The invention relates to 2apos; fucosyllactose fermentation supplementary material nitrogen source production method

Abstract

The invention discloses a preparation method of a supplementary nitrogen source for fermentation of 2 '-fucosyllactose, belongs to the technical field of 2'-fucosyllactose, and solves two major problems of significant increase of fermentation cost, excessive accumulation of proteins, peptides and amino acids in a fermentation tank and serious foam caused by additional addition of yeast powder. Comprising the following steps: aiming at the defects of high cost and more foam caused by additional addition of yeast powder in 2 '-FL fermentation, an alternative scheme of ammonia water and folic acid is adopted as a supplementary material nitrogen source, so that additional yeast powder can be avoided, the raw material cost is reduced, and the use amount of foam and a defoaming agent caused by protein accumulation is reduced.

Description

Technical Field

[0001] This invention belongs to the field of 2'-fucosylated lactