A method for reducing foam in a fermentation process of l-tyrosine

Abstract

This invention provides a method for reducing foam during L-tyrosine fermentation. By adding an appropriate amount of L-tyrosine seed crystals during the initial stage of L-tyrosine crystallization, when a large amount of foam is generated during fermentation, the crystals are enlarged, accelerating the crystallization rate. Simultaneously, ultrasonic-assisted crystallization is used to further enlarge the crystals and optimize their crystal form, thereby reducing excessive foaming during fermentation caused by L-tyrosine crystallization. This method solves the problem of severe foaming in the fermentation broth due to small crystals during L-tyrosine fermentation. It also reduces the consumption of defoaming agents (the effective component of which is a surfactant), avoiding the negative impacts of excessive surfactant use during cell growth, and has significant application value.

Description

Technical Field

[0001] This invention relates to the field of amino acid production technology by fermentation, and in particular to a method for reducing foaming during L-tyrosine fermentation. Background Technology

[0002] L-Tyrosine is an aromatic amino acid that plays a vital role in human and animal metabolism and growth, and has wide applications in medicine, food, and chemical industries. In medicine, L-tyrosine can regulate mood and stimulate brain activity in the human body. In the food and feed industry, L-tyrosine is an important food and feed additive. In the chemical industry, L-tyrosine can be used in various novel polymers, coatings, adhesives, pharmaceuticals, bio-cosmetics, and health and nutrition products.

[0003] The main methods for producing L-tyrosine include protein hydrolysate extraction, chemical synthesis, enzymatic methods, and microbial fermentation. Microbial fermentation offers advantages over other methods, such as a shorter production cycle, lower energy consumption, and environmental friendliness. It is the most promising method for large-scale L-tyrosine production, emerging alongside the development of metabolic engineering and synthetic biology in recent years. However, during fermentation, tyrosine crystallization easily generates foam, leading to insufficient oxygen supply, affecting cell growth, and posing a significant risk of contamination. Therefore, current production methods typically use defoamers to reduce foaming. However, this method not only increases production costs, but also, since the active ingredient in defoamers is a surfactant, excessive addition can inhibit cell growth, thus affecting the yield and conversion rate of L-tyrosine. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for reducing foam during L-tyrosine fermentation.

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0006] A method for reducing foam during L-tyrosine fermentation involves adding tyrosine seed crystals when a large amount of foam is generated during the early stage of L-tyrosine fermentation, and using ultrasound-assisted tyrosine crystallization to increase crystal size, reduce foam generation, and enhance strain activity.

[0007] Preferably, in the above-mentioned method for reducing foam during L-tyrosine fermentation, when the L-tyrosine yield reaches 2 g / L in the early stage of fermentation, 0.2-0.5 g / L of pure tyrosine is added as tyrosine seed crystals, and the ultrasonic frequency for ultrasonic-assisted tyrosine crystallization is 12-24 kHz, and the ultrasonic time is 0.5 h-1 h.

[0008] Preferably, the method for reducing foam during the L-tyrosine fermentation process described above includes the following specific steps:

[0009] (1) Activation culture: Two generations of tyrosine-producing bacteria E.coli TYR03 were passaged on slant and activated culture was performed;

[0010] (2) Seed culture: All activated strains were inoculated into the seed tank to obtain seed solution, and the seed culture medium in the seed tank was adjusted with ammonia water and the pH was maintained at 6.7-7.0;

[0011] (3) Fermentation culture: Inoculate 15-20% of seed liquid into the fermenter, culture continuously, and add feed in between to obtain fermentation broth. Adjust the fermentation medium in the fermenter with ammonia water and maintain the pH at 6.7-7.0. Control the dissolved oxygen at 40-60%. Use defoaming electrodes to monitor the amount of foam in the fermenter in real time, and add defoaming agent in automatic mode using a peristaltic pump. When the L-tyrosine yield reaches 2g / L in the early stage of fermentation (this is the period when L-tyrosine fermentation produces a lot of foam), add 0.2-0.5g / L of pure tyrosine as tyrosine seed crystals, and use ultrasound-assisted crystallization. The ultrasound frequency is 12-24kHz and the ultrasound time is 0.5h-1h.

