CN117384770B - Method for continuous flow production of single cell protein - Google Patents

Abstract

The invention provides a method for continuously producing single-cell protein, which comprises the following steps: (1) Fermenting and culturing yarrowia lipolytica in the presence of a main culture medium; (2) Fermenting and culturing until the OD value of the thalli is 400-450, discharging, and adding an auxiliary culture medium into the thalli after discharging is finished, and continuing fermenting and culturing; (3) Repeating the step (2), and centrifuging the material obtained by discharging to obtain the single-cell protein; the carbon source of the main culture medium comprises acetate and anhydrous glucose; in the main culture medium, the mass concentration of acetate is 14-20 g/L, and the mass percentage of anhydrous glucose is 3-5%; in the invention, the method can continuously produce single-cell proteins and can also improve the yield of single-cell proteins.

Description

Method for continuous flow production of single cell protein

Technical Field

The invention belongs to the technical field of preparation of biological materials, and particularly relates to a method for continuously producing single-cell proteins.

Background

The continuous fermentation method is also called as continuous culture method, and refers to a fermentation method in which sterilized fresh liquid culture medium is continuously added into a fermentation tank at a certain flow rate and fermentation liquid is discharged from the fermentation tank at the same flow rate when the batch liquid submerged culture is carried out to the later stage of logarithmic growth of microorganisms. The continuous fermentation method is characterized in that: the operation time of cleaning, charging, sterilizing, inoculating, can placing and the like in batch method culture and fermentation can be reduced, the manpower and material resources are saved, the cost is reduced, and the production efficiency is improved; the device has the advantages of small volume, low investment, capability of adopting multistage continuous culture and fermentation, more perfect and reasonable device configuration, convenient mechanical and automatic control, and belongs to a method for culturing and fermenting in a stable state, thereby providing a good environment for the high-speed growth of microorganisms in a constant state and facilitating the study of the physiological, biochemical and genetic characteristics of the microorganisms.

The continuous long-time culture and fermentation are easy to cause strain variation, degradation, mixed bacteria pollution and other problems. The culture does not change along with the environmental conditions of fermentation, including temperature, pH, culture concentration, product concentration, dissolved oxygen, oxidation-reduction potential and the like, so that the logarithmic growth phase of the microorganism in the batch method culture can be infinitely prolonged, the stable growth rate and specific growth rate of the microorganism are maintained, and the cell concentration, total cell quantity and volume of culture solution in a fermentation tank are kept constant. The product produced by continuous culture and fermentation has stable performance. However, if the operation is improper, the newly added culture medium is not easy to be completely mixed with the original culture medium, and the culture and fermentation process is affected.

In semi-continuous fermentation, two methods are available depending on the mode of feeding the mash. The first method is to connect a group of several fermenters so that the first three fermenters are kept in a continuous fermentation state. When starting to put into production, the first tank is connected with the yeast, and then the mash is continuously added into the tank and the tank is always in a main fermentation state. After the first tank is filled, the mash flows into the second tank, and the mash is fed into the first tank and the second tank respectively, and the two tanks are kept in a main fermentation state all the time. And after the second tank is filled up, naturally flowing into the third tank. After the third tank is fully filled, the liquid flows into the fourth tank. After the fourth tank is filled, the third tank is changed into the fifth tank, the fifth tank is changed into the sixth tank after being filled, and so on. After fermentation in the fourth, fifth, sixth and so on tanks, the fermentation is carried out and distilled. And cleaning and sterilizing the fermentation tank after the fermentation tank is emptied, and repeating the above steps. After the three fermentation tanks are continuously used for a period of time, the feeding of the mash is stopped, the mash is fermented until the mash is sent to distillation, and the mash is cleaned and sterilized. At this time, the other two or three empty tanks can be used as the first three tanks, and the mother liquor is added to the mash. A new cycle is started. The method is characterized in that the first three tanks are in a continuous main fermentation state, and the later is in a post fermentation state.

The second method is that 7-8 tanks form a group of tanks, and overflow pipes are used between each tank to be connected in series from the upper part of the previous tank to the bottom of the next tank: during production, 1/3 of the volume of the yeast in the fermentation tank is prepared, the first fermentation tank is added, then the mash is fed in a flowing mode under the condition that the main fermentation state is kept, after the fermentation tank is full, the mash flows into the second tank through the overflow pipe, and when the mash in the second tank is added to 1/3 of the volume, the mash is added to the second tank instead. After the second tank is filled, the second tank flows into the third tank, then the second tank operation is repeated until the last tank, and finally the mature beer is distilled one by one from the first tank to the last tank. The semi-continuous fermentation method has the advantage that the preparation of the yeast is omitted; the fermentation time is shortened; the equipment utilization rate is high; but has the disadvantage of requiring high requirements for aseptic operation.

Fed batch fermentation has been widely used for amino acids, growth hormone, antibiotics, vitamins, enzyme proteins, organic solvents, organic acids, nucleotides, polymers, and the like. Major advantages of fed-batch fermentation include: the inhibition of the substrate and the process is released, the optimal fermentation state is achieved as much as possible, the conversion rate of the product is improved, and the like.

Continuous culture is industrially mainly used for producing microbial cells, primary metabolites and metabolites related to energy production and cell proliferation, such as yeast, single cell proteins, alcohol, and treating industrial sewage, etc. However, due to the relatively industrialized membrane equipment technology, bacterial variation and pollution in the production process, the technology cannot break through in other fields, but is still worth exploring from the development point of view.

The single cell protein has extremely important function in the feed and food industry, and the development and production of the single cell protein have wider prospects in China. The SCP can be produced in a large scale and in a factory fast way relative to the long production period of the terrestrial animal and plant protein source, and can change waste into valuable and promote the development of recycling economy. Related studies have also shown that SCP has a certain feasibility in replacing fish meal or fish oil in aquatic feeds.

