahydrate 0.05. The pH is 6.50.

The composition of the fermentation medium is (g/L): glucose 150, yeast extract powder 20, sodium chloride 0.01, sodium acetate 0.5, ammonium citrate 0.2, potassium dihydrogen phosphate 0.4, magnesium sulfate heptahydrate 0.2, manganese sulfate heptahydrate 0.05. The pH is 6.00.

The composition of the feeding medium is (g/L): glucose 420, yeast extract powder 20, sodium chloride 0.01, sodium acetate 0.5, ammonium citrate 0.2, potassium dihydrogen phosphate 0.4, magnesium sulfate heptahydrate 0.2, heptahydrate Manganese sulfate 0.05. The pH is 6.00.

In the present invention, with MgO as neutralizing agent, the step of feeding batch fermentation to produce L-magnesium lactate is as follows:

(1) Slant culture: Lactobacillus rhamnosus CGMCC 1.2134 was inoculated on a solid slant medium, and cultured at 42° C. for 24 hours.

(2) Seed culture: Transfer the strains obtained in step (1) to 100ml seed medium under aseptic conditions, cultivate the primary seed solution at 42°C and 180rpm for 24 hours, and then add 20ml of the primary seed solution Transfer to 180ml of fresh seed medium under sterile conditions, and cultivate the secondary seed solution at 42°C and 180rpm for 12h. 15 g/L of calcium carbonate was added to the seed medium as a neutralizing agent to maintain a constant pH.

(3) Fermentation culture: the secondary seed liquid obtained in step (2) is inserted into 2L of fermentation medium according to 10% (v/v) inoculation amount, and at a stirring speed of 200rpm, at a fermentation temperature of 42°C, feed the mass percentage concentration Control pH 6.25 ± 0.05 for 15% MgO. When the glucose was exhausted for the first time, the feed medium was added to increase the glucose concentration to 80g/L, and the fermentation ended when the glucose was exhausted again.

As shown in Figure 2, feed-batch fermentation produces L-magnesium lactate in a 5L fermenter, the fermentation time is 65h, the lactic acid output in the lower tank is 144g/L, the conversion rate of sugar and acid is 96.1%, and the production rate of L-lactic acid is 2.15g/L/h.

Example 2

1. Effect of crystallization temperature on crystallization of L-magnesium lactate in fermentation broth

Obtain the fermented liquid that L-lactic acid content is 140g/L with feeding-batch fermentation mode, fermented liquid is divided into 250ml Erlenmeyer flask by every bottle 100ml, Erlenmeyer flask is placed in different temperatures (temperature gradient is 25-42°C) for 120 minutes, and measure the change of lactic acid content in the supernatant after crystal precipitation at different temperatures, and set up 3 parallel experiments under each temperature condition.

As shown in Table 1, the concentration of lactic acid in the crystallization supernatant increases with the increase of temperature, and the fitting equation (R ² =0.996) obtained by crystallization of magnesium lactate is:

C=78.03-0.542T+0.0113T ² ;

Where C represents the concentration of lactic acid in the supernatant (g/L), and T represents the crystallization temperature (° C.). As the temperature decreased, the lower the concentration of lactic acid in the supernatant, the higher the removal efficiency of lactic acid by crystallization. However, it is found by comparison that only 4.90% more magnesium lactate crystals are obtained at 25°C than at 42°C, and the crystallization is carried out at the fermentation temperature (42°C), which will not greatly reduce the lactic acid removal rate, and at the same time due to saving cooling, re- The heating process can make the process more energy-saving and time-saving. For this reason, 42°C is selected as the crystallization temperature in this process.

2. Effect of product concentration on crystallization of L-magnesium lactate in fermentation broth

Obtain the magnesium lactate fermented liquid of different L-lactic acid content (110-160g/L) with feeding-batch fermentation mode, the fermented liquid under specific concentration is packed in the 250ml Erlenmeyer flask by 100ml of every bottle, conical The bottle was placed at 42°C for 120 minutes, and the change of lactic acid content in the supernatant after crystal precipitation was measured under different product concentrations. Three parallel experiments were set up under each concentration condition.

