A method for producing erythritol through continuous fermentation

Abstract

The present invention discloses a method for producing erythritol through continuous fermentation. It relates to the field of fermentation engineering technology. The method comprises placing a seed liquid in a culture medium and establishing a continuous fermentation mechanism with CO2 feedback control. The present invention produces erythritol by continuous fermentation, and in the late fermentation stage, feeds are added and discharged simultaneously to maintain a constant glucose and volume of the fermentation liquid. In the continuous fermentation stage, the feed is discharged at the same rate as the feed. On the one hand, by releasing part of the bacterial cells, the growth rate of the bacterial cells is reduced, the consumption of the substrate glucose by bacterial growth is reduced, and the conversion rate of erythritol is improved. On the other hand, the bacterial cells can be reused multiple times, shortening the fermentation cycle of new tanks. In addition, combined with tail gas analysis, the yeast growth status is timely judged. Based on the tail carbon value as the control parameter, the respiratory status of the bacterial cells during the fermentation process is analyzed. By adjusting the feed flow acceleration rate, the bacterial cell growth rate and respiratory frequency are effectively controlled, thereby promoting an increase in the conversion rate of glucose to erythritol.

Description

Technical Field

[0001] The present invention relates to the technical field of fermentation engineering, and more particularly to a method for producing erythritol through continuous fermentation. Background Art

[0002] Erythritol is widely used as a low-calorie sweetener and a diluent for high-intensity sweeteners. It can be used in chocolate, baked goods, candy, table sugar, soft drinks, etc.

[0003] Generally speaking, erythritol can be produced by biological extraction, chemical synthesis and microbial fermentation. The biological extraction method refers to the extraction of erythritol from plants such as seaweed and moss by certain chemical and physical means. The method is cumbersome, the process is complex, the cost is high, and the yield is low. The chemical synthesis method generally refers to the treatment of starch or cellulose materials oxidized by periodic acid by chemical reduction of raw materials to obtain erythritol. The synthesis route is long, the reaction conditions are strict, the pollution problem is prominent, and the quality of the obtained product is unstable. There are many types of by-products, and purification and separation are difficult. The microbial fermentation method refers to the production of erythritol by microbial metabolic fermentation under a suitable culture environment. It has obvious production advantages and is the most ideal method for producing erythritol. However, the yield of erythritol by microbial fermentation in the prior art is low.

[0004] Therefore, how to provide a method for increasing the yield of erythritol produced by microbial fermentation is a problem that those skilled in the art urgently need to solve. Summary of the Invention

[0005] In view of this, the present invention provides a method for producing erythritol by continuous fermentation.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for producing erythritol through continuous fermentation, comprising the following steps:

[0008] (1) placing a 10% inoculum of Candida lipolytica seed solution into a sterilized fermentation medium;

[0009] (2) Establishing a CO2 feedback control continuous fermentation mechanism:

[0010] 1) Bacteria growth stage: adjust the speed to control the oxygen consumption rate of Candida lipolytica to 0.4-0.7 molO2 / L / h and the tail carbon to 0.3-0.4%. After 6-8 hours of fermentation, Candida lipolytica enters the logarithmic growth phase and the tail carbon value rises parabolically to 2.4-2.6%. After 40-50 hours of fermentation, the OD 600 =30-35, the tail carbon content decreases from 2.4-2.6% to 2.2-2.4%, entering the erythritol production stage;

[0011] 2) Erythritol production stage: fermentation to 60-70h, OD 600 =45-50, the glucose concentration in the tank dropped to 15 g / L, and feeding and discharging of the fermentation liquid were started. The initial flow rate was 2.75-2.95 g / L / h, and the glucose concentration in the tank was controlled at 10-15 g / L. The fermentation liquid discharge rate was kept consistent with the feed flow rate;

[0012] 3) Continuous fermentation stage: According to the CO2 data in the tail gas, the flow addition rate is adjusted: when the tail carbon value is between 2.2% and 2.4%, the flow rate is maintained at 2.75-2.95 g / L / h; when the tail carbon value is greater than 2.4%, the flow rate is reduced to 2.5-2.75 g / L / h; when the tail carbon value is less than 2.2%, the flow rate is increased to 2.95-3.1 g / L / h; when the fermentation reaches 280-285 hours, the flow addition rate is stopped, and the fermentation is continued to 300-310 hours. When the glucose concentration in the tank is less than 5 g / L, the fermentation is terminated to obtain erythritol fermentation liquid.

