A process for efficiently fermenting, separating and extracting L-phenylalanine

Abstract

The invention relates to the technical field of producing L-phenylalanine, and in particular to a process for efficiently fermenting, separating and extracting L-phenylalanine, comprising the following steps: S1: controlling pH and DO in a fermenter, and performing biological fermentation and conversion of L-phenylalanine by fermentation bacteria, and the bottom liquid after conversion enters a membrane separation unit with the fermentation bacteria for solid-liquid separation to obtain a clarified raw material and a solid feed liquid, and the solid feed liquid is returned to the fermenter; S2: obtaining a decolorized raw material; S3: obtaining a concentrated raw material; S4: passing the concentrated raw material into a vacuum freeze dryer to obtain L-phenylalanine crystals, and crushing the L-phenylalanine crystals for standby use; the invention provides a process for efficiently fermenting, separating and extracting L-phenylalanine, which can greatly improve the extraction efficiency of L-phenylalanine: through the layout design of a process device and the optimization of a process flow, the extraction efficiency of L-phenylalanine can be significantly improved.

Description

Technical Field

[0001] The invention relates to the field of L-phenylalanine production and chemical engineering technology, and in particular to a process for efficiently fermenting, separating and extracting L-phenylalanine. Background Art

[0002] L-phenylalanine is a physiologically active aromatic amino acid. It is one of the essential amino acids that cannot be synthesized by humans and animals. It is also an important component of aspartame, a new sweetener with high sweetness and low calories. As an important biochemical product, L-Phe has a wide range of applications in food, feed additives, and medicine.

[0003] The preparation methods of L-phenylalanine mainly include microbial fermentation and microbial enzyme method. Among them, microbial fermentation is more widely used. However, the traditional production process has problems such as scattered equipment, complicated process, low extraction efficiency and low product purity.

[0004] For example, application number 202311214928.5 provides a new method for separating and extracting L-alanine from L-alanine fermentation broth. In order to achieve a higher product purity, steps including inactivation treatment, solid-liquid separation, clarified liquid treatment, vacuum distillation concentration, cooling crystallization, centrifugation, ethanol elution and vacuum drying are set. In addition, the existing production process mostly adopts batch production. For example, the separation and extraction of the produced L-phenylalanine in the technical solution of CN116656581A are relatively conventional, which will lead to a significant reduction in the production efficiency of L-phenylalanine during the process due to excessive accumulation of L-phenylalanine. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a process for efficiently fermenting, separating and extracting L-phenylalanine.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A process for efficiently fermenting, separating and extracting L-phenylalanine comprises the following steps:

[0008] S1: Control pH and DO in the fermentation tank, and convert L-phenylalanine through biological fermentation by fermentation bacteria. After the conversion, the bottom liquid enters the membrane separation unit with the fermentation bacteria for solid-liquid separation to obtain clarified raw materials and solid liquid, and the solid liquid is returned to the fermentation tank;

[0009] S2: adding a special small-pore resin to the clarified raw material obtained by the above separation to decolorize it, thereby obtaining a decolorized raw material;

[0010] S3: Concentrating the decolorized raw material using a reverse osmosis membrane to obtain a concentrated raw material;

[0011] S4: passing the concentrated raw material into a vacuum freeze dryer to obtain L-phenylalanine crystals, and then crushing the L-phenylalanine crystals for later use.

[0012] As a further technical solution, the fermentation bacteria in S1 are: ATCC21673 Corynebacterium glutamicum obtained by screening:

[0013] The screening method is:

[0014] ATCC21673 Corynebacterium glutamicum lyophilized powder is added to a modified MRS liquid medium at a 1% inoculation rate, and cultured at 37°C until the logarithmic growth phase, i.e., after culturing for 24 hours, 10% of the culture solution after culture is inoculated into a fresh modified MRS liquid medium, and cultured in a 40°C environment for 24 hours, and then 10% of the culture solution after culture is inoculated into a fresh modified MRS liquid medium, and cultured in a 45°C environment for 24 hours, and then 10% of the culture solution after culture is inoculated into a fresh modified MRS liquid medium, and cultured in a 50°C environment for 24 hours, and the desired fermentation bacteria are obtained from the culture solution after culture;

[0015] Improved MRS liquid medium: peptone 10.5g, yeast extract 4.2g, beef extract 10.5g, glucose 21.0g, sodium acetate 5.5g, dipotassium hydrogen phosphate 2.0g, diammonium citrate 2.0g, Tween-80 0.1mL, magnesium sulfate heptahydrate 0.58g, manganese sulfate tetrahydrate 0.025g, Centella asiatica extract 3g, distilled water 1000mL; adjust pH to 6.5, sterilize at 121℃ for 20min;