The degree of supersaturation related to the product concentration is another important factor affecting crystallization besides the crystallization time. During the fed-batch fermentation process of magnesium lactate, when the lactic acid concentration reaches 110g/L, a small amount of magnesium lactate appears in the fermentation broth crystals, these small amounts of crystals can be used as seeds during the crystallization process, thereby avoiding the need for external seeds. As shown in Table 2, as the concentration of lactic acid in the fermentation broth increased, the amount of seed crystals formed during the fermentation process gradually increased, which promoted the formation of magnesium lactate crystals, thereby gradually reducing the concentration of lactic acid in the supernatant. However, when the lactic acid concentration is greater than 150g/L, the acid production rate of the bacteria decreases significantly. Therefore, in this process, 140g/L lactic acid is selected as the initial concentration of crystallization.

3. Effect of crystallization time on crystallization of L-magnesium lactate in fermentation broth

Obtain the magnesium lactate fermented liquid of L-lactic acid content 140g/L with feeding-batch fermentation mode, divide into 250ml Erlenmeyer flask by every bottle 100ml, place Erlenmeyer flask at 42 ℃, under different time (0- 300min) to stand still, and measure the change of lactic acid content in the supernatant after crystal precipitation under different standing times, and set 3 parallel experiments under each time condition.

As shown in Table 3, under the conditions of initial lactic acid concentration of 140g/L and crystallization temperature of 42°C, the lactic acid content in the supernatant dropped rapidly by 46.4% within 120 minutes, and then the lactic acid content in the supernatant decreased as the crystallization time prolongs The trend gradually slowed down, reflecting the reduced rate of formation of magnesium lactate crystals. Therefore, choose 120min to be the crystallization time of magnesium lactate in this technology.

Example 3

The medium used in this embodiment is as follows:

The composition of solid slant medium is (g/L): glucose 20, yeast extract 5, soybean peptone 10, beef extract 10, sodium chloride 10, sodium acetate 5, ammonium citrate 2, magnesium sulfate heptahydrate 0.2, sulfuric acid heptahydrate Manganese 0.05, agar 15. The pH is 6.50.

The composition of the liquid seed medium is (g/L): glucose 40, peptone 10, yeast extract powder 10, sodium chloride 0.01, sodium acetate 0.5, ammonium citrate 0.2, potassium dihydrogen phosphate 0.4, magnesium sulfate heptahydrate 0.2, Manganese sulfate heptahydrate 0.05. The pH is 6.50.

The composition of the fermentation medium is (g/L): glucose 150, yeast extract powder 20, sodium chloride 0.01, sodium acetate 0.5, ammonium citrate 0.2, potassium dihydrogen phosphate 0.4, magnesium sulfate heptahydrate 0.2, manganese sulfate heptahydrate 0.05. The pH is 6.00.

The composition of the feeding medium is (g/L): glucose 420, yeast extract powder 20, sodium chloride 0.01, sodium acetate 0.5, ammonium citrate 0.2, potassium dihydrogen phosphate 0.4, magnesium sulfate heptahydrate 0.2, heptahydrate Manganese sulfate 0.05. The pH is 6.00.

In the present invention, use MgO as neutralizing agent, adopt the step of fermentation separation coupling mode to produce L-magnesium lactate as follows:

(1) Slant culture: Lactobacillus rhamnosus CGMCC 1.2134 was inoculated on a solid slant medium, and cultured at 42° C. for 24 hours.

(2) Seed culture: Transfer the strains obtained in step (1) to 100ml seed medium under aseptic conditions, cultivate the primary seed solution at 42°C and 180rpm for 24 hours, and then add 20ml of the primary seed solution Transfer to 180ml of fresh seed medium under sterile conditions, and cultivate the secondary seed solution at 42°C and 180rpm for 12h. 15 g/L of calcium carbonate was added to the seed medium as a neutralizing agent to maintain a constant pH.

(3) Fermentation culture: the secondary seed liquid of step (2) gained is inserted into 2L fermentation medium according to 10% (v/v) inoculum size, at stirring speed 200rpm, under fermentation temperature 42 ℃, feed 15%MgO Control pH 6.25 ± 0.05. The repeated batch fermentation process includes multiple fermentation cycles and magnesium lactate online removal process. In a single fermentation cycle, when the glucose is depleted for the first time, the feed medium is added to keep the glucose concentration below 30g/L, as much as possible reduce the glucose suppression effect. When the lactic acid concentration reached 140±5g/L and the glucose was exhausted, the fermentation process was suspended. Add 15% MgO to increase the pH of the fermentation broth in the fermenter and stabilize it to 7.0, preparing for the next step of crystallization.

(4) Product separation: pump 80-85% of the volume of the above supersaturated magnesium lactate fermentation liquid into the crystallization tank, crystallize and precipitate at 42°C for 120 minutes, pump the supernatant back to the fermentation tank for reuse, and control the reuse rate of fermentation wastewater At 60-70%, a high concentration of magnesium lactate crystals is simultaneously obtained. After the crystallization supernatant was transported back to the fermenter, the feed medium was added to the fermenter to increase the glucose concentration to 70g/L and then enter the next round of fermentation acid production and online removal cycle. A total of four in situ magnesium lactate removals were performed.

Among them, the output of L-lactic acid after the first fed-batch fermentation was 144g/L, the conversion rate of sugar and acid was 96.1%, and the space-time yield was 2.15g/L ^/ h. After the pH of the feed solution in the fermenter was adjusted and stabilized to 7.0, 2.5 L of the feed liquid was pumped into the crystallization tank (heated at 42° C.), and 0.5 L of the feed liquid remained in the fermenter. After the fermentation liquid was crystallized and precipitated in the crystallization tank for 120 minutes, 1.5L supernatant was pumped back to the fermentation tank. The reuse rate of the fermentation wastewater was 64.52%, and the removal rate of lactic acid was 76.67%. After adding 0.71 L of feed medium, enter the second feed-batch fermentation process.

After the second fed-batch fermentation, the L-lactic acid output was 142g/L, the sugar-acid conversion rate was 98.16%, and the space-time yield was 2.18g/L ^/ h. After the pH of the feed liquid in the fermenter was adjusted and stabilized to 7.0, 2.6L of feed liquid was pumped into the crystallization tank (heated at 42°C), and 0.5L of feed liquid remained in the fermenter. After the fermentation liquid was crystallized and precipitated in the crystallization tank for 120 minutes, 1.5L supernatant was pumped back to the fermentation tank. The reuse rate of the fermentation wastewater was 65.57%, and the removal rate of lactic acid was 77.80%. After adding 0.70 L of feed medium, enter the third feed-batch fermentation process.

After the third fed-batch fermentation, the L-lactic acid output was 143g/L, the sugar-acid conversion rate was 96.31%, and the space-time yield was 2.17g/L ^/ h. After the pH of the feed liquid in the fermenter was adjusted and stabilized to 7.0, 2.55 L of feed liquid was pumped into the crystallization tank (heated at 42° C.), and 0.5 L of feed liquid remained in the fermenter. After the fermentation liquid was crystallized and precipitated in the crystallization tank for 120 minutes, 1.5L supernatant was pumped back to the fermentation tank. The reuse rate of the fermentation wastewater was 63.49%, and the removal rate of lactic acid was 77.10%. After adding 0.78 L of feed medium, enter the fourth feed-batch fermentation process.

After the fourth fed-batch fermentation, the L-lactic acid output was 148g/L, the sugar-acid conversion rate was 95.05%, and the space-time yield was 2.41g/L ^/ h. After the pH of the feed solution in the fermenter was adjusted and stabilized to 7.0, 2.65 L of the feed solution was pumped into the crystallization tank (heated at 42° C.), and 0.5 L of the feed solution remained in the fermenter. After the fermentation broth was crystallized and precipitated in the crystallization tank for 120 minutes, 1.5L of the supernatant was pumped back to the fermentation tank. The reuse rate of the fermentation wastewater was 62.50%, and the removal rate of lactic acid was 79.10%. After adding 0.80 L of feed medium, enter the fifth feed-batch fermentation process.