[0013] By adopting the preparation process of the present invention, the erythritol concentration in the fermentation broth in step 3) reaches 245-252 g / L, and the conversion rate is calculated to be 71-72%.

[0014] Furthermore, the tail carbon data is collected and analyzed online in real time on the intake and exhaust gases using Sunny Optical Hengping tail gas mass spectrometer.

[0015] Preferably: Step (1) Preparation method of Candida lipolytica seed solution:

[0016] 21) Primary seed solution culture: Prepare a primary seed culture medium, inoculate Candida lipolytica and culture at a constant temperature to obtain a primary seed solution;

[0017] 22) Secondary seed liquid culture: prepare a secondary seed tank culture medium, inoculate the primary seed liquid into the secondary seed tank culture medium, and culture the Candida lipolytica seed liquid.

[0018] Preferably: in step 21), the primary seed culture medium comprises 5 g / L yeast powder, 10 g / L glucose, and 2 g / L diammonium phosphate; the inoculation amount is 1%; in step 22), the secondary seed tank culture medium comprises 5 g / L yeast powder, 20 g / L glucose, and 1 g / L diammonium phosphate, and the inoculation amount is 5%.

[0019] Preferred conditions after inoculation in step (1): the fermentation tank is adjusted to pH 4.0 with 15% ammonia water, the ventilation ratio is 1VVM, the fermentation tank pressure is 0.025 MPa, and the DO is maintained at 40-50%;

[0020] Fermentation medium: glucose 350 g / L, yeast powder 5 g / L, potassium dihydrogen phosphate 10 g / L, sodium chloride 5 g / L, magnesium sulfate heptahydrate 0.247 g / L, diammonium hydrogen phosphate 5 g / L.

[0021] Preferably, the fermentation medium is prepared by:

[0022] 51) Weigh and sterilize: yeast powder, potassium dihydrogen phosphate, sodium chloride, glucose, and magnesium sulfate heptahydrate, dissolve in water, transfer to a fermentation tank, and sterilize;

[0023] 52) Cooling: Place the sterilized fermentation tank in a cool place;

[0024] 53) Mixing: Weigh diammonium phosphate, dissolve it in water, sterilize it, and add the sterilized diammonium phosphate to the fermenter before inoculation to obtain the fermentation medium.

[0025] Furthermore, the mass volume ratio of yeast powder, potassium dihydrogen phosphate, sodium chloride, glucose, magnesium sulfate heptahydrate, diammonium hydrogen phosphate and the fermentation medium after inoculation is: 50g:100g:50g:3500g:2.47g:50g:10L.

[0026] Preferably, a portion of the fermentation broth discharged during the feeding process of step (2) is used as a new tank of Candida lipolytica seed liquid and is inoculated into a new sterilized fermentation medium at a rate of 10% to continue culturing, and steps 1) to 3) are repeated, and the remaining portion is mixed with the erythritol fermentation broth in step 3) and processed together.

[0027] Preferably, the ingredients of the feed in step (2) are: 500 g / L glucose, 0.21 g / L magnesium sulfate heptahydrate, and 1.2 g / L nitrogen source.

[0028] Furthermore, the nitrogen source 1.2 g / L is converted according to the nitrogen content of each raw material.

[0029] Preferred nitrogen sources include, but are not limited to, urea, diammonium phosphate, yeast powder or peptone.

[0030] Preferably, the erythritol fermentation broth obtained in step 3) is centrifuged, decolorized, filtered, cross-linked, separated, concentrated, and crystallized to obtain finished erythritol.

[0031] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a method for producing erythritol by continuous fermentation, which achieves the following technical effects:

[0032] (1) The present invention uses a continuous fermentation method to produce erythritol. In the late fermentation stage, the feed is added and discharged simultaneously to maintain a constant glucose content and volume in the fermentation broth. During this stage, the fermentation broth is diluted by adding new feed, which reduces the concentration of by-products in the fermentation broth, thereby reducing the inhibitory effect of by-products on erythritol production. In addition, the present invention introduces some magnesium sulfate and nitrogen source while adding fresh sugar solution, thereby increasing the osmotic pressure of the fermentation broth, further reducing the formation of by-products, and increasing the yield and purity of erythritol, reaching a purity of 100%.