Therefore, developing a method with high single-cell protein yield, avoiding resource waste, saving cost and being capable of continuously producing single-cell protein is a problem to be solved in the field.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention aims to provide a method for continuously producing single-cell proteins, which can continuously produce single-cell proteins, can also improve the yield of single-cell proteins and avoid resource waste and waste.

To achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a method for continuous flow production of a single cell protein, the method comprising:

(1) Fermenting and culturing yarrowia lipolytica in the presence of a main culture medium; (2) Fermenting and culturing until the OD value of the thalli is 400-450, discharging, and adding an auxiliary culture medium into the thalli after the discharging is finished, and continuing fermenting and culturing; (3) Repeating the step (2), and centrifuging the material obtained by discharging to obtain the single-cell protein; the nitrogen source of the main culture medium comprises acetate and anhydrous glucose; in the main culture medium, the mass concentration of acetate is 14-20 g/L (for example, 14. 14g/L, 14.2 g/L, 14.5 g/L, 14.8 g/L, 15g/L, 15.2 g/L, 15.5 g/L, 15.8 g/L, 16g/L, 16.2 g/L, 16.5 g/L, 16.8 g/L, 17g/L, 17.2 g/L, 17.5 g/L, 17.8 g/L, 18g/L, 18.2 g/L, 18.5 g/L, 18.8 g/L, 19 g/L, 19.2 g/L, 19.5 g/L, 19.8 g/L, 20g/L and the like), and the mass percentage of anhydrous glucose is 3-5% (for example, 3.2%, 3.4%, 4.4%, and the like).

According to the method, yarrowia lipolytica is fermented and cultured in the presence of a main culture medium with a specific formula, then the yarrowia lipolytica is cultured to a certain period, the discharging is carried out, an auxiliary culture medium is supplemented, and the steps are repeated, so that continuous production of single-cell proteins is realized, the single-cell proteins obtained by the method are high in yield, resource waste and waste are avoided, the auxiliary culture medium is supplemented in a specific period, and high-yield single-cell proteins can be obtained in a relatively short fermentation time.

In the present invention, the OD of the cells is 400 to 450, and may be 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, or the like, for example.

In the invention, the OD value of the thallus is less than 400, which can lead to the reduction of the yield of single cell protein; greater than 450 may result in an extended fermentation time.

Preferably, the acetate salt comprises sodium acetate.

Preferably, the main culture medium in step (1) further comprises the following components: 2.2-3 wt% (e.g., 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, etc.), 100-200 mg/L (e.g., 100/L, 105/L, 110/L, 115/L, 120/L, 125/L, 130/L, 135/L, 140/L, 145/L, 150/L, 155/L, 160/L, 165/L, 170/L, 175/L, 180/L, 185/L, 190/L, 195/L, 200/L, etc.), 150-250 mg/L (e.g., 150/L, 155/L, 160/L, 165/L, 170/L, 175/L, 180/L) 185/L, 190/L, 195/L, 200/L, 205/L, 210/L, 215/L, 220/L, 225/L, 230/L, 235/L, 240/L, 245/L, 250/L, etc.), pyridoxine 250-350 mg/L (e.g., 250/L, 255/L, 260/L, 265/L, 270/L, 275/L, 280/L, 285/L, 290/L, 295/L, 300/L, 305/L, 310/L, 315/L, 320/L, 325/L, 330/L, 335/L, 340/L, 345/L, 350/L, etc.), biotin 30-50 mg/L (e.g., 30/L, 32/L, 34/L, etc.), biotin, 36 mg/L, 38/L, 40/L, 42/L, 44/L, 46/L, 48/L, 50/L, etc.), 700 to 900mg/L (for example, 700/L, 710/L, 720/L, 730/L, 740/L, 750/L, 760/L, 770/L, 780/L, 790/L, 800/L, 810/L, 820/L, 830/L, 840/L, 850/L, 860/L, 870/L, 880/L, 890/L, 900/L), riboflavin 150 to 250mg/L (for example, 150/L, 155/L, 160/L, 165/L, 170/L, 175/L, 180/L, 185/L, 190/L) 195/L, 200/L, 205/L, 210/L, 215/L, 220/L, 225/L, 230/L, 235/L, 240/L, 245/L, 250/and the like), inositol 5-7 g/L (e.g., 5g/L, 5.2g/L, 5.4g/L, 5.6g/L, 5.8g/L, 6g/L, 6.2g/L, 6.4g/L, 6.6g/L, 6.8g/L, 7g/L and the like, etc.), p-aminobenzoic acid 100-200 mg/L (e.g., 100/L, 105/L, 110/L, 115/L, 120/L, 125/L, 130/L, 135/L, 140/L, 145/L, 150/L, 155/L, etc.), p-aminobenzoic acid, 160mg/L, 165 mg/L, 170 mg/L, 175 mg/L, 180mg/L, 185 mg/L, 190 mg/L, 195 mg/L, 200mg/L, etc.).