[0016] Each L of fermentation medium contains:

[0017] Glucose 24.5g, ammonium sulfate 4.2g, ferrous sulfate heptahydrate 0.072g, L-tyrosine 0.3g, sodium dihydrogen phosphate 2.8g, calcium chloride dihydrate 0.018g, magnesium sulfate heptahydrate 2.5g, vitamin B1 0.078g, sodium chloride 1.1g, yeast powder 2.2g, kanamycin sulfate 0.03g, glycerol 1.3g, peptone 4g, auxiliary additive solution 30-40mL, pH 6.9±0.1.

[0018] As a further technical solution, the auxiliary additive liquid contains per L: Centella asiatica extract Mg, manganese sulfate heptahydrate 2.0g, aluminum sulfate 18hydrate 0.25g, zinc sulfate heptahydrate 1.2g, boric acid 0.5-0.6g, cobalt sulfate heptahydrate 0.74g, sodium molybdate dihydrate 2-3g, copper sulfate pentahydrate 0.25g, nickel sulfate hexahydrate 0.21g;

[0019] Where, M = X + 0.5Y;

[0020] Where M is the mass of Centella asiatica extract;

[0021] X is the mass of sodium molybdate dihydrate;

[0022] Y is the mass of boric acid.

[0023] As a further technical solution, the conditions of S1 fermentation bacteria are as follows: the residence time of fermentation bacteria is greater than 30 days, gas is introduced into the bottom through a vertically inserted tubular ceramic microporous aeration head to achieve stirring and DO, pH is controlled between 6-6.5, DO is controlled between 0.32-0.35mmolO2 / L, and the fermentation temperature is 35-37℃;

[0024] Tubular ceramic microporous aeration head every 0.2-0.5m 2 Insert 1, the average pore size of the membrane is 10-100nm;

[0025] A gas collection device is set on the top of the fermentation tank.

[0026] As a further technical solution, the membrane separation unit uses a ceramic flat membrane with a pore size of 30-60nm. The bottom is connected to a gas collection device for membrane flushing. The membrane flushing gas volume is 40-60L / m per minute. 2 ; For every Q minutes of filtration, backwash Q / 20 minutes, and the backwash flow rate is 1.5-3 times the filtration flow rate.

[0027] As a further technical solution, the pore size of the special small-pore resin is 3 to 6 nm;

[0028] The method for preparing the special small-pore resin is as follows:

[0029] First, the oil component and the water component are prepared separately;

[0030] The oil component and the water component are mixed together in a volume ratio of 8:1-1.5 to obtain a mixed oil-water system;

[0031] Then, methylene blue is added dropwise to the mixed oil-water system, wherein the amount of methylene blue added is 0.12% of the volume of the mixed oil-water system, the stirring speed is adjusted to 500 r / min, the temperature is adjusted to 50° C., stirring is carried out for 1.5 hours, the temperature is adjusted to 70° C., stirring is continued for 4 hours, the temperature is adjusted to 78° C., stirring is continued for 2 hours, and finally the temperature is adjusted to 83° C., heat preservation is carried out for 4 hours, the temperature is lowered to 38° C., and the mixture is allowed to stand for 1 hour, and then filtered, washed, and vacuum dried for 24 hours to obtain the product.

[0032] As a further technical solution, the oil component preparation method is:

[0033] Adding monomer, toluene, n-heptane, crosslinking agent and initiator to the reaction kettle in sequence, stirring and mixing evenly to obtain an oil component;

[0034] Wherein, the weight ratio of the monomer, toluene, n-heptane, crosslinking agent and initiator is: 22-25: 1-1.2: 0.5-0.6: 0.2-0.3: 0.1-0.15;

[0035] The monomers are methyl acrylate, methyl acrylate, and allyl glycidyl ether, wherein methyl acrylate, methyl acrylate, and allyl glycidyl ether are mixed in a mass ratio of 4:2:1;

[0036] The cross-linking agent is tetraethylene glycol dimethacrylate;

[0037] The initiator is azobisisobutyronitrile;

[0038] The water component preparation method comprises: adding sodium chloride to deionized water, stirring and mixing uniformly to obtain a sodium chloride solution, adding polyvinyl pyrrolidone, polyvinyl alcohol, and gelatin to the sodium chloride solution, stirring for 30 minutes, and then ultrasonically dispersing for 10 minutes to obtain;

[0039] Among them, the mass fraction of sodium chloride is 10%;

[0040] The mixing mass ratio of sodium chloride solution, polyvinyl pyrrolidone, polyvinyl alcohol and gelatin is 100:1.2:5.