After the fifth fed-batch fermentation, the output of L-lactic acid was 150g/L, the conversion rate of sugar and acid was 96.43%, and the space-time yield was 1.85g/L ^/ h. In five batches of repeated batches of fermentation (Table 4) including four product in-situ removal processes, a total of 1643.07 g of L-lactic acid was produced, and the total sugar-acid conversion rate was 94.5%.

As shown in table 5, find after feeding-batch fermentation and fermentation separation coupling process contrast, adopt the method provided by the present invention to produce magnesium lactate, in product yield, space-time yield and the lower tank concentration of each circulation and Under the similar premise of fed-batch fermentation, water consumption can be saved by 40%, inorganic salt consumption can be saved by 41%, yeast extract powder consumption can be saved by 43%, and the total output of L-lactic acid in the coupling process of fermentation separation is the same as single fed-batch fermentation 3.82 times of the process (430.0g).

Example 4

The medium used in this embodiment is as follows:

The composition of solid slant medium is (g/L): glucose 20, yeast extract 5, soybean peptone 10, beef extract 10, sodium chloride 10, sodium acetate 5, ammonium citrate 2, magnesium sulfate heptahydrate 0.2, sulfuric acid heptahydrate Manganese 0.05, agar 15. The pH is 6.50.

The composition of the liquid seed medium is (g/L): glucose 40, peptone 10, yeast extract powder 10, sodium chloride 0.01, sodium acetate 0.5, ammonium citrate 0.2, potassium dihydrogen phosphate 0.4, magnesium sulfate heptahydrate 0.2, Manganese sulfate heptahydrate 0.05. The pH is 6.50.

The composition of the fermentation medium is (g/L): glucose 145, yeast extract powder 25, sodium chloride 0.01, sodium acetate 0.5, ammonium citrate 0.2, potassium dihydrogen phosphate 0.4, magnesium sulfate heptahydrate 0.2, manganese sulfate heptahydrate 0.05. The pH is 6.00.

The composition of the feeding medium is (g/L): glucose 450, yeast extract powder 15, sodium chloride 0.01, sodium acetate 0.5, ammonium citrate 0.2, potassium dihydrogen phosphate 0.4, magnesium sulfate heptahydrate 0.2, heptahydrate Manganese sulfate 0.05. The pH is 6.00.

In the present invention, use MgO as neutralizing agent, adopt the step of fermentation separation coupling mode to produce L-magnesium lactate as follows:

(1) Slant culture: Lactobacillus rhamnosus CGMCC 1.2134 was inoculated on a solid slant medium, and cultured at 42° C. for 24 hours.

(2) Seed culture: Transfer the strains obtained in step (1) to 100ml seed medium under aseptic conditions, cultivate the primary seed liquid at 42°C and 180rpm for 24h, and then add 50ml of the primary seed liquid Transfer to 450ml of fresh seed medium under sterile conditions, and cultivate the secondary seed solution at 42°C and 180rpm for 12h. 15 g/L of calcium carbonate was added to the seed medium as a neutralizing agent to maintain a constant pH.

(3) Fermentation culture: the secondary seed liquid of step (2) gained is inserted into 5L fermentation medium according to 10% (v/v) inoculum size, at stirring speed 220rpm, under fermentation temperature 42 ℃, feed 15%MgO Control pH 6.25 ± 0.05. The repeated batch fermentation process includes multiple fermentation cycles and magnesium lactate online removal process. In a single fermentation cycle, when the glucose is depleted for the first time, the feed medium is added to keep the glucose concentration below 30g/L, as much as possible reduce the glucose suppression effect. When the lactic acid concentration reached 140±5g/L and the glucose was exhausted, the fermentation process was suspended. Add 15% MgO to increase the pH of the fermentation broth in the fermenter and stabilize it to 7.0, preparing for the next step of crystallization.

(4) Product separation: pump 80-85% of the volume of the above supersaturated magnesium lactate fermentation liquid into the crystallization tank, crystallize and precipitate at 42°C for 120 minutes, pump the supernatant back to the fermentation tank for reuse, and control the reuse rate of fermentation wastewater At 60-70%, a high concentration of magnesium lactate crystals is simultaneously obtained. After the crystallization supernatant was transported back to the fermenter, the feed medium was added to the fermenter to increase the glucose concentration to 70g/L and then enter the next round of fermentation acid production and online removal cycle. A total of four in situ magnesium lactate removals were performed.