[0012] Preferably, in the above method for reducing foam during L-tyrosine fermentation, in step (3), when the L-tyrosine yield reaches 2 g / L in the early stage of fermentation, 0.4 g / L of pure L-tyrosine is added as tyrosine seed crystals, and ultrasonicated at a frequency of 16 kHz for 0.5 h.

[0013] Preferably, in the method for reducing foam during L-tyrosine fermentation, the slant culture medium used in step (1) is: glucose 1-2 g / L, peptone 6-10 g / L, beef extract 6-10 g / L, yeast extract 3-5 g / L, sodium chloride 3-5 g / L, agar powder 15-30 g / L, and the remainder is water.

[0014] Preferably, in the above method for reducing foam during L-tyrosine fermentation, the seed culture medium used in step (2) is: glucose 15-30 g / L, yeast extract powder 3-6 g / L, ammonium sulfate 1-5 g / L, potassium dihydrogen phosphate 1-5 g / L, anhydrous magnesium sulfate 0.5-2 g / L, ferrous sulfate heptahydrate 20-40 mg / L, manganese sulfate monohydrate 10-30 mg / L, V H 0.1-0.5 mg / L, V B1 0.5-1 mg / L, trace element mixture 1-2 ml / L, defoamer 0.5 g / L, the remainder is water.

[0015] Preferably, in the method for reducing foam during L-tyrosine fermentation described above, the fermentation medium used in step (3) is: glucose 20-40 g / L, yeast extract powder 3-5 g / L, ammonium sulfate 1-5 g / L, potassium dihydrogen phosphate 1-5 g / L, anhydrous magnesium sulfate 0.5-2 g / L, ferrous sulfate heptahydrate 30-60 mg / L, manganese sulfate monohydrate 20-40 mg / L, V H 0.1-0.5 mg / L, V B1 0.5-1 mg / L, trace element mixture 1-2 ml / L, the remainder is water.

[0016] Preferably, in the above-mentioned method for reducing foam during L-tyrosine fermentation, the trace element mixture contains ammonium molybdate 0.28 mg / L, boric acid 5 mg / L, and CoCl2. . 6H2O 1.4mg / L, MnSO4 . H2O 0.5 mg / L, CuSO4 . 7H2O 0.5 mg / L, ZnSO4 . 7H2O 0.6mg / L, the remainder is water (the above components are weighed as solids and dissolved in 1L of water, and stored at 4℃).

[0017] Preferably, in the above-mentioned method for reducing foam during L-tyrosine fermentation, the strategy for adding tyrosine seed crystals is to dry-heat sterilize the tyrosine seed crystals at 160°C for 2 hours and then add them to the fermenter through a heat exchanger.

[0018] Preferably, in the above-mentioned method for reducing foam during L-tyrosine fermentation, the ultrasonic-assisted crystallization is performed by inserting an ultrasonic rod into an ultrasonic jacket to perform ultrasonic-assisted crystallization on the fermentation broth.

[0019] Preferably, the method for reducing foam during the L-tyrosine fermentation process described above includes the following fermentation conditions: inoculum size of 600 ml seed culture, fermentation volume of 3 L, culture temperature of 37°C, pH of 6.8-7.0, rotation speed linked to dissolved oxygen, and fermentation time of 35 h.

[0020] Beneficial effects:

[0021] The above-mentioned method for reducing foam during L-tyrosine fermentation involves adding an appropriate amount of L-tyrosine seed crystals during the initial stage of L-tyrosine crystallization, when a large amount of foam is generated during L-tyrosine fermentation, to increase the crystal size and accelerate the crystallization rate. At the same time, ultrasonic-assisted crystallization is used to increase the crystal size and optimize the crystal form, thereby reducing the problem of excessive foam during the fermentation process caused by L-tyrosine crystallization.