[0041] As a further technical solution, the reverse osmosis membrane adopts a 4-6 stage series mode, and the concentration multiple is more than 100 times.

[0042] As a further technical solution, the cold trap temperature of the vacuum freeze dryer is not lower than -50°C under no-load, and the water capture capacity should be ≥10kg / 24h.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] The present invention provides a process for efficiently fermenting, separating and extracting L-phenylalanine, which can greatly improve the extraction efficiency of L-phenylalanine: through the layout design of the process device and the optimization of the process flow, the extraction efficiency of L-phenylalanine can be significantly improved.

[0045] Ability to improve product purity: Using a special small-pore resin for decolorization, combined with subsequent separation steps, it can effectively remove impurities and pigments in the fermentation broth, thereby improving the purity of the L-phenylalanine product.

[0046] Environmental protection and energy saving: The entire production process adopts biological fermentation and physical separation methods to reduce pollution to the environment; at the same time, it optimizes energy utilization and achieves energy saving and consumption reduction.

[0047] The present invention introduces a certain amount of auxiliary additive liquid into the culture medium, and the coordinated effect of the multiple active ingredients provided by the auxiliary additive liquid not only improves the cell growth rate and cell vitality, but also effectively enhances the absorption effect of the fermentation bacteria on trace elements, thereby improving the yield of L-phenylalanine and the sugar-acid conversion efficiency.

[0048] In the process of increasing the fermentation yield of L-phenylalanine, in addition to adding auxiliary additives, the fermentation conditions were also optimized, including controlling appropriate temperature, pH value, ventilation volume and other parameters to maintain DO within an appropriate range. The coordination of multiple parameters greatly improved the sugar-acid conversion rate and significantly increased the yield of L-phenylalanine.

[0049] By introducing a certain amount of glycerol as an auxiliary carbon source and fermenting with glucose, the yield and production intensity of L-phenylalanine can be increased. This is because glycerol has a lower by-product generation rate and a higher product-to-substrate mass yield when used as a carbon source.

[0050] The present invention can obtain L-phenylalanine fermentation bacteria with higher fermentation performance by screening ATCC21673 Corynebacterium glutamicum, further improve the fermentation yield of L-phenylalanine, and the fermentation bacteria obtained after screening not only have better temperature resistance, but also can adapt to a wider temperature range, and can maintain the activity of enzymes in the fermentation bacteria and the stability of metabolic functions for a long time, so that the substrate can be more efficiently converted into L-alanine. During the fermentation process, the fermentation performance of the fermentation bacteria will not be significantly reduced due to the increase in temperature, thereby avoiding the problem that the conventional fermentation bacteria easily cause large fluctuations in the fermentation efficiency of L-phenylalanine due to the narrow temperature adaptability. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0052] Figure 1 A diagram of the apparatus for the process of efficiently fermenting, separating and extracting L-phenylalanine;

[0053] Figure 2 This is the L-phenylalanine fermentation time and yield curve. DETAILED DESCRIPTION

[0054] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] The preparation method of Centella asiatica extract is as follows:

[0056] First, the Centella asiatica leaves were cleaned, and then mixed with water at a solid-liquid ratio of 1:10, and pulped for 30 minutes to obtain Centella asiatica slurry;

[0057] The pH value of the Centella asiatica slurry is adjusted to 8.5 with a 5% sodium hydroxide solution, and then the temperature is adjusted to 50°C and kept warm for 4 hours. The pH value of the Centella asiatica slurry is then adjusted to neutral. An 80% ethanol solution in an amount three times its volume is added to the Centella asiatica slurry, and the mixture is stirred at 800 r / min for 4 hours, and then centrifuged at 5500 r / min for 15 minutes, filtered to obtain a filtrate, and the filtrate is spray-dried to obtain a Centella asiatica extract.