Preferably, the main culture medium in step (1) further comprises the following components: boric acid350~500mL/L (e.g., 350/L, 360/L, 370/L, 380/L, 390/L, 400/L, 410/L, 420/L, 430/L, 440/L, 450/L, 460/L, 470/L, 480/L, 490/L, 500/L, etc.), 100-200 mg/L (e.g., 100/L, 105/L, 110/L, 115/L, 120/L, 125/L, 130/L, 135/L, 140/L, 145/L, 150/L, 155/L, 160/L, 165/L, 170/L, 175/L, 180/L, 185/L, 190/L, 195/L, 200mg/L, etc.), and the like 250-400 mg/L (for example, 250/L, 255/L, 260/L, 265/L, 270/L, 275/L, 280/L, 285/L, 290/L, 295/L, 300/L, 305/L, 310/L, 315/L, 320/L, 325/L, 330/L, 335/L, 340/L, 345/L, 350/L, 355/L, 360/L, 365/L, 370/L, 375/L, 380/L, 385/L, 390/L, 395/L, 400/L, etc.), 150-250 mg/L (for example, 150/L, 155/L, 160/L, 165/L, 170/L, etc.), sodium manganate, 175 mg/L, 180/L, 185/L, 190/L, 195/L, 200/L, 205/L, 210/L, 215/L, 220/L, 225/L, 230/L, 235/L, 240/L, 245/L, 250/L, etc.), potassium iodide 0.005 to 0.015mg/L (which may be, for example, 0.005/L, 0.006/L, 0.007/L, 0.008/L, 0.009/L, 0.01/L, 0.011/L, 0.012/L, 0.013/L, 0.014/L, 0.015/L, etc.), calcium chloride 0.005 to 0.015g/L (0.005/L, 0.006/L, 0.007/L, 0.008/L, 0.009/L, 0.01/L, 0.015/L, etc.) 0.011/L, 0.012/L, 0.013/L, 0.014/L, 0.015/L, etc.), 0.02-0.08 g/L (e.g., 0.02/L, 0.025/L, 0.03/L, 0.035/L, 0.04/L, 0.045/L, 0.05/L, 0.055/L, 0.06/L, 0.065/L, 0.07/L, 0.075/L, 0.08/L, etc.), 0.2-0.8 g/L (e.g., 0.2/L, 0.25/L, 0.3/L, 0.35/L, 0.4/L, 0.45/L, 0.5/L, 0.55/L, 0.6/L, 0.65/L, 0.7/L, 0.75/L, 0.8/L, etc.), and sodium chloride.

In the invention, the main culture medium is compounded with specific content by each component, so that the yield of single cell protein is high and the fermentation time is short; the main medium composition, which is not defined by the present invention, may result in reduced single cell protein production or prolonged fermentation time.

Preferably, the pH value of the main culture medium is 6 to 8, for example, 6, 6.5, 7, 7.5, 8, etc.

In the present invention, the pH value of the main culture medium is adjusted by using 28% ammonia water and 50% acetic acid.

Preferably, the yarrowia lipolytica in the step (1) is obtained by sequentially performing amplification culture on a primary yeast extract peptone glucose medium (YPD), a secondary yeast extract peptone glucose medium and a tertiary yeast extract peptone glucose medium.

Preferably, the OD value of the cells obtained by the amplification culture of the primary yeast extract peptone glucose medium is 5 to 8, and for example, 5, 5.2, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.4, 7.6, 7.8, 8 and the like can be used.

Preferably, the OD value of the cells obtained by amplification culture in a secondary yeast extract peptone glucose medium is 12 to 18, and may be, for example, 12, 13, 14, 15, 16, 17, 18, etc.

Preferably, the OD value of the thallus obtained by amplification culture of the tertiary yeast extract peptone glucose culture medium is 40-65, and can be 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64 and the like.

Preferably, the fermentation culture of step (1) is performed in a fermenter.

According to the invention, the unfrozen yarrowia lipolytica frozen stock solution is inoculated into a primary YPD shake flask, the temperature is 30-37 ℃, the rotating speed is 160-200 revolutions per minute, the yarrowia lipolytica frozen stock solution is cultured until the OD grows to 5.2-7.6, then the yarrowia lipolytica frozen stock solution is inoculated into a secondary YPD culture medium, the yarrowia lipolytica frozen stock solution is cultured until the OD value of a thallus is 12-18, then the yarrowia lipolytica frozen stock solution is inoculated into a tertiary YPD culture medium, the thallus OD value of the thallus is 40-65, and then the yarrowia lipolytica frozen stock solution is inoculated into a fermentation tank for fermentation culture.

Preferably, the co-medium in step (2) comprises 15-30 g/L sodium acetate (e.g. 15g/L, 16g/L, 17g/L, 18g/L, 19 g/L, 20g/L, 21 g/L, 22g/L, 23 g/L, 24g/L, 25g/L, 26 g/L, 27 g/L, 28g/L, 29 g/L, 30g/L, etc.), yeast base nitrogen source (YNB) 10-30 g/L (e.g. cocoaIn the form of 10/L, 11/L, 12/L, 13/L, 14/L, 15/L, 16/L, 17/L, 18/L, 19/L, 20/L, 21/L, 22/L, 23/L, 24/L, 25/L, 26/L, 27/L, 28/L, 29/L, 30/L, etc.), 600 to 800mg/L (for example, 600/L, 610/L, 620/L, 630/L, 640/L, 650/L, 660/L, 670/L, 680/L, 690/L, 700/L, 710/L, 720/L, 730/L, 740/L, 750/L, 760/L, 770/L, 780/L, 790/L, 800/L, etc.), and the like 450-550 mg/L (for example, 450/L, 460/L, 470/L, 480/L, 490/L, 500/L, 510/L, 520/L, 530/L, 540/L, 550/L, etc.), 0.03-0.05 mg/L (for example, 0.03/L, 0.032/L, 0.034/L, 0.036/L, 0.038/L, 0.04/L, 0.042/L, 0.044/L, 0.046/L, 0.048/L, 0.05/L, etc.), 0.06-0.08 g/L (for example, 0.06/L, 0.062/L, 0.064/L, 0.066/L, 0.068/L, 0.07/L, 0.072/L, etc.), and the like, 0.074 g/L, 0.076 g/L, 0.078 g/L, 0.08g/L, etc.), magnesium sulfate 0.08-0.1 g/L (e.g., may be 0.08g/L, 0.081 g/L, 0.082 g/L, 0.083 g/L, 0.084 g/L, 0.085 g/L, 0.086 g/L, 0.087 g/L, 0.088 g/L, 0.089 g/L, 0.09g/L, 0.091 g/L, 0.092 g/L, 0.094 g/L, 0.096 g/L, 0.098 g/L, 0.1g/L, etc.), sodium chloride 0.6-0.8 g/L (e.g., may be 0.6g/L, 0.62/L, 0.086 g/L, 0.66/L, 0.37/L, 52, 0.37/L, 72/L, 0.37/L, 52, 0.37/L, etc.), hydrogen, 0.37/L, g/L, etc0.2 to 1.5 g/L (for example, 0.2g/L, 0.3 g/L, 0.4g/L, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1g/L, 1.1 g/L, 1.2 g/L, 1.3 g/L, 1.4 g/L, 1.5 g/L, etc.) and 0.5 to 3 g/L (for example, 0.5g/L, 0.6g/L, 0.8g/L, 1g/L, 1.2 g/L, 1.4 g/L, 1.6 g/L, 1.8 g/L, 2.2 g/L, 2.4 g/L, 2.6 g/L, 2.8g/L, 2.37/L, g/L, etc.) of monopotassium phosphate, etc.).