[0022] The method solves the problem of severe foaming in the fermentation broth caused by the fine crystals of L-tyrosine during fermentation; at the same time, it reduces the consumption of defoamer (the effective ingredient is a surfactant), avoiding the negative effects of excessive use of surfactant during cell growth, and has important application value. Detailed Implementation

[0023] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0024] The tyrosine-producing strains used in the following examples were all E. coli TYR03, purchased from the Metabolic Engineering Laboratory of Tianjin University of Science and Technology. The trace element mixture used contained ammonium molybdate 0.28 mg / L, boric acid 5 mg / L, and CoCl2. . 6H2O 1.4 mg / L, MnSO4 . H2O 0.5mg / L, CuSO4 . 7H2O 0.5mg / L, ZnSO4 . 7H2O 0.6 mg / L, the remainder being water (the above components were weighed as solids and dissolved in 1 L of water, then stored at 4℃). The fermentation conditions were: inoculum size 600 ml seed culture, fermentation volume 3 L, culture temperature 37℃, pH 6.8-7.0, agitation speed linked to dissolved oxygen, and fermentation time 35 h.

[0025] Example 1

[0026] A method for reducing foam during L-tyrosine fermentation, comprising the following steps:

[0027] (1) Activation culture: Take out the E. coli TYR03 preservation tube of tyrosine-producing bacteria from the -80℃ freezer, pass it to two generations of slant culture, and activate culture. The slant culture medium used includes: glucose 1g / L, peptone 6g / L, beef extract 6g / L, yeast extract powder 5g / L, sodium chloride 3g / L, and agar powder 25g / L.

[0028] (2) Seed culture: All activated strains were inoculated into a seed tank to obtain seed culture. The seed culture medium used was: glucose 30 g / L, yeast extract powder 3 g / L, ammonium sulfate 3 g / L, potassium dihydrogen phosphate 4 g / L, anhydrous magnesium sulfate 1.5 g / L, ferrous sulfate heptahydrate 20 mg / L, manganese sulfate monohydrate 20 mg / L, V H 0.4 mg / L, V B1 0.5 mg / L, 2 ml / L of trace element mixture, two drops of antifoaming agent (about 0.5 g / L), and adjust the culture medium with ammonia water to maintain the pH at 6.7–7.0;

[0029] (3) Fermentation culture: Inoculate 15% seed culture into the fermenter and culture continuously. Dissolved oxygen is controlled at 40%. The pH of the fermenter is adjusted and maintained at 7.0 with ammonia water. The amount of foam in the fermenter is monitored in real time using an antifoaming electrode, and antifoaming agent is added automatically using a peristaltic pump. The system is 3L. The fermentation culture medium is: glucose 25g / L, yeast extract powder 3g / L, ammonium sulfate 3g / L, potassium dihydrogen phosphate 4g / L, anhydrous magnesium sulfate 2g / L, ferrous sulfate heptahydrate 40mg / L, manganese sulfate monohydrate 30mg / L, V H 0.2 mg / L, V B1 0.5 mg / L, trace element mixture 2 ml / L.

[0030] Example 2

[0031] A method for reducing foam during L-tyrosine fermentation, referring to Example 1, involves adding 0.2 g / L of L-tyrosine seed crystals when the L-tyrosine yield reaches 2 g / L during fermentation.

[0032] Example 3

[0033] A method for reducing foam in the L-tyrosine fermentation process, referring to Example 2, but with the amount of L-tyrosine seed crystals added in Example 2 increased from 0.2 g / L to 0.3 g / L.

[0034] Example 4

[0035] A method for reducing foam in the L-tyrosine fermentation process, referring to Example 3, but with the amount of L-tyrosine seed crystals added in Example 3 increased from 0.3 g / L to 0.4 g / L.

[0036] Example 5

[0037] A method for reducing foam in the L-tyrosine fermentation process, referring to Example 4, but with the amount of L-tyrosine seed crystals added in Example 4 increased from 0.4 g / L to 0.5 g / L.

[0038] Table 1 Comparison of different seed crystal addition amounts with yield and defoamer usage in the examples

[0039] Example number 1 2 3 4 5 Defoamer usage (ml) 6.6 4.6 3.5 2.8 2.9 Yield (g / L) 49.6 52.1 52.6 53.1 53.2

[0040] As shown in Table 1, the yields of Examples 2, 3, 4, and 5 were relatively similar; the amount of defoamer used in Examples 4 and 5 was lower, while the amount of seed crystals added in Example 4 was even less than that in Example 5. Therefore, the amount of L-tyrosine seed crystals added in subsequent examples was 0.4 g / L.