[0033] (2) The present invention discharges the material at the same rate as the feeding during the continuous fermentation stage. On the one hand, by releasing part of the bacterial cells, the growth rate of the bacterial cells is reduced, the consumption of the substrate glucose by the bacterial cells is reduced, and the conversion rate of erythritol is improved; on the other hand, the released fermentation liquid can also be used as the seed liquid of a new fermentation tank, realizing the repeated use of the bacterial cells, reducing the preparation process of the seed liquid, shortening the fermentation cycle of the new tank, and simplifying the production process.

[0034] (3) The present invention combines tail gas analysis to timely judge the yeast growth situation. Based on the CO2 (tail carbon) value in the tail gas as a control parameter, the respiratory status of the bacteria during the fermentation process is analyzed. By adjusting the feed flow acceleration rate, the growth rate and respiratory frequency of the bacteria are effectively controlled, preventing the bacteria from consuming too much glucose during growth and respiration, and promoting the increase in the conversion rate of glucose to erythritol. During the continuous fermentation stage, the tail carbon value increases, indicating that the nutrients of Candida lipolytica are too abundant, the bacteria consume too much glucose during respiration, and the conversion of glucose to erythritol is reduced. It is necessary to reduce the feed flow acceleration to inhibit yeast growth; the tail carbon value decreases, indicating that the nutrients of Candida lipolytica are reduced, the bacteria respiration is inhibited, and the production of erythritol increases. It is necessary to appropriately increase the feed flow acceleration to maintain the glucose concentration and promote product production. According to the change of the tail carbon trend, the feed flow acceleration rate can be quickly adjusted to reduce the large accumulation of glucose in the fermentation broth, reduce the bacterial concentration of Candida lipolytica, and increase the yield of erythritol, achieving efficient conversion of glucose to erythritol with a conversion rate of 72%. DETAILED DESCRIPTION

[0035] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of protection of the present invention.

[0036] The embodiment of the present invention discloses a method for producing erythritol by continuous fermentation.

[0037] In the examples, Candida lipolytica was purchased from Shandong Food Fermentation Industry Research and Design Institute. All unmentioned raw materials were conventional raw materials, and all unmentioned methods were conventional experimental methods, which will not be described in detail here.

[0038] Example 1

[0039] Preparation of seed solution

[0040] (1) Culture of primary seed solution (150 mL / 500 mL triangular flask)

[0041] Prepare a first-level seed culture medium (yeast powder 5g / L, glucose 10g / L, diammonium phosphate 2g / L, 115℃, sterilize for 20min), inoculate Candida lipolytica at 1% inoculum, and culture in a constant temperature shaker at 35℃, 200rpm for 24h to obtain a first-level seed solution, and measure its OD 600 =5~6.

[0042] (2) Secondary seed solution (3L / 5L triangular flask) culture

[0043] Prepare a secondary seed tank culture medium (5 g / L yeast powder, 20 g / L glucose, 1 g / L diammonium phosphate), transfer the primary seed solution to the secondary seed tank culture medium at a 5% inoculum volume, control the culture temperature to 35°C, the culture time to 16 h, the DO40-50%, adjust the pH value to 5.0 with 15% ammonia water, the ventilation ratio to 1 VVM, the fermentation tank pressure to 0.025 MPa, culture for 15-16 h, and obtain the Candida lipolytica seed solution. Measure the OD 600 =10~12.

[0044] Example 2

[0045] Preparation of fermentation medium

[0046] ① Weigh and sterilize: 50g yeast powder, 100g potassium dihydrogen phosphate, 50g sodium chloride, 3500g glucose, and 2.47g magnesium sulfate heptahydrate. Dissolve them in appropriate amount of water and transfer them to a fermentation tank. Adjust the volume to 8.9L and sterilize at 115℃ for 20min.

[0047] ②Cooling: Place the sterilized fermentation tank at room temperature and cool it to 35°C;

[0048] ③ Mixing: Weigh 50 g of diammonium hydrogen phosphate, dilute to 100 mL with water, sterilize at 115°C for 20 min, and add the sterilized diammonium hydrogen phosphate to the fermentation tank before inoculation to obtain fermentation medium (at the time of inoculation, 1 L of Candida lipolytica seed liquid, a total of 10 L).

[0049] Erythritol was produced by fermentation using the Candida lipolytica seed solution prepared in Example 1 and the fermentation medium prepared in Example 2, as shown in Examples 3 to 7 and Comparative Examples 1 to 4.