[0058] The following are specific embodiments:

[0059] Example 1

[0060] A process for efficiently fermenting, separating and extracting L-phenylalanine comprises the following steps:

[0061] S1: Control pH and DO in fermentation tank 1, and convert L-phenylalanine by fermentation bacteria. After conversion, the bottom liquid enters membrane separation unit 2 with the fermentation bacteria for solid-liquid separation to obtain clarified raw materials and solid liquid, and the solid liquid is returned to fermentation tank 1. The glucose concentration in fermentation tank 1 drops to 1.0g / L, and glucose is added; the fermentation bacteria are: ATCC21673 Corynebacterium glutamicum obtained by screening:

[0062] The screening method is:

[0063] ATCC21673 Corynebacterium glutamicum lyophilized powder is added to a modified MRS liquid medium at a 1% inoculation rate, and cultured at 37°C until the logarithmic growth phase, i.e., after culturing for 24 hours, 10% of the culture solution after culture is inoculated into a fresh modified MRS liquid medium, and cultured in a 40°C environment for 24 hours, and then 10% of the culture solution after culture is inoculated into a fresh modified MRS liquid medium, and cultured in a 45°C environment for 24 hours, and then 10% of the culture solution after culture is inoculated into a fresh modified MRS liquid medium, and cultured in a 50°C environment for 24 hours, and the desired fermentation bacteria are obtained from the culture solution after culture;

[0064] Improved MRS liquid medium: peptone 10.5g, yeast extract 4.2g, beef extract 10.5g, glucose 21.0g, sodium acetate 5.5g, dipotassium hydrogen phosphate 2.0g, diammonium citrate 2.0g, Tween-80 0.1mL, magnesium sulfate heptahydrate 0.58g, manganese sulfate tetrahydrate 0.025g, Centella asiatica extract 3g, distilled water 1000mL; adjust pH to 6.5, sterilize at 121℃ for 20min;

[0065] Each L of fermentation medium contains:

[0066] Glucose 24.5g, ammonium sulfate 4.2g, ferrous sulfate heptahydrate 0.072g, L-tyrosine 0.3g, sodium dihydrogen phosphate 2.8g, calcium chloride dihydrate 0.018g, magnesium sulfate heptahydrate 2.5g, vitamin B1 0.078g, sodium chloride 1.1g, yeast powder 2.2g, kanamycin sulfate 0.03g, glycerol 1.3g, peptone 4g, auxiliary additive solution 30mL, pH 6.9±0.1.

[0067] Each L of the auxiliary additive solution contains: 2.25 g of Centella asiatica extract, 2.0 g of manganese sulfate heptahydrate, 0.25 g of aluminum sulfate 18hydrate, 1.2 g of zinc sulfate heptahydrate, 0.5 g of boric acid, 0.74 g of cobalt sulfate heptahydrate, 2 g of sodium molybdate dihydrate, 0.25 g of copper sulfate pentahydrate, and 0.21 g of nickel sulfate hexahydrate;

[0068] The fermentation conditions are as follows: the fermentation bacteria stay time is 35 days, gas is introduced into the bottom through a vertically inserted tubular ceramic microporous aeration head to achieve stirring and DO, pH is controlled between 6-6.5, DO is controlled between 0.32-0.35mmolO2 / L, and the fermentation temperature is 36℃;

[0069] Tubular ceramic microporous aeration head every 0.2m 2 Insert 1, the average pore size of the membrane is 30nm;

[0070] A gas collecting device 3 is arranged on the top of the fermentation tank 1 .

[0071] The membrane separation unit 2 uses a ceramic flat membrane with a pore size of 30 nm. The bottom is connected to the gas collection device 3 for membrane flushing. The membrane flushing gas volume is 50 L / m per minute. 2 ; For every Q minutes of filtration, the backwash is Q / 20 minutes, and the backwash flow rate is twice the filtration flow rate.

[0072] S2: adding the clarified raw material obtained by the above separation to a special small-pore resin 4 for decolorization to obtain a decolorized raw material; the pore size of the special small-pore resin is 5 nm;

[0073] The method for preparing the special small-pore resin is as follows:

[0074] First, the oil component and the water component are prepared separately;

[0075] The oil component and the water component are mixed together in a volume ratio of 8:1 to obtain a mixed oil-water system;

[0076] Then, methylene blue is added dropwise to the mixed oil-water system, wherein the amount of methylene blue added is 0.12% of the volume of the mixed oil-water system, the stirring speed is adjusted to 500 r / min, the temperature is adjusted to 50° C., stirring is carried out for 1.5 hours, the temperature is adjusted to 70° C., stirring is continued for 4 hours, the temperature is adjusted to 78° C., stirring is continued for 2 hours, and finally the temperature is adjusted to 83° C., heat preservation is carried out for 4 hours, the temperature is lowered to 38° C., and the mixture is allowed to stand for 1 hour, and then filtered, washed, and vacuum dried for 24 hours to obtain the product.