[0041] Example 6

[0042] A method for reducing foam during L-tyrosine fermentation, comprising the following steps:

[0043] (1) Activation culture: Take out the E. coli TYR03 tyrosine-producing bacteria preservation tube from the -80℃ freezer, pass it to two generations of slant culture, and activate culture. The slant culture medium used includes: glucose 1-2g / L, peptone 6-10g / L, beef extract 6-10g / L, yeast extract powder 3-5g / L, sodium chloride 3-5g / L, and agar powder 15-30g / L.

[0044] (2) Seed culture: All activated strains were inoculated into a seed tank to obtain seed culture. The seed culture medium used was: glucose 20 g / L, yeast extract powder 3 g / L, ammonium sulfate 3 g / L, potassium dihydrogen phosphate 4 g / L, anhydrous magnesium sulfate 1.5 g / L, ferrous sulfate heptahydrate 20 mg / L, manganese sulfate monohydrate 20 mg / L, V H 0.4 mg / L, V B1 0.5 mg / L, 2 ml / L of trace element mixture, two drops of defoamer, and adjust the culture medium with ammonia water to maintain the pH at 6.7–7.0;

[0045] (3) Fermentation culture: 15% seed culture was inoculated into the fermenter and continuously cultured. Dissolved oxygen was controlled at 40%. The pH of the fermenter was adjusted and maintained at 7.0 with ammonia water. The amount of foam in the fermenter was monitored in real time using an antifoaming electrode, and antifoaming agent was added automatically using a peristaltic pump in 3L system. When the L-tyrosine yield reached 2g / L during fermentation, 0.4g / L of L-tyrosine seed crystals were added. After adding L-tyrosine seed crystals, the fermentation broth was crystallized using an ultrasonic rod at a frequency of 12kHz for 0.5h. The fermentation culture medium was: glucose 25g / L, yeast extract powder 3g / L, ammonium sulfate 3g / L, potassium dihydrogen phosphate 4g / L, anhydrous magnesium sulfate 2g / L, ferrous sulfate heptahydrate 40mg / L, manganese sulfate monohydrate 30mg / L, V H 0.2 mg / L, V B1 0.5 mg / L, trace element mixture 2 ml / L.

[0046] Example 7

[0047] A method for reducing foam in an L-tyrosine fermentation process, referring to Example 6, but with the ultrasonic frequency increased from 12 kHz to 16 kHz.

[0048] Example 8

[0049] A method for reducing foam in an L-tyrosine fermentation process, referring to Example 7, but with the ultrasonic frequency increased from 16 kHz to 20 kHz.

[0050] Example 9

[0051] A method for reducing foam in an L-tyrosine fermentation process, referring to Example 8, but with the ultrasonic frequency increased from 20 kHz to 24 kHz.

[0052] Table 2 Comparison of different ultrasonic frequencies, defoamer usage, and yield.

[0053] Example No. 6 7 8 9 Defoamer usage (ml) 0.9 0.6 0.6 0.9 Yield (g / L) 54.6 54.8 52.9 51.7

[0054] As shown in Table 2, the amount of defoamer used in Examples 6, 7, 8, and 9 was relatively low, with Example 7 having the highest yield. Therefore, the ultrasonic frequency in subsequent examples was 16 kHz.

[0055] Example 10

[0056] A method for reducing foam in L-tyrosine fermentation process, referring to Example 6, differs only in the ultrasonic time, which is 1 hour in Example 10.

[0057] Table 3 Comparison of the effects of different ultrasonic times on defoamer usage, bacterial OD, and yield.

[0058] Example No. 4 7 10 Ultrasound time (h) 0 0.5 1 Defoamer usage (ml) 2.8 0.6 0.5 <![CDATA[OD 600 ]]> 100.5 110.8 98.4 Yield (g / L) 53.1 54.8 50.3

[0059] Table 3 shows that the highest yield was achieved with 0.5 hours of ultrasonic treatment, which required less defoamer and did not have a negative impact on bacterial growth.

[0060] In summary, by adding 0.4 g / L of L-tyrosine seed crystals and sonicating at 16 kHz for 0.5 hours during the early stage of L-tyrosine fermentation when the yield reaches 2.0 g / L, the amount of foam during fermentation can be effectively reduced, solving the problem of decreased cell activity caused by defoamers and increasing the yield of L-tyrosine.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. The construction steps of the strain of the present invention are not in any particular order. All improvements and modifications made by those skilled in the art based on the method of the present invention or the method of the present invention are considered to be within the scope of protection of the present invention.