[0050] Example 3

[0051] The fermentation medium was inoculated with OD 600 =10.3 of Candida lipolytica seed liquid, the fermentation tank was initially adjusted to pH 4.0 with ammonia (15%), the ventilation ratio was 1VVM, the fermentation tank pressure was 0.025Mpa, and DO (dissolved oxygen) was maintained at 40-50%. At the beginning of fermentation, the bacteria entered the lag phase and grew slowly. The speed was adjusted to control the oxygen consumption rate of Candida lipolytica to 0.4molO2 / L / h, and the tail carbon was maintained at 0.3%. After 8 hours, Candida lipolytica entered the logarithmic growth phase, and the tail carbon value rose to 2.5% in the form of a parabola; after 40 hours of fermentation, OD 600 =35, the tail carbon value dropped from 2.45% to 2.4%, entering the erythritol production stage.

[0052] Fermentation to 65h, OD 600 =45, the glucose concentration in the tank dropped to 15 g / L, the tail carbon value was 2.4%, and feed was started (glucose 500 g / L, magnesium sulfate heptahydrate 0.21 g / L, urea 2.58 g / L), with an initial flow rate of 2.75 g / L / h. The glucose concentration in the tank was controlled at 10-15 g / L, and the fermentation liquid discharge rate was kept consistent with the feed flow rate;

[0053] Continuous fermentation stage: fermentation to 102h, tail carbon increased to 2.7%, reduce the feed flow acceleration rate to 2.5g / L / h; fermentation to 145h, tail carbon decreased to 2.13%, increase the feed flow acceleration rate to 3.1g / L / h; fermentation to 203h, tail carbon increased to 2.67%, reduce the feed flow acceleration rate to 2.6g / L / h; fermentation to 261h, tail carbon decreased to 2.1%, increase the feed flow acceleration rate to 2.97g / L / h; fermentation to 282h, stop feeding, fermentation to 300h, OD 600 =50, the glucose concentration in the tank was 2.5 g / L, and the fermentation was completed. The erythritol concentration in the fermentation broth reached 245 g / L, and the conversion rate was calculated to be 71.0%.

[0054] Example 4

[0055] The fermentation medium was inoculated with OD 600=11 Candida lipolytica seed liquid, the fermentation tank was initially adjusted to pH 4.0 with ammonia (15%), the ventilation ratio was 1VVM, the fermentation tank pressure was 0.025Mpa, and DO was maintained at 40-50%. At the beginning of fermentation, the bacteria entered the lag phase and grew slowly. The speed was adjusted to control the oxygen consumption rate of Candida lipolytica to 0.55molO2 / L / h, and the tail carbon was maintained at 0.34%. After 7h, Candida lipolytica entered the logarithmic growth phase, and the tail carbon value rose to 2.4% in the form of a parabola; after 40h of fermentation, OD 600 =33, the tail carbon content dropped from 2.43% to 2.37%, entering the erythritol production stage.

[0056] Fermentation to 60h, OD 600 =47, the glucose concentration in the tank dropped to 15 g / L, the tail carbon value was 2.36%, and feeding was started (glucose 500 g / L, magnesium sulfate heptahydrate 0.21 g / L, urea 2.58 g / L), with an initial flow rate of 2.85 g / L / h. The glucose concentration in the tank was controlled at 10-15 g / L, and the fermentation liquid discharge rate was kept consistent with the feed flow rate;

[0057] Continuous fermentation stage: fermentation to 100h, tail carbon increased to 2.6%, reduce the feed flow acceleration rate to 2.65g / L / h; fermentation to 146h, tail carbon decreased to 2.08%, increase the feed flow acceleration rate to 3.0g / L / h; fermentation to 189h, tail carbon decreased to 1.96%, increase the feed flow acceleration rate to 3.1g / L / h; fermentation to 246h, tail carbon increased to 2.75%, reduce the feed flow acceleration rate to 2.5g / L / h; fermentation to 280h, stop feeding, fermentation to 302h, OD 600 =47, the glucose concentration in the tank was 2.0 g / L, and the fermentation was completed. The erythritol concentration in the fermentation broth reached 247 g / L, and the conversion rate was calculated to be 71.3%.

[0058] Example 5

[0059] The fermentation medium was inoculated with OD 600 =10.5 of Candida lipolytica seed liquid, the fermentation tank was initially adjusted to pH 4.0 with ammonia (15%), the ventilation ratio was 1VVM, the fermentation tank pressure was 0.025Mpa, and DO was maintained at 40-50%. At the beginning of fermentation, the bacteria entered the lag phase and grew slowly. The speed was adjusted to control the oxygen consumption rate of Candida lipolytica to 0.70molO2 / L / h, and the tail carbon was maintained at 0.4%. After 6 hours, Candida lipolytica entered the logarithmic growth phase, and the tail carbon value rose to 2.6% in the form of a parabola; after 40 hours of fermentation, OD 600 =30, the tail carbon content dropped from 2.45% to 2.35%, entering the erythritol production stage.

[0060] Fermentation to 70h, OD 600 =50, the glucose concentration in the tank dropped to 15 g / L, the tail carbon value was 2.28%, and feeding was started (glucose 500 g / L, magnesium sulfate heptahydrate 0.21 g / L, urea 2.58 g / L), with an initial flow rate of 2.95 g / L / h. The glucose concentration in the tank was controlled at 10-15 g / L, and the fermentation liquid discharge rate was kept consistent with the feed flow rate;

[0061] Continuous fermentation stage: fermentation to 110h, tail carbon increased to 2.42%, reduce the feed flow acceleration rate to 2.75g / L / h; fermentation to 156h, tail carbon decreased to 2.25%, increase the feed flow acceleration rate to 2.95g / L / h; fermentation to 200h, tail carbon decreased to 1.99%, increase the feed flow acceleration rate to 3.05g / L / h; fermentation to 262h, tail carbon increased to 2.15%, reduce the feed flow acceleration rate to 2.97g / L / h; fermentation to 285h, stop feeding, fermentation to 302h, OD 600 =47.2, the glucose concentration in the tank was 3.0 g / L, and the fermentation was completed. The erythritol concentration in the fermentation broth reached 252 g / L, and the conversion rate was calculated to be 72.0%.

[0062] Example 6

[0063] The fermentation medium was inoculated with OD 600 =11.5 Candida lipolytica seed liquid, the fermentation tank was initially adjusted to pH 4.0 with ammonia (15%), the ventilation ratio was 1VVM, the fermentation tank pressure was 0.025Mpa, and DO was maintained at 40-50%. At the beginning of fermentation, the bacteria entered the lag phase and grew slowly. The speed was adjusted to control the oxygen consumption rate of Candida lipolytica to 0.45molO2 / L / h, and the tail carbon was maintained at 0.35%. After 7.5h, Candida lipolytica entered the logarithmic growth phase, and the tail carbon value rose parabolically to 2.53%; after 40h of fermentation, OD 600 =34, the tail carbon content dropped from 2.5% to 2.2%, entering the erythritol production stage.

[0064] Fermentation to 63h, OD 600 =45, the glucose concentration in the tank dropped to 15 g / L, the tail carbon value was 2.37, and feed was started (glucose 500 g / L, magnesium sulfate heptahydrate 0.21 g / L, diammonium phosphate 5.64 g / L), with an initial flow rate of 2.80 g / L / h. The glucose concentration in the tank was controlled at 10-15 g / L, and the fermentation liquid discharge rate was kept consistent with the feed flow rate;

[0065] Continuous fermentation stage: fermentation to 98h, tail carbon increased to 2.59%, reduce the feed flow acceleration rate to 2.60g / L / h; fermentation to 150h, tail carbon decreased to 2.05%, increase the feed flow acceleration rate to 3.05g / L / h; fermentation to 202h, tail carbon increased to 2.36%, reduce the feed flow acceleration rate to 2.85g / L / h; fermentation to 247h, tail carbon maintained at 2.35%, feed flow acceleration rate unchanged; fermentation to 283h, stop feeding, fermentation to 305h, OD 600 =48.5, the glucose concentration in the tank was 4.7 g / L, and the fermentation was completed. The erythritol concentration in the fermentation broth reached 250 g / L, and the conversion rate was calculated to be 71.7%.

[0066] Example 7

[0067] The fermentation medium was inoculated with OD 600 =11.3 of Candida lipolytica seed liquid, the fermentation tank was initially adjusted to pH 4.0 with ammonia (15%), the ventilation ratio was 1VVM, the fermentation tank pressure was 0.025Mpa, and DO was maintained at 40-50%. At the beginning of fermentation, the bacteria entered the lag phase and grew slowly. The speed was adjusted to control the oxygen consumption rate of Candida lipolytica to 0.50molO2 / L / h, and the tail carbon was maintained at 0.33%. After 7.5h, Candida lipolytica entered the logarithmic growth phase, and the tail carbon value rose parabolically to 2.43%; after 40h of fermentation, OD 600 =34.5, the tail carbon content dropped from 2.53% to 2.4%, and the erythritol production stage began.

[0068] Fermentation to 67h, OD 600 =48, the glucose concentration in the tank dropped to 15 g / L, the tail carbon value was 2.35%, and feed was started (glucose 500 g / L, magnesium sulfate heptahydrate 0.21 g / L, yeast powder 1.2 g / L), with an initial feed rate of 2.80 g / L / h. The glucose concentration in the tank was controlled at 10-15 g / L, and the fermentation liquid discharge rate was kept consistent with the feed feed rate;

[0069] Continuous fermentation stage: fermentation to 105h, tail carbon increased to 2.71%, reduce the feed flow acceleration rate to 2.65g / L / h; fermentation to 162h, tail carbon decreased to 2.0%, increase the feed flow acceleration rate to 2.97g / L / h; fermentation to 206h, tail carbon increased to 2.15%, reduce the feed flow acceleration rate to 2.96g / L / h; fermentation to 249h, tail carbon increased to 2.47%, reduce the feed flow acceleration rate to 2.73g / L / h; fermentation to 285h, stop feeding, fermentation to 304h, OD 600 =46.5, the glucose concentration in the tank was 4.0 g / L, and the fermentation was completed. The erythritol concentration in the fermentation liquid reached 249 g / L, and the conversion rate was calculated to be 71.5%.

[0070] Comparative experiment

[0071] The main difference between the comparative example and the example is that the comparative example uses constant dissolved oxygen to control the fermentation process and uses glucose concentration feedback in the fermenter to adjust the feeding rate after feeding. The example uses tail carbon analysis to control the fermentation process and adjust the feeding rate. Clearly, tail carbon analysis is superior to constant dissolved oxygen and glucose concentration feedback.

[0072] Comparative Example 1

[0073] Inoculate at 10% of the inoculum volume. 600 =10.7 seed liquid, the fermentation tank was initially adjusted to pH 4.0 with ammonia (15%), the ventilation ratio was 1VVM, the fermentation tank pressure was 0.025Mpa, and the DO was maintained at 40-50%. When the bacteria grew to the DO of the fermentation tank of 12-18%, the speed and air volume were adjusted to control the DO to be maintained at 12-18%; fermentation was continued for 45h, and the OD 600 =32.4, entering the erythritol production stage, adjusting the speed and air volume to control DO to maintain at 20-30%.

[0074] Fermentation to 62h, OD 600 =45, the glucose concentration in the tank dropped to 15g / L, and feeding was started (glucose 500g / L), with an initial flow rate of 2.75g / L / h; continued fermentation, adjusted the flow rate to control the glucose concentration in the tank to 10-15g / L, and the fermentation liquid discharge rate was consistent with the feed flow rate; fermentation was stopped after 280h, and feeding was stopped after 314h, OD 600 =63.4, the glucose concentration in the tank was 4.5 g / L, and the fermentation was completed. The erythritol concentration in the fermentation liquid reached 232 g / L, and the conversion rate was calculated to be 66.5%.

[0075] Comparative Example 2

[0076] Inoculate at 10% of the inoculum volume. 600 =11.6 seed liquid, the fermentation tank was initially adjusted to pH 4.0 with ammonia (15%), the ventilation ratio was 1VVM, the fermentation tank pressure was 0.025Mpa, and the dissolved oxygen DO was maintained at 40-50%. When the bacteria grew to the fermentation tank DO of 12-18%, the speed and air volume were adjusted to control the DO to be maintained at 12-18%; fermentation was continued for 43 hours, and OD 600 =33.7, entering the erythritol production stage, adjusting the speed and air volume to control DO to maintain at 20-30%.

[0077] Fermentation to 63h, OD 600=43.2, the glucose concentration in the tank dropped to 10g / L, and feeding was started (glucose 500g / L), with an initial flow rate of 2.95g / L / h; continued fermentation, adjusted the flow rate to control the glucose concentration in the tank to 10-15g / L, and the fermentation liquid discharge rate was consistent with the feed flow rate; fermentation was stopped for 280h, feeding was stopped, and fermentation was continued for 322h, OD 600 =62.3, the glucose concentration in the tank was 4.0 g / L, and the fermentation was completed. The erythritol concentration in the fermentation liquid reached 232 g / L, and the conversion rate was calculated to be 66.3%.

[0078] Comparative Example 3

[0079] Inoculate at 10% of the inoculum volume. 600 =10.8 seed liquid, the fermentation tank was initially adjusted to pH 4.0 with ammonia (15%), the ventilation ratio was 1VVM, the fermentation tank pressure was 0.025Mpa, and the DO was maintained at 40-50%. When the bacteria grew to the DO of the fermentation tank of 12-18%, the speed and air volume were adjusted to control the DO to be maintained at 12-18%; fermentation was continued for 44 hours, and the OD 600 =35.2, entering the erythritol production stage, adjusting the speed and air volume to control DO to maintain at 20-30%.

[0080] Fermentation to 65h, OD 600 =44.2, the glucose concentration in the tank dropped to 15g / L, and feeding was started (glucose 500g / L, magnesium sulfate heptahydrate 0.21g / L, yeast powder 1.2g / L), with a flow rate of 2.75g / L / h; the fermentation was continued, and the flow rate was adjusted to control the glucose concentration in the tank to 10-15g / L. The fermentation liquid discharge rate was kept consistent with the feed flow rate; the fermentation was continued for 280h, and the feeding was stopped. The fermentation was continued for 313h, and the OD 600 =58.8, the glucose concentration in the tank was 4.2 g / L, and the fermentation was completed. The erythritol concentration in the fermentation broth reached 238 g / L, and the conversion rate was calculated to be 68.2%.

[0081] Comparative Example 4

[0082] Inoculate at 10% of the inoculum volume. 600 =11.7 seed liquid, the fermentation tank was initially adjusted to pH 4.0 with ammonia (15%), the ventilation ratio was 1VVM, the fermentation tank pressure was 0.025Mpa, and the DO was maintained at 40-50%. When the bacteria grew to the DO of the fermentation tank of 12-18%, the speed and air volume were adjusted to control the DO to be maintained at 12-18%; fermentation was continued for 43 hours, and the OD 600 =32.3, entering the erythritol production stage, adjusting the speed and air volume to control DO to maintain at 20-30%.

[0083] Fermentation to 64h, OD600 =42.5, the glucose concentration in the tank dropped to 13g / L, and feeding was started (glucose 500g / L, magnesium sulfate heptahydrate 0.21g / L, urea 2.58g / L), with a flow rate of 2.85g / L / h; the fermentation was continued, and the flow rate was adjusted to control the glucose concentration in the tank to 10-15g / L. The fermentation liquid discharge rate was kept consistent with the feed flow rate; the fermentation was continued for 280h, and the feeding was stopped. The fermentation was continued for 315h, and the OD 600 =60.4, the glucose concentration in the tank was 2.4 g / L, and the fermentation was completed. The erythritol concentration in the fermentation broth reached 242 g / L, and the conversion rate was calculated to be 69.4%.

[0084] Fermentation broth post-treatment

[0085] The erythritol fermentation broths obtained in Examples 3 to 7 and Comparative Examples 1 to 4 were treated as follows to obtain finished erythritol products.

[0086] (1) The fermentation broth was centrifuged at 4000 rpm for 40 min to separate the supernatant;

[0087] (2) Decolorization: Heat to 80°C for 10 min to inactivate the enzyme, and use activated carbon for 10 min for decolorization;

[0088] (3) Filtration: Plate and frame filtration to remove impurities;

[0089] (4) Ionization: Cool down to 40°C, filter the liquid and pass it through cation and anion resins to remove inorganic salts in the liquid;

[0090] (5) Separation: The liquid after separation is filtered through a 500-day nanofiltration membrane to filter out macromolecular substances with a molecular weight of about 100 to 500;

[0091] (6) Concentration and crystallization: The separated erythritol clear solution is concentrated at 80° C. to a content of more than 50%, and then cooled and crystallized;

[0092] (7) Separation and drying: The separator separates the erythritol crystals from the liquid, and then fluidized bed drying is performed to obtain the finished erythritol product;

[0093] (8) Erythritol packaging.

[0094] The detailed results are shown in Table 1:

[0095] Table 1

[0096]

[0097]

[0098] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0099] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for producing erythritol by continuous fermentation, characterized in that: The following steps are involved: (1) Place the Candida lipolytica seed solution at a 10% inoculum into the sterilized fermentation medium; (2) Establishing a CO2 feedback regulation continuous fermentation mechanism: 1) Bacterial growth stage: The speed was adjusted to control the oxygen consumption rate of Candida lipolytica to 0.4~0.7 molO2 / L / h and the tail carbon to 0.3~0.4%. After 6~8 h of fermentation, Candida lipolytica entered the logarithmic growth phase and the tail carbon value increased parabolically to 2.4~2.6%. After 40~50 h of fermentation, the OD 600 =30~35, tail carbon decreases from 2.4~2.6% to 2.2~2.4%, entering the erythritol production stage; 2) Erythritol production stage: fermentation for 60-70 h, OD 600 =45~50, the glucose concentration in the tank dropped to 15 g / L, and feeding and discharging of fermentation liquid were started. The initial flow rate was 2.75~2.95 g / L / h, and the glucose concentration in the tank was controlled at 10~15 g / L. The fermentation liquid discharge rate was kept consistent with the feed flow rate. 3) Continuous fermentation stage: According to the CO2 data in the tail gas, the feed rate was adjusted: when the tail carbon value was between 2.2 and 2.4%, the feed rate was maintained at 2.75 to 2.95 g / L / h; when the tail carbon value was greater than 2.4%, the feed rate was reduced to 2.5 to 2.75 g / L / h; when the tail carbon value was less than 2.2%, the feed rate was increased to 2.95 to 3.1 g / L / h; when the fermentation lasted for 280 to 285 h, the feed rate was stopped, and the fermentation was continued for 300 to 310 h. When the glucose concentration in the tank was less than 5 g / L, the fermentation was terminated and the erythritol fermentation liquid was obtained.

2. The method for producing erythritol by continuous fermentation according to claim 1, wherein: Step (1) Preparation method of Candida lipolytica seed solution: 21) Primary seed solution culture: Prepare a primary seed culture medium, inoculate it with Candida lipolytica and culture it at a constant temperature to obtain the primary seed solution; 22) Secondary seed liquid culture: Prepare secondary seed tank culture medium, inoculate the primary seed liquid into the secondary seed tank culture medium, and culture the Candida lipolytica seed liquid.

3. The method for producing erythritol by continuous fermentation according to claim 2, wherein: Step 21) The primary seed culture medium comprises 5 g / L yeast powder, 10 g / L glucose, and 2 g / L diammonium phosphate; the inoculation amount is 1%; Step 22) The secondary seed tank culture medium comprises 5 g / L yeast powder, 20 g / L glucose, and 1 g / L diammonium phosphate; the inoculation amount is 5%.

4. The method for producing erythritol by continuous fermentation according to claim 3, wherein: Conditions after inoculation in step (1): the fermenter was adjusted to pH 4.0 with 15% ammonia water, the ventilation ratio was 1 VVM, the fermenter pressure was 0.025 MPa, and the DO was maintained at 40-50%; The fermentation medium includes: 350 g / L glucose, 5 g / L yeast powder, 10 g / L potassium dihydrogen phosphate, 5 g / L sodium chloride, 0.247 g / L magnesium sulfate heptahydrate, and 5 g / L diammonium hydrogen phosphate.

5. The method for producing erythritol by continuous fermentation according to claim 4, wherein: The preparation method of the fermentation medium is: 51) Weigh and sterilize: yeast powder, potassium dihydrogen phosphate, sodium chloride, glucose, and magnesium sulfate heptahydrate, dissolve in water, transfer to a fermentation tank, and sterilize; 52) Cooling: Place the sterilized fermenter in a cool place; 53) Mixing: Weigh diammonium phosphate, dissolve in water, sterilize, and add the sterilized diammonium phosphate to the fermenter before inoculation to obtain the fermentation medium.

6. The method for producing erythritol by continuous fermentation according to claim 5, wherein: A portion of the fermentation liquid discharged during the feeding process of step (2) is used as the Candida lipolytica seed liquid of a new tank and is inoculated into a new sterilized fermentation medium at a rate of 10% to continue culturing, and steps 1) to 3) are repeated. The remaining portion is mixed with the erythritol fermentation liquid in step 3) and processed together.

7. The method for producing erythritol by continuous fermentation according to claim 6, wherein: The ingredients of the feed in step (2) are: 500 g / L glucose, 0.21 g / L magnesium sulfate heptahydrate, and 1.2 g / L nitrogen source.

8. The method for producing erythritol by continuous fermentation according to claim 7, wherein: The nitrogen source includes urea, diammonium phosphate, yeast powder or peptone.

9. The method for producing erythritol by continuous fermentation according to claim 8, wherein: Step 3) The obtained erythritol fermentation broth is centrifuged, decolorized, filtered, cross-linked, separated, concentrated, and crystallized to obtain finished erythritol.

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