[0077] The oil component preparation method is as follows:

[0078] Adding monomer, toluene, n-heptane, crosslinking agent and initiator to the reaction kettle in sequence, stirring and mixing evenly to obtain an oil component;

[0079] Wherein, the weight ratio of the monomer, toluene, n-heptane, crosslinking agent and initiator is: 22:1:0.5:0.2:0.1;

[0080] The monomers are methyl acrylate, methyl acrylate, and allyl glycidyl ether, wherein methyl acrylate, methyl acrylate, and allyl glycidyl ether are mixed in a mass ratio of 4:2:1;

[0081] The cross-linking agent is tetraethylene glycol dimethacrylate;

[0082] The initiator is azobisisobutyronitrile;

[0083] The water component preparation method comprises: adding sodium chloride to deionized water, stirring and mixing uniformly to obtain a sodium chloride solution, adding polyvinyl pyrrolidone, polyvinyl alcohol, and gelatin to the sodium chloride solution, stirring for 30 minutes, and then ultrasonically dispersing for 10 minutes to obtain;

[0084] Among them, the mass fraction of sodium chloride is 10%;

[0085] The mixing mass ratio of sodium chloride solution, polyvinyl pyrrolidone, polyvinyl alcohol and gelatin is 100:1.2:5.

[0086] S3: The decolorized raw material is concentrated by using a reverse osmosis membrane 5 to obtain a concentrated raw material; the reverse osmosis membrane adopts a 6-stage series mode, and the concentration multiple is more than 100 times;

[0087] The reverse osmosis membrane used is BW2540, purchased from Qinsen High-Tech in Hunan, China, with an effective membrane area of ​​2.6m 2 .

[0088] S4: passing the concentrated raw material into a vacuum freeze dryer 6 to obtain L-phenylalanine crystals, and crushing the L-phenylalanine crystals for later use;

[0089] The cold trap temperature of the vacuum freeze dryer should not be lower than -50℃ when no load, and the water capture capacity should be ≥10kg / 24h.

[0090] Example 2

[0091] Based on Example 1, the difference from the technical solution of Example 1 is that each L of the fermentation medium contains:

[0092] Glucose 24.5g, ammonium sulfate 4.2g, ferrous sulfate heptahydrate 0.072g, L-tyrosine 0.3g, sodium dihydrogen phosphate 2.8g, calcium chloride dihydrate 0.018g, magnesium sulfate heptahydrate 2.5g, vitamin B1 0.078g, sodium chloride 1.1g, yeast powder 2.2g, kanamycin sulfate 0.03g, glycerol 1.3g, peptone 4g, auxiliary additive solution 30mL, pH 6.9±0.1.

[0093] Each L of the auxiliary additive solution contains: 2.77 g of Centella asiatica extract, 2.0 g of manganese sulfate heptahydrate, 0.25 g of aluminum sulfate 18hydrate, 1.2 g of zinc sulfate heptahydrate, 0.54 g of boric acid, 0.74 g of cobalt sulfate heptahydrate, 2.5 g of sodium molybdate dihydrate, 0.25 g of copper sulfate pentahydrate, and 0.21 g of nickel sulfate hexahydrate;

[0094] Example 3

[0095] Based on Example 1, the difference from the technical solution of Example 1 is that each L of the fermentation medium contains:

[0096] Glucose 24.5g, ammonium sulfate 4.2g, ferrous sulfate heptahydrate 0.072g, L-tyrosine 0.3g, sodium dihydrogen phosphate 2.8g, calcium chloride dihydrate 0.018g, magnesium sulfate heptahydrate 2.5g, vitamin B1 0.078g, sodium chloride 1.1g, yeast powder 2.2g, kanamycin sulfate 0.03g, glycerol 1.3g, peptone 4g, auxiliary additive solution 30mL, pH 6.9±0.1.

[0097] Each L of the auxiliary additive solution contains: 3.3 g of Centella asiatica extract, 2.0 g of manganese sulfate heptahydrate, 0.25 g of aluminum sulfate 18hydrate, 1.2 g of zinc sulfate heptahydrate, 0.6 g of boric acid, 0.74 g of cobalt sulfate heptahydrate, 3 g of sodium molybdate dihydrate, 0.25 g of copper sulfate pentahydrate, and 0.21 g of nickel sulfate hexahydrate;

[0098] Example 4

[0099] Based on Example 1, the difference from the technical solution of Example 1 is that the method for preparing the special small-pore resin is:

[0100] First, the oil component and the water component are prepared separately;

[0101] The oil component and the water component are mixed together in a volume ratio of 8:1.5 to obtain a mixed oil-water system;

[0102] Then, methylene blue is added dropwise to the mixed oil-water system, wherein the amount of methylene blue added is 0.12% of the volume of the mixed oil-water system, the stirring speed is adjusted to 500 r / min, the temperature is adjusted to 50° C., stirring is carried out for 1.5 hours, the temperature is adjusted to 70° C., stirring is continued for 4 hours, the temperature is adjusted to 78° C., stirring is continued for 2 hours, and finally the temperature is adjusted to 83° C., heat preservation is carried out for 4 hours, the temperature is lowered to 38° C., and the mixture is allowed to stand for 1 hour, and then filtered, washed, and vacuum dried for 24 hours to obtain the product.

[0103] The oil component preparation method is as follows:

[0104] Adding monomer, toluene, n-heptane, crosslinking agent and initiator to the reaction kettle in sequence, stirring and mixing evenly to obtain an oil component;

[0105] Wherein, the weight ratio of the monomer, toluene, n-heptane, crosslinking agent and initiator is: 25:1.2:0.6:0.3:0.15;

[0106] The monomers are methyl acrylate, methyl acrylate, and allyl glycidyl ether, wherein methyl acrylate, methyl acrylate, and allyl glycidyl ether are mixed in a mass ratio of 4:2:1;

[0107] The cross-linking agent is tetraethylene glycol dimethacrylate;

[0108] The initiator is azobisisobutyronitrile;

[0109] The water component preparation method comprises: adding sodium chloride to deionized water, stirring and mixing uniformly to obtain a sodium chloride solution, adding polyvinyl pyrrolidone, polyvinyl alcohol, and gelatin to the sodium chloride solution, stirring for 30 minutes, and then ultrasonically dispersing for 10 minutes to obtain;

[0110] Among them, the mass fraction of sodium chloride is 10%;

[0111] The mixing mass ratio of sodium chloride solution, polyvinyl pyrrolidone, polyvinyl alcohol and gelatin is 100:1.2:.

[0112] Comparative Example 1: This comparative example is basically the same as Example 1, except that no Centella asiatica extract is added to the auxiliary additive liquid.

[0113] Comparative Example 2: This comparative example is basically the same as Example 1, except that the Centella asiatica extract in the auxiliary additive liquid is replaced by the Herba Lycopodii extract, and the extraction method remains unchanged.

[0114] Comparative Example 3:

[0115] This comparative example is basically the same as Example 1, except that the amount of Centella asiatica extract added in the auxiliary additive liquid does not satisfy M=X+0.5Y, specifically: 2.0 g of Centella asiatica extract, 0.5 g of boric acid, and 2 g of sodium molybdate dihydrate.

[0116] Comparative Example 4

[0117] This comparative example is basically the same as Example 1, except that the dedicated small-pore resin is replaced by D101 type macroporous resin.

[0118] Comparative Example 5

[0119] This comparative example is basically the same as Example 1, except that ATCC21673 Corynebacterium glutamicum is not screened.

[0120] Comparative Example 6

[0121] This comparative example is basically the same as Example 1, except that no Centella asiatica extract is added to the modified MRS liquid culture medium used in the screening process of ATCC21673 Corynebacterium glutamicum.

[0122] test:

[0123] L-phenylalanine was produced according to the process of the embodiment and the comparative example, and the L-phenylalanine yield of each group was compared. The total fermentation time of the experiment was 55h:

[0124] Table 1

[0125]

[0126]

[0127] It can be seen from Table 1 that the process of the present invention for producing L-phenylalanine has the characteristics of high yield and high fermentation efficiency. It can be seen from the embodiment and comparative examples 1 and 2 that the Centella asiatica extract in the auxiliary additive liquid in the fermentation medium has a certain effect of improving the fermentation efficiency and the yield of L-phenylalanine, while the use of other ingredients to replace the Centella asiatica extract has a disappearance of the improvement effect. It can be seen from Example 3 that the addition amount of the Centella asiatica extract in the auxiliary additive liquid does not satisfy M=X+0.5Y, which will slightly reduce the yield of L-phenylalanine.

[0128] According to the process of the embodiment and the comparative example, L-phenylalanine was produced. The total fermentation time of the experiment was 55 hours. The total sugar consumption and sugar-acid conversion rate were compared:

[0129] Table 2

[0130]

[0131]

[0132] It can be seen from Table 2 that the process of the present invention can significantly improve the fermentation efficiency of L-phenylalanine, has a higher sugar-acid conversion rate, and reduces the cost.

[0133] The decolorization rates of the fermentation clarified raw materials in the comparative example and comparative example 4 are compared:

[0134] The fermented clarified raw materials of Example 1 and Comparative Example 4 were subjected to full wavelength scanning under an ultraviolet spectrophotometer, and it was found that the maximum absorption peak appeared at a wavelength of 360 nm. 360 nm was used as the subsequent detection wavelength to measure the absorbance of the fermented clarified raw material solution before and after decolorization, and the decolorization rate was calculated by substituting it into the formula;

[0135] Decolorization rate (%) = (D0-D1) / D0×100%;

[0136] Where D0 is the absorbance of the fermented clarified raw material before decolorization, and D1 is the absorbance of the fermented clarified raw material after decolorization;

[0137] Table 3

[0138] Decolorization rate % Example 1 92.5 Example 2 92.1 Example 3 92.3 Example 4 92.7 Comparative Example 4 72.3

[0139] It can be seen from Table 3 that the special small-pore resin prepared by the present invention has a higher decolorization rate.

[0140] Taking Example 1 as the basic sample, the L-alanine production curves at different times are compared. Figure 2 .

[0141] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A process for efficiently fermenting, separating and extracting L-phenylalanine, characterized in that: The following steps are involved: S1: Control pH and DO in the fermentation tank, and convert L-phenylalanine through biological fermentation by fermentation bacteria. After the conversion, the bottom liquid enters the membrane separation unit with the fermentation bacteria for solid-liquid separation to obtain clarified raw materials and solid liquid, and the solid liquid is returned to the fermentation tank; S2: adding a special small-pore resin to the clarified raw material obtained by the above separation to decolorize it, thereby obtaining a decolorized raw material; S3: Concentrating the decolorized raw material using a reverse osmosis membrane to obtain a concentrated raw material; S4: passing the concentrated raw material into a vacuum freeze dryer to obtain L-phenylalanine crystals, and then crushing the L-phenylalanine crystals for later use.

2. A process for efficient fermentation, separation and extraction of L-phenylalanine according to claim 1, characterized in that: The fermentation bacteria in S1 are: ATCC21673 Corynebacterium glutamicum obtained by screening: The screening method is: ATCC21673 Corynebacterium glutamicum lyophilized powder is added to a modified MRS liquid medium at a 1% inoculation rate, and cultured at 37°C until the logarithmic growth phase, i.e., after culturing for 24 hours, 10% of the culture solution after culture is inoculated into a fresh modified MRS liquid medium, and cultured in a 40°C environment for 24 hours, and then 10% of the culture solution after culture is inoculated into a fresh modified MRS liquid medium, and cultured in a 45°C environment for 24 hours, and then 10% of the culture solution after culture is inoculated into a fresh modified MRS liquid medium, and cultured in a 50°C environment for 24 hours, and the desired fermentation bacteria are obtained from the culture solution after culture; Improved MRS liquid medium: peptone 10.5g, yeast extract 4.2g, beef extract 10.5g, glucose 21.0g, sodium acetate 5.5g, dipotassium hydrogen phosphate 2.0g, diammonium citrate 2.0g, Tween-80 0.1mL, magnesium sulfate heptahydrate 0.58g, manganese sulfate tetrahydrate 0.025g, Centella asiatica extract 3g, distilled water 1000mL; adjust pH to 6.5, sterilize at 121℃ for 20min; Each L of fermentation medium contains: Glucose 24.5g, ammonium sulfate 4.2g, ferrous sulfate heptahydrate 0.072g, L-tyrosine 0.3g, sodium dihydrogen phosphate 2.8g, calcium chloride dihydrate 0.018g, magnesium sulfate heptahydrate 2.5g, vitamin B1 0.078g, sodium chloride 1.1g, yeast powder 2.2g, kanamycin sulfate 0.03g, glycerol 1.3g, peptone 4g, auxiliary additive solution 30-40mL, pH 6.9±0.

1.

3. A process for efficient fermentation, separation and extraction of L-phenylalanine according to claim 2, characterized in that: The auxiliary additive liquid contains: Centella asiatica extract Mg, manganese sulfate heptahydrate 2.0g, aluminum sulfate 18hydrate 0.25g, zinc sulfate heptahydrate 1.2g, boric acid 0.5-0.6g, cobalt sulfate heptahydrate 0.74g, sodium molybdate dihydrate 2-3g, copper sulfate pentahydrate 0.25g, nickel sulfate hexahydrate 0.21g per liter; Where, M = X + 0.5Y; Where M is the mass of Centella asiatica extract; X is the mass of sodium molybdate dihydrate; Y is the mass of boric acid.

4. The process for efficiently fermenting, separating and extracting L-phenylalanine according to claim 1, characterized in that: The conditions for S1 fermentation bacteria are as follows: the residence time of the fermentation bacteria is greater than 30 days, gas is introduced into the bottom through a vertically inserted tubular ceramic microporous aeration head to achieve stirring and DO, pH is controlled between 6-6.5, DO is controlled between 0.32-0.35mmolO2 / L, and the fermentation temperature is 35-37℃; Tubular ceramic microporous aeration head every 0.2-0.5m 2 Insert 1, the average pore size of the membrane is 10-100nm; A gas collection device is set on the top of the fermentation tank.

5. The process for efficiently fermenting, separating and extracting L-phenylalanine according to claim 1, characterized in that: The membrane separation unit uses a ceramic flat membrane with a pore size of 30-60nm. The bottom is connected to a gas collection device for membrane flushing. The membrane flushing gas volume is 40-60L / m per minute. 2 ; For every Q minutes of filtration, the backwash is Q / 20 minutes, and the backwash flow rate is 1.5-3 times the filtration flow rate.

6. The process for efficiently fermenting, separating and extracting L-phenylalanine according to claim 1, characterized in that: The pore size of the special small-pore resin in S2 is 3 to 6 nm; The method for preparing the special small-pore resin is as follows: First, the oil component and the water component are prepared separately; The oil component and the water component are mixed together in a volume ratio of 8:1-1.5 to obtain a mixed oil-water system; Then, methylene blue is added dropwise to the mixed oil-water system, wherein the amount of methylene blue added is 0.12% of the volume of the mixed oil-water system, the stirring speed is adjusted to 500 r / min, the temperature is adjusted to 50° C., stirring is carried out for 1.5 hours, the temperature is adjusted to 70° C., stirring is continued for 4 hours, the temperature is adjusted to 78° C., stirring is continued for 2 hours, and finally the temperature is adjusted to 83° C., heat preservation is carried out for 4 hours, the temperature is lowered to 38° C., and the mixture is allowed to stand for 1 hour, and then filtered, washed, and vacuum dried for 24 hours to obtain the product.

7. A process for efficient fermentation, separation and extraction of L-phenylalanine according to claim 6, characterized in that: The oil component preparation method is: Adding monomer, toluene, n-heptane, crosslinking agent and initiator to the reaction kettle in sequence, stirring and mixing evenly to obtain an oil component; Wherein, the weight ratio of the monomer, toluene, n-heptane, crosslinking agent and initiator is: 22-25: 1-1.2: 0.5-0.6: 0.2-0.3: 0.1-0.15; The monomers are methyl acrylate, methyl acrylate, and allyl glycidyl ether, wherein methyl acrylate, methyl acrylate, and allyl glycidyl ether are mixed in a mass ratio of 4:2:1; The cross-linking agent is tetraethylene glycol dimethacrylate; The initiator is azobisisobutyronitrile; The water component preparation method comprises: adding sodium chloride to deionized water, stirring and mixing uniformly to obtain a sodium chloride solution, adding polyvinyl pyrrolidone, polyvinyl alcohol, and gelatin to the sodium chloride solution, stirring for 30 minutes, and then ultrasonically dispersing for 10 minutes to obtain; Among them, the mass fraction of sodium chloride is 10%; The mixing mass ratio of sodium chloride solution, polyvinyl pyrrolidone, polyvinyl alcohol and gelatin is 100:1.2:

5.

8. The process for efficiently fermenting, separating and extracting L-phenylalanine according to claim 1, characterized in that: The reverse osmosis membrane described in S3 adopts a 4-6 stage series mode.

9. A process for efficient fermentation, separation and extraction of L-phenylalanine according to claim 8, characterized in that: The cold trap temperature of the vacuum freeze dryer is not lower than -50°C under no-load, and the water capture capacity should be ≥10kg / 24h.

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