In the invention, the auxiliary culture medium is compounded by the components according to the specific content, so that the single-cell protein has high yield and short fermentation time.

In the invention, the mass concentration or mass percentage content of each component of the main culture medium and the auxiliary culture medium is calculated by the volume of water being 1L.

Preferably, the volume of the discharging material in the step (2) is 0.4-0.5 times of the total volume, for example, 0.4 times, 0.42 times, 0.44 times, 0.46 times, 0.48 times, 0.5 times, and the like.

Preferably, the volume of the auxiliary medium added in the step (2) is 0.4-0.45 times of the total volume before discharging, for example, 0.4 times, 0.41 times, 0.42 times, 0.44 times, 0.45 times, and the like.

In the invention, the centrifugal equipment comprises a disc type centrifugal machine, wherein the feeding speed during centrifugation is 0.5-2L/min, and slag is discharged every half hour to obtain bacterial slag and supernatant; and (3) refluxing the supernatant to the fermentation broth for re-centrifugation, and sterilizing and filtering after at least three times of centrifugation, wherein the obtained supernatant can be prepared into an auxiliary culture medium again, so that resource waste is avoided.

In the invention, the disc centrifuge further comprises a step of cleaning the disc centrifuge before feeding, wherein the cleaning rotating speed is 9000-10000 rpm, the water pressure is 0.1-0.2 MPa, the cleaning time is 10-30 s, and then the deslagging period and the deslagging time are set, and the feeding is started; the initial feeding speed is controlled below 2L/min, and the flow rate is regulated after the supernatant liquid flows out normally. Reducing the feeding speed can reduce the solid content in the supernatant fluid, and the smaller the flow is, the better the separation effect is; the slag discharging condition of the equipment is attended at any time, and the slag discharging period and the slag discharging time are adjusted according to the material property; the deslagging period represents deslagging frequency, and deslagging time represents primary deslagging amount; the higher the solid content in the material is, the shorter the deslagging period is, and the longer the deslagging time is; the slag fixing water content can be reduced by prolonging the slag discharging period; if the initial feeding speed is too high, the retention time of the materials in the rotary drum is too short, the separation effect is not ideal, the slag can be manually discharged in advance, the rotary drum is emptied, then the feeding speed is reduced, and the separation is continued; the discharged sludge can be mixed with the materials and then separated for the second time.

Preferably, after the centrifugation in the step (3), the method further comprises the steps of diluting and low-temperature treatment of the bacterial residues obtained by centrifugation, and spray drying.

Preferably, the dilution includes mixing the obtained bacterial residues with water until the dry weight concentration of the bacterial bodies is 60-80 g/L, for example, 60 g/L, 62 g/L, 64 g/L, 66 g/L, 68 g/L, 70 g/L, 72 g/L, 74 g/L, 76 g/L, 78 g/L, 80 g/L and the like.

Preferably, the temperature of the low-temperature treatment is 2-5 ℃, for example, 2 ℃, 3 ℃, 4 ℃, 5 ℃ and the like; the time is 12-16 h, for example, 12h, 13 h, 14h, 15 h, 16h and the like.

In the invention, the low-temperature treatment is helpful for the thallus to release protein and improve the content of crude protein in single-cell protein.

Preferably, the feed rate of the spray drying is 4.5-6L/h, for example, 4.5L/h, 4.6L/h, 4.7L/h, 4.8L/h, 4.9L/h, 5L/h, 5.1L/h, 5.2L/h, 5.3L/h, 5.4L/h, 5.5L/h, 5.6L/h, 5.7L/h, 5.8L/h, 5.9L/h, 6L/h and the like; the air inlet temperature is 120-160 ℃, for example, 120 ℃, 130 ℃, 140 ℃, 150 ℃, 160 ℃ and the like; the frequency is 320-380 Hz, such as 320 Hz, 330 Hz, 340Hz, 350 Hz, 360Hz, 370 Hz, 380Hz, etc.

Preferably, the wall material of the single cell protein comprises at least one of methylcellulose, alginate or calcium phosphate;

preferably, the time interval for harvesting the single-cell protein is 20-40 min, for example, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, 32 min, 34 min, 36 min, 38 min, 40 min and the like.