In the L-magnesium lactate fermentation separation coupling process carried out in a 10L tank, the L-lactic acid concentration reached 142g/L, and the fermentation separation coupling process produced a total of 4108g of L-lactic acid, the fermentation time was 206h, and the average space-time yield of L-lactic acid was 2.23g/L L/h, the conversion rate of sugar and acid is 95.2%.

Example 5

The medium used in this embodiment is as follows:

The composition of solid slant medium is (g/L): glucose 20, yeast extract 5, soybean peptone 10, beef extract 10, sodium chloride 10, sodium acetate 5, ammonium citrate 2, magnesium sulfate heptahydrate 0.2, sulfuric acid heptahydrate Manganese 0.05, agar 15. The pH is 6.50.

The composition of the liquid seed medium is (g/L): glucose 40, peptone 10, yeast extract powder 10, sodium chloride 0.01, sodium acetate 0.5, ammonium citrate 0.2, potassium dihydrogen phosphate 0.4, magnesium sulfate heptahydrate 0.2, Manganese sulfate heptahydrate 0.05. The pH is 6.50.

The composition of the fermentation medium is (g/L): glucose 140, yeast extract powder 20, sodium chloride 0.01, sodium acetate 0.5, ammonium citrate 0.2, potassium dihydrogen phosphate 0.4, magnesium sulfate heptahydrate 0.2, manganese sulfate heptahydrate 0.05. The pH is 6.00.

The composition of the feeding medium is (g/L): glucose 500, yeast extract powder 15, sodium chloride 0.01, sodium acetate 0.5, ammonium citrate 0.2, potassium dihydrogen phosphate 0.4, magnesium sulfate heptahydrate 0.2, heptahydrate Manganese sulfate 0.05. The pH is 6.00.

In the present invention, use MgO as neutralizing agent, adopt the step of fermentation separation coupling mode to produce L-magnesium lactate as follows:

(1) Slant culture: Lactobacillus rhamnosus CGMCC 1.2134 was inoculated on a solid slant medium, and cultured at 42° C. for 24 hours.

(2) Seed culture: Transfer the strains obtained in step (1) to 100ml seed medium under aseptic conditions, cultivate the primary seed liquid at 42°C and 180rpm for 24h, and then add 50ml of the primary seed liquid Transfer to 400ml of fresh seed medium under sterile conditions, and cultivate the secondary seed solution at 42°C and 180rpm for 12h. 15 g/L of calcium carbonate was added to the seed medium as a neutralizing agent to maintain a constant pH.

(3) Fermentation culture: the secondary seed liquid of step (2) gained is inserted into 15L fermentation medium according to 3% (v/v) inoculum, at stirring speed 240rpm, under 42 ℃ of fermentation temperature, feed 15%MgO Control pH 6.25 ± 0.05. The repeated batch fermentation process includes multiple fermentation cycles and magnesium lactate online removal process. In a single fermentation cycle, when the glucose is depleted for the first time, the feed medium is added to keep the glucose concentration below 30g/L, as much as possible reduce the glucose suppression effect. When the lactic acid concentration reached 140±5g/L and the glucose was exhausted, the fermentation process was suspended. Add 15% MgO to increase the pH of the fermentation broth in the fermenter and stabilize it to 7.0, preparing for the next step of crystallization.

(4) Product separation: pump 80-85% of the volume of the above supersaturated magnesium lactate fermentation liquid into the crystallization tank, crystallize and precipitate at 42°C for 120 minutes, pump the supernatant back to the fermentation tank for reuse, and control the reuse rate of fermentation wastewater At 60-70%, a high concentration of magnesium lactate crystals is simultaneously obtained. After the crystallization supernatant was transported back to the fermenter, the feed medium was added to the fermenter to increase the glucose concentration to 70g/L and then enter the next round of fermentation acid production and online removal cycle. A total of four in situ magnesium lactate removals were performed.