Claims

1. A method for reducing foam during L-tyrosine fermentation, characterized in that: The specific steps are as follows: (1) Activation culture: Tyrosine-producing bacteria E. coli TYR03 Two generations of slant culture were used for activation. (2) Seed culture: All activated strains were inoculated into the seed tank to obtain seed solution, and the seed culture medium in the seed tank was adjusted with ammonia water and the pH was maintained at 6.7~7.0; (3) Fermentation culture: Inoculate 15-20% of seed liquid into the fermenter, culture continuously, and add feed in between to obtain fermentation broth. Adjust the fermentation medium in the fermenter with ammonia water and maintain the pH at 6.7-7.

0. Control the dissolved oxygen at 40-60%. Use defoaming electrodes to monitor the amount of foam in the fermenter in real time, and add defoaming agent in automatic mode using a peristaltic pump. When the L-tyrosine yield reaches 2g / L in the early stage of fermentation, add 0.2-0.5g / L of pure tyrosine as tyrosine seed crystals, and use ultrasound-assisted crystallization with an ultrasound frequency of 12-24kHz and an ultrasound time of 0.5h-1h.

2. The method for reducing foam during L-tyrosine fermentation according to claim 1, characterized in that: In step (3), when the L-tyrosine yield reaches 2 g / L in the early stage of fermentation, 0.4 g / L of pure L-tyrosine is added as tyrosine seed crystals, and the mixture is ultrasonicated at a frequency of 16 kHz for 0.5 h.

3. The method for reducing foam during L-tyrosine fermentation according to claim 1, characterized in that: The slant culture medium used in step (1) is: glucose 1-2g / L, peptone 6-10g / L, beef extract 6-10g / L, yeast extract powder 3-5g / L, sodium chloride 3-5g / L, agar powder 15-30g / L, and the remainder is water.

4. The method for reducing foam during L-tyrosine fermentation according to claim 1, characterized in that: The seed culture medium used in step (2) is: glucose 15-30 g / L, yeast extract powder 3-6 g / L, ammonium sulfate 1-5 g / L, potassium dihydrogen phosphate 1-5 g / L, anhydrous magnesium sulfate 0.5-2 g / L, ferrous sulfate heptahydrate 20-40 mg / L, manganese sulfate monohydrate 10-30 mg / L, V H 0.1-0.5 mg / L, V B1 0.5-1 mg / L, trace element mixture 1-2 ml / L, defoamer 0.5 g / L, the remainder is water.

5. The method for reducing foam during L-tyrosine fermentation according to claim 1, characterized in that: The fermentation medium used in step (3) is: glucose 20-40 g / L, yeast extract powder 3-5 g / L, ammonium sulfate 1-5 g / L, potassium dihydrogen phosphate 1-5 g / L, anhydrous magnesium sulfate 0.5-2 g / L, ferrous sulfate heptahydrate 30-60 mg / L, manganese sulfate monohydrate 20-40 mg / L, V H 0.1-0.5 mg / L, V B1 0.5-1 mg / L, trace element mixture 1-2 ml / L, the remainder is water.

6. The method for reducing foam during L-tyrosine fermentation according to claim 4 or 5, characterized in that: The trace element mixture contains ammonium molybdate 0.28 mg / L, boric acid 5 mg / L, CoCl2·6H2O 1.4 mg / L, MnSO4·H2O 0.5 mg / L, CuSO4·7H2O 0.5 mg / L, ZnSO4·7H2O 0.6 mg / L, and the remainder is water.

7. The method for reducing foam during L-tyrosine fermentation according to claim 1, characterized in that: The strategy for adding tyrosine seed crystals is to dry-heat sterilize the tyrosine seed crystals at 160°C for 2 hours and then add them to the fermenter after passing them through a heat ring.

8. The method for reducing foam during L-tyrosine fermentation according to claim 1, characterized in that: The fermentation conditions were as follows: inoculum volume 600ml seed culture, fermentation volume 3L, culture temperature 37℃, pH 6.8-7.0, rotation speed and dissolved oxygen linkage, and fermentation time 35h.