As a preferred technical solution of the present invention, the method includes:

(1) Inoculating yarrowia lipolytica with the OD value of the thalli of 40-65 into a fermentation tank for fermentation culture in the presence of a main culture medium; the main culture medium comprises 14-20 g/L of sodium acetate, 3-5 wt% of anhydrous glucose, 2.2-3 wt% of yeast powder, 100-200 mg/L of vitamin B, 150-250 mg/L of nicotinic acid, 250-350 mg/L of pyridoxine, 30-50 mg/L of biotin, 700-900 mg/L of calcium pantothenate, 150-250 mg/L of riboflavin, 5-7 g/L of inositol, 100-200 mg/L of p-aminobenzoic acid and boric acid350-500 mL/L, 100-200 mg/L of manganese sulfate, 250-400 mg/L of zinc sulfate, 150-250 mg/L of sodium manganate, 0.005-0.015 mg/L of potassium iodide, 0.00-0.015 g/L of calcium chloride, 0.02-0.08 g/L of magnesium sulfate and 0.2-0.8 g/L of sodium chloride;

(2) Fermenting and culturing until the OD value of the thalli is 400-450, discharging, and after discharging is finished, adding an auxiliary culture medium into a fermentation tank, and continuing fermenting and culturing; the auxiliary culture medium comprises 15-30 g/L of sodium acetate, 10-30 g/L of yeast basic nitrogen source, 600-800 mg/L of zinc sulfate, 450-550 mg/L of sodium manganate, 0.03-0.05 mg/L of potassium iodide, 0.06-0.08 g/L of calcium chloride, 0.08-0.1 g/L of magnesium sulfate, 0.6-0.8 g/L of sodium chloride and dipotassium hydrogen phosphate0.2-1.5 g/L and 0.5-3 g/L of potassium dihydrogen phosphate;

(3) And (3) repeating the step (2), centrifuging the discharged material, diluting the obtained bacterial residues with water until the dry weight concentration of the bacterial bodies is 60-80 g/L, placing the bacterial residues at 2-5 ℃ for 12-16 h, then performing spray drying, wherein the feeding speed of the spray drying is 4.5-6L/h, the air inlet temperature is 120-160 ℃, the frequency is 320-380 Hz, taking at least one of methylcellulose, alginate or calcium phosphate as a wall material, and harvesting single-cell proteins every 20-40 min to obtain the single-cell proteins.

In the invention, the fermentation liquor obtained by discharging each time can be centrifuged, and the fermentation liquor obtained by continuous production can be uniformly centrifuged, so that continuous production of single-cell proteins is realized by continuously discharging and supplementing (namely supplementing auxiliary culture medium), and the production period of the invention is at least 30 days of continuous fermentation.

The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.

Compared with the prior art, the invention has the beneficial effects that:

according to the method, the yarrowia lipolytica is fermented and cultured by adopting the main culture medium with a specific formula, then the yarrowia lipolytica is cultured to a certain period, the material is discharged and the auxiliary culture medium is supplemented, and the steps are repeated, so that continuous production of single-cell protein is realized, the single-cell protein yield obtained by the method is high, resource waste and waste are avoided, and the auxiliary culture medium is supplemented in a specific period, so that high-yield single-cell protein can be obtained in a relatively short fermentation time; the unit yield of single cell protein is more than or equal to 25g/L/h.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.

In the following embodiments of the invention, the dry weight, yield and crude protein content were tested as follows:

(1) Dry weight of single cell protein: the unit is g/L

Weighing 1.00L fermentation liquor, centrifuging at 8000 rpm for 10 min to obtain thallus, adding 1L water to re-suspend uniformly, centrifuging at 8000 rpm for 10 min again to obtain wet thallus, and weighing the wet thallus with the mass of m1; 5.000 g of the dried bacterial cells are taken from m1 and dried at 103+/-2 ℃ according to the method of national standard GB/T6435-2014 'determination of moisture in feed', and the weight m2 (accurate to 4 decimal places) of the dried bacterial cells is obtained.

Cell dry weight dcw=m2/5.000xm1/1.00

(2) Yield: the unit is g/(L.h)

Yield = dry weight of single cell protein +.time of high density fermentation (fermentation cycle).

(3) Crude protein content

According to national standard GB/T6432-2018 ' Kai's method for determining crude protein in feed ', the crude protein content (mass fraction) is obtained through digestion, ammonia distillation and titration.

In the following embodiments of the invention, yarrowia lipolytica species, purchased from China industry microbiological culture Collection center, accession number: CICC 32291; the components of the formulated medium are all commercially available chemicals.

Culturing seed liquid: and (3) inoculating the unfrozen yarrowia lipolytica frozen stock solution into a primary YPD shake flask at the temperature of 32 ℃ at the rotating speed of 180 revolutions per minute, culturing until the OD grows to 6.4, inoculating into a secondary YPD culture medium at the temperature of 32 ℃ at the rotating speed of 180 revolutions per minute, culturing until the OD grows to 15, inoculating into a tertiary YPD culture medium at the temperature of 32 ℃ at the rotating speed of 180 revolutions per minute, and culturing until the OD value grows to 52 to obtain amplified yarrowia lipolytica for later use.

In the invention, the term "OD value" refers to the OD value of fermentation broth, which is obtained by dilution and ultraviolet spectrophotometry (dilution to the lowest effective value which can be detected by an ultraviolet spectrophotometer), and specifically comprises the following steps: the OD at 600 nm of the fermentation broth was measured using an ultraviolet spectrophotometer (Alpha-1106, shanghai spectral element instruments Co., ltd.).

Example 1

The embodiment provides a method for continuously producing single-cell protein, which is characterized by comprising the following steps:

(1) Inoculating yarrowia lipolytica subjected to amplification culture into a 1000L fermentation tank in the presence of a main culture medium for fermentation culture; the main culture medium has a volume of 400L, and comprises sodium acetate 18g/L, anhydrous glucose 4wt%, yeast powder 2.5 wt%, vitamin B1 150mg/L, nicotinic acid 200mg/L, pyridoxine 300mg/L, biotin 40mg/L, calcium pantothenate 800mg/L, riboflavin 200mg/L, inositol 6g/L, p-aminobenzoic acid 150mg/L, and boric acid420mL/L, 150mg/L of manganese sulfate, 320mg/L of zinc sulfate, 200mg/L of sodium manganate, 0.01mg/L of potassium iodide, 0.01g/L of calcium chloride, 0.05g/L of magnesium sulfate and 0.5g/L of sodium chloride; the pH value of the main culture medium is 7.4;