In the L-magnesium lactate fermentation separation coupling process carried out in a 30L tank, the L-lactic acid concentration reached 140g/L, a total of 12.30Kg L-lactic acid was obtained during the fermentation separation coupling process, the fermentation time was 210h, and the average space-time yield of L-lactic acid was 2.15 g/L/h, the sugar-acid conversion rate is 94.1%.

Claims (9)

1. a kind of method producing magnesium lactate based on crystallisation Integrated process, it is characterised in that include the following steps：

(1) inclined-plane culture：It is trained using 1.2134 inclined-planes rhamnose lactic acid bacteria Lactobacillus rhamnosus CGMCC It supports, slant strains cultural method is to be inoculated with Lactobacillus rhamnosus CGMCC 1.2134 in a manner of scribing line Onto solid slope culture medium, 24-48h is cultivated at 37-42 DEG C；

(2) seed culture：By the strain transfer after inclined-plane culture to seed culture medium, cultivated under 37-42 DEG C, 120-220rpm Then primary seed solution is accessed fresh seeds culture medium by 24-36h again by 10% inoculum concentration, at 37-42 DEG C, 120- Secondary seed solution 8-15h is cultivated under 220rpm；In liquid seed culture medium add 15g/L calcium carbonate as neutralizer to maintain Constant pH；

(3) fermented and cultured：The secondary seed solution that seed growth phase is obtained accesses fermentation training according to 3-10% (v/v) inoculum concentration Base is supported, at speed of agitator 180-240rpm, 37-42 DEG C of fermentation temperature, stream plus MgO control pH 5.5-6.5；

(4) situ product detaches：It is fermenting repeatedly comprising the online removal of multiple magnesium lactates and feed supplement cycle during Batch fermentation Lactic acid concn reaches 140-160g/L, and glucose exhaust after suspend lactic fermentation, stream plus MgO, make zymotic fluid pH in fermentation tank Rise and stablize to 6.5-7.5, prepares for the crystallization sedimentation of next step；The above supersaturated magnesium lactate zymotic fluid is pumped into crystallization Tank realizes separation of solid and liquid under fermentation temperature in a manner of crystalline deposit, by the supernatant blowback fermentation tank reuse comprising thalline, The magnesium lactate crystalline solid of high concentration is obtained simultaneously；

During Integrated process, magnesium lactate crystallization temperature is 42 DEG C, crystallization time 120min；Batch fermentation process repeatedly In, fermentation lactic acid initial concentration is 140g/L.

2. according to the method described in claim 1, it is characterized in that：MgO is added in such a way that stream adds in fermentation process to adjust For pH 6.25 ± 0.05, the MgO concentration is 10wt%-20wt%.

3. according to the method described in claim 1, it is characterized in that：Fermentation medium initial glucose content is 100-160g/ L, yeast extract content are 10-30g/L.

4. according to the method described in claim 1, it is characterized in that：During feed supplement-Batch fermentation, mended after glucose exhausts Feed supplement is added with culture medium to glucose content to be 60-80g/L；Lactic acid final concentration is 110- after feed supplement-Batch fermentation 160g/L。

5. according to the method described in claim 4, it is characterized in that：Feed supplement is 300-500g/L with culture medium glucose content, Yeast extract content is 10-30g/L.

6. according to the method described in claim 1, it is characterized in that：Repeatedly during Batch fermentation, in each fermentation cycle knot Shu Hou, adding MgO makes pH rise and stablizes to 7.0；The MgO concentration is 10wt%-20wt%.

7. according to the method described in claim 1, it is characterized in that：Repeatedly during Batch fermentation, mended after glucose exhausts Feed supplement is added with culture medium to glucose content to be 60-80g/L, a concentration of 140g/L of fermentation lactic acid.

8. according to the method described in claim 1, it is characterized in that：During Integrated process, by zymotic fluid total volume 80-85% is pumped into crystallizing tank, and magnesium lactate crystallization temperature is 42 DEG C, crystallization time 120min.

9. according to the method described in claim 1, it is characterized in that：During Integrated process, fermentation waste water reclamation rate control System is in 60-70%.