(2) Fermenting and culturing until the OD value of the thalli is 420, discharging, wherein the discharging volume is 300L, and after the discharging is finished, adding 300L of auxiliary culture medium into a fermentation tank, and continuing fermenting and culturing; the auxiliary culture medium comprises 22g/L of sodium acetate, 20g/L of yeast basic nitrogen source, 700mg/L of zinc sulfate, 500mg/L of sodium manganate, 0.04mg/L of potassium iodide, 0.07g/L of calcium chloride, 0.09g/L of magnesium sulfate, 0.7g/L of sodium chloride and dipotassium hydrogen phosphate0.8g/L and 1.6 g/L of monopotassium phosphate;

(3) Repeating the step (2), continuously producing single-cell proteins, carrying out disc centrifugation on the materials obtained by discharging, centrifuging the supernatant obtained by centrifugation for three times, and then sterilizing and filtering the supernatant, wherein the supernatant can be used for preparing an auxiliary culture medium again; diluting the bacterial residues obtained by centrifugation with water until the dry weight concentration of the bacterial residues is 70 g/L, placing at 4 ℃ for 16 hours, then performing spray drying, wherein the feeding speed of the spray drying is 5L/h, the air inlet temperature is 140 ℃, the frequency is 340Hz, taking calcium phosphate as a wall material, and harvesting single-cell proteins every 30 minutes to obtain the single-cell proteins.

Example 2

The embodiment provides a method for continuously producing single-cell protein, which is characterized by comprising the following steps:

(1) Inoculating yarrowia lipolytica subjected to amplification culture into a 1000L fermentation tank in the presence of a main culture medium for fermentation culture; the main culture medium has a volume of 400L, and comprises 16g/L sodium acetate, 3wt% of anhydrous glucose, 2.2 wt% of yeast powder, 120mg/L vitamin B1, 240mg/L nicotinic acid, 340mg/L pyridoxine, 32mg/L biotin, 750mg/L calcium pantothenate, 160mg/L riboflavin, 5.5g/L inositol, 120mg/L para-aminobenzoic acid, and boric acid480mL/L, 120mg/L of manganese sulfate, 260mg/L of zinc sulfate, 240mg/L of sodium manganate, 0.013mg/L of potassium iodide, 0.014g/L of calcium chloride, 0.02g/L of magnesium sulfate and 0.2g/L of sodium chloride; the pH value of the main culture medium is 6.8;

(2) Fermenting and culturing until the OD value of the thalli is 400, discharging, wherein the discharging volume is 350L, and after discharging, adding 300L of auxiliary culture medium into a fermentation tank, and continuing fermenting and culturing; the auxiliary culture medium comprises 28g/L of sodium acetate, 12g/L of yeast basic nitrogen source, 650mg/L of zinc sulfate, 540mg/L of sodium manganate, 0.03mg/L of potassium iodide, 0.08g/L of calcium chloride, 0.1g/L of magnesium sulfate, 0.6g/L of sodium chloride and dipotassium hydrogen phosphate0.5g/L and 2.8g/L of monopotassium phosphate;

(3) Repeating the step (2), continuously producing single-cell proteins, carrying out disc centrifugation on the materials obtained by discharging, centrifuging the supernatant obtained by centrifugation for three times, and then sterilizing and filtering the supernatant, wherein the supernatant can be used for preparing an auxiliary culture medium again; diluting the bacterial residues obtained by centrifugation with water until the dry weight concentration of the bacterial residues is 60 g/L, placing at 4 ℃ for 12 hours, then performing spray drying, wherein the feeding speed of the spray drying is 5.5L/h, the air inlet temperature is 150 ℃, the frequency is 360Hz, taking methylcellulose as a wall material, and harvesting single-cell proteins every 30 minutes to obtain the single-cell proteins.

Example 3

The embodiment provides a method for continuously producing single-cell protein, which is characterized by comprising the following steps:

(1) Inoculating yarrowia lipolytica subjected to amplification culture into a 1000L fermentation tank in the presence of a main culture medium for fermentation culture; the main culture medium has a volume of 400L, and comprises 20g/L sodium acetate, 5wt% of anhydrous glucose, 2.8 wt% of yeast powder, 180mg/L vitamin B, 150mg/L nicotinic acid, 260mg/L pyridoxine, 48mg/L biotin, 850mg/L calcium pantothenate, 240mg/L riboflavin, 6.5g/L inositol, 180mg/L p-aminobenzoic acid, and boric acid360mL/L, 180mg/L of manganese sulfate, 380mg/L of zinc sulfate, 160mg/L of sodium manganate, 0.08mg/L of potassium iodide, 0.008g/L of calcium chloride, 0.08g/L of magnesium sulfate and 0.8g/L of sodium chloride; the pH value of the main culture medium is 7.2;

(2) Fermenting and culturing until the OD value of the thalli is 400, discharging, wherein the discharging volume is 350L, and after discharging, adding 300L of auxiliary culture medium into a fermentation tank, and continuing fermenting and culturing; the auxiliary culture medium comprises 16g/L of sodium acetate, 28g/L of yeast basic nitrogen source, 760mg/L of zinc sulfate, 480mg/L of sodium manganate, 0.05mg/L of potassium iodide, 0.06g/L of calcium chloride, 0.08g/L of magnesium sulfate, 0.8g/L of sodium chloride and dipotassium hydrogen phosphate1.5 g/L and potassium dihydrogen phosphate 0.5g/L;

(3) Repeating the step (2), continuously producing single-cell proteins, carrying out disc centrifugation on the materials obtained by discharging, centrifuging the supernatant obtained by centrifugation for three times, and then sterilizing and filtering the supernatant, wherein the supernatant can be used for preparing an auxiliary culture medium again; diluting the bacterial residues obtained by centrifugation with water until the dry weight concentration of the bacterial residues is 80 g/L, placing at 4 ℃ for 14h, then performing spray drying, wherein the feeding speed of the spray drying is 4.5L/h, the air inlet temperature is 120 ℃, the frequency is 380Hz, taking methylcellulose as a wall material, and harvesting single-cell proteins every 30 min to obtain the single-cell proteins.

Example 4

This example provides a method for continuous production of single cell proteins, which differs from example 1 only in that the main medium has a volume of 400L, including 18g/L sodium acetate, 4wt% anhydrous dextrose, 4wt% yeast powder, and vitamin B1 80mg/L, 130mg/L of nicotinic acid, 220mg/L of pyridoxine, 60mg/L of biotin, 600mg/L of calcium pantothenate, 120mg/L of riboflavin, 4g/L of inositol, 80mg/L of para-aminobenzoic acid and boric acid600mL/L, 220mg/L of manganese sulfate, 420mg/L of zinc sulfate, 260mg/L of sodium manganate, 0.017mg/L of potassium iodide, 0.002g/L of calcium chloride, 0.01g/L of magnesium sulfate and 0.1g/L of sodium chloride, and the other steps were the same as in example 1.

Example 5

This example provides a method for continuous production of single cell proteins, which differs from example 1 only in that the volume of the main medium is 400L, including 18g/L sodium acetate, 4wt% anhydrous dextrose, 2 wt% yeast powder, 220mg/L vitamin B1, 260mg/L niacin, 360mg/L pyridoxine, 20mg/L biotin, 950mg/L calcium pantothenate, 260mg/L riboflavin, 8g/L inositol, 220mg/L para-aminobenzoic acid, boric acid300mL/L, 80mg/L of manganese sulfate, 240mg/L of zinc sulfate, 140mg/L of sodium manganate, 0.002mg/L of potassium iodide, 0.017g/L of calcium chloride, 0.1g/L of magnesium sulfate and 1g/L of sodium chloride, and the other steps were the same as in example 1.

Example 6

This example provides a method for continuous production of single cell proteins, which differs from example 1 only in that the co-culture medium comprises 32g/L sodium acetate, 8g/L yeast basal nitrogen source, 550mg/L zinc sulfate, 440mg/L sodium manganate, 0.02mg/L potassium iodide, 0.1g/L calcium chloride, 0.12g/L magnesium sulfate, 0.5g/L sodium chloride, dipotassium hydrogen phosphate0.1 The g/L and potassium dihydrogen phosphate 0.4g/L were the same as in example 1.

Example 7

This example provides a method for continuous production of single cell proteins, which differs from example 1 only in that the co-culture medium comprises 12g/L sodium acetate, 35g/L yeast basal nitrogen source, 850mg/L zinc sulfate, 580mg/L sodium manganate, 0.07mg/L potassium iodide, 0.04g/L calcium chloride, 0.06g/L magnesium sulfate, 0.9g/L sodium chloride, dipotassium hydrogen phosphate1.7 The g/L and the potassium dihydrogen phosphate 3.2g/L were the same as in example 1.

Example 8

This example provides a method for continuous production of single cell proteins, which differs from example 1 only in that boric acid is not present in the main medium, the amounts of the other components are adjusted so that the ratio of the other components is maintained, the total amount of the medium is unchanged, and the other steps are the same as in example 1.

Example 9

This example provides a method for continuous production of single cell proteins which differs from example 1 only in that zinc sulfate in the main medium is replaced with an equivalent amount of cobalt chloride hexahydrate, and all other steps are the same as in example 1.

Comparative example 1

This comparative example provides a method for continuous production of single cell proteins, which differs from example 1 only in that in step (2) fermentation culture is performed until the OD value of the cell is 350, and in that the cell is discharged, and in the other steps, the same as in example 1 is performed.

Comparative example 2

This comparative example provides a method for continuous production of single cell protein, which differs from example 1 only in that the mass concentration of sodium acetate in the main medium is 12g/L, the content of anhydrous glucose is adjusted so that the total mass of sodium acetate and anhydrous glucose is unchanged, and the other steps are the same as in example 1.

Comparative example 3

This comparative example provides a method for continuous production of single cell proteins, which differs from example 1 only in that the mass concentration of sodium acetate in the main medium is 24g/L, and the other steps are the same as in example 1.

The yields and crude protein contents of single-cell proteins obtained by the methods provided in examples 1 to 10 and comparative examples 1 to 3 are shown in Table 1.

TABLE 1

As can be seen from Table 1, the continuous flow method for producing single-cell protein provided by the invention can ensure uninterrupted production of single-cell protein by controlling the formula of the main culture medium, fermenting to a certain period for discharging and feeding, and simultaneously controlling the fed culture medium to adopt a specific formula, and continuously producing 350L fermentation liquor per hour by using 1000L fermentation tank 4 to produce at least 42kg single-cell protein product by continuously discharging and feeding; greatly reduces the generation of wastes and recycles resources.

As can be seen from a comparison of examples 1 and 4-9, the method provided by the present invention, in which the main medium and the auxiliary medium are not of specific composition, results in a decrease in the yield of single cell proteins and in the crude protein content.

As can be seen from comparison of example 1 and comparative examples 1-3, the method provided by the invention has an OD value of 350 for discharging, and does not grow to the maximum rate, so that the yield is low and the crude protein content is low; sodium acetate is not limited in scope, and can lead to slow or inhibited cell growth, both of which can lead to reduced yields and crude protein content.

While the foregoing is directed to embodiments of the present invention, other and further details of the invention may be had by the present invention, it should be understood that the foregoing description is merely illustrative of the present invention and that no limitations are intended to the scope of the invention, except insofar as modifications, equivalents, improvements or modifications are within the spirit and principles of the invention.

Claims (8)

1. A method for continuous production of single cell proteins, the method comprising:

(1) Fermenting and culturing yarrowia lipolytica in the presence of a main culture medium;

(2) Fermenting and culturing until the OD value of the thalli is 400-450, discharging, and adding an auxiliary culture medium into the thalli after the discharging is finished, and continuing fermenting and culturing;

(3) Repeating the step (2), and centrifuging the material obtained by discharging to obtain the single-cell protein;

the main culture medium comprises 14-20 g/L of sodium acetate, 3-5% of anhydrous glucose, 2.2-3 wt% of yeast powder, 100-200 mg/L of vitamin B, 150-250 mg/L of nicotinic acid, 250-350 mg/L of pyridoxine, 30-50 mg/L of biotin, 700-900 mg/L of calcium pantothenate, 150-250 mg/L of riboflavin, 5-7 g/L of inositol, 100-200 mg/L of para-aminobenzoic acid, 350-500 mL/L of boric acid, 100-200 mg/L of manganese sulfate, 250-400 mg/L of zinc sulfate, 150-250 mg/L of sodium manganate, 0.005-0.015 mg/L of potassium iodide, 0.00-0.015 g/L of calcium chloride, 0.02-0.08 g/L of magnesium sulfate and 0.2-0.8 g/L of sodium chloride;

the auxiliary culture medium in the step (2) comprises 15-30 g/L of sodium acetate, 10-30 g/L of yeast basic nitrogen source, 600-800 mg/L of zinc sulfate, 450-550 mg/L of sodium manganate, 0.03-0.05 mg/L of potassium iodide, 0.06-0.08 g/L of calcium chloride, 0.08-0.1 g/L of magnesium sulfate, 0.6-0.8 g/L of sodium chloride, 0.2-1.5 g/L of dipotassium hydrogen phosphate and 0.5-3 g/L of monopotassium phosphate;

and (3) after the centrifugation, the step of diluting and low-temperature treating the bacterial residues obtained by the centrifugation and spray drying.

2. The method according to claim 1, wherein the pH of the main culture medium is 6-8.

3. The method of claim 1, wherein the yarrowia lipolytica of step (1) is obtained by sequentially subjecting a primary yeast extract peptone glucose medium, a secondary yeast extract peptone glucose medium, and a tertiary yeast extract peptone glucose medium to amplification culture;

the OD value of the thalli obtained by amplification culture of the primary yeast extract peptone glucose culture medium is 5-8;

the OD value of the thalli obtained by amplification culture of the secondary yeast extract peptone glucose culture medium is 12-18;

the OD value of the thalli obtained by amplification culture of the tertiary yeast extract peptone glucose culture medium is 40-65;

the fermentation culture of the step (1) is carried out in a fermentation tank.

4. The method of claim 1, wherein the volume of the discharged material in the step (2) is 0.4-0.5 times of the total volume;

and (3) adding the auxiliary culture medium in the step (2) in a volume which is 0.4-0.45 times of the total volume before discharging.

5. The method of claim 1, wherein the dilution comprises mixing the resulting bacterial sludge with water to a bacterial dry weight concentration of 60-80 g/L;

the temperature of the low-temperature treatment is 2-5 ℃ and the time is 12-16 h.

6. The method according to claim 1, wherein the spray drying is carried out at a feed rate of 4.5-6 l/h, an inlet air temperature of 120-160 ℃ and a frequency of 320-380 hz.

7. The method of claim 1, wherein the wall material of the single cell protein is at least one of methylcellulose, alginate, or calcium phosphate;

the time interval for harvesting the single cell protein is 20-40 min.

8. The method according to claim 1, characterized in that the method comprises:

(1) Inoculating yarrowia lipolytica with the OD value of the thalli of 40-65 into a fermentation tank for fermentation culture in the presence of a main culture medium; the main culture medium comprises 14-20 g/L of sodium acetate, 3-5 wt% of anhydrous glucose, 2.2-3 wt% of yeast powder, 1-200 mg/L of vitamin B, 150-250 mg/L of nicotinic acid, 250-350 mg/L of pyridoxine, 30-50 mg/L of biotin, 700-900 mg/L of calcium pantothenate, 150-250 mg/L of riboflavin, 5-7 g/L of inositol, 100-200 mg/L of para-aminobenzoic acid, 350-500 mL/L of boric acid, 100-200 mg/L of manganese sulfate, 250-400 mg/L of zinc phosphate, 150-250 mg/L of sodium manganate, 0.005-0.015 mg/L of potassium iodide, 0.005-0.015 g/L of calcium chloride, 0.02-0.08 g/L of magnesium sulfate and 0.2-0.8 g/L of sodium chloride;

(2) Fermenting and culturing until the OD value of the thalli is 400-450, discharging, and after discharging is finished, adding an auxiliary culture medium into a fermentation tank, and continuing fermenting and culturing; the auxiliary culture medium comprises 15-30 g/L of sodium acetate, 10-30 g/L of yeast basic nitrogen source, 600-800 mg/L of zinc sulfate, 450-550 mg/L of sodium manganate, 0.03-0.05 mg/L of potassium iodide, 0.06-0.08 g/L of calcium chloride, 0.08-0.1 g/L of magnesium sulfate, 0.6-0.8 g/L of sodium chloride, 0.2-1.5 g/L of dipotassium hydrogen phosphate and 0.5-3 g/L of potassium dihydrogen phosphate;

(3) And (3) repeating the step (2), centrifuging the discharged material, diluting the obtained bacterial residues with water until the dry weight concentration of the bacterial bodies is 60-80 g/L, placing the bacterial residues at 2-5 ℃ for 12-16 h, then performing spray drying, wherein the feeding speed of the spray drying is 4.5-6L/h, the air inlet temperature is 120-160 ℃, the frequency is 320-380 Hz, taking at least one of methylcellulose, alginate or calcium phosphate as a wall material, and harvesting single-cell proteins every 20-40 min to obtain the single-cell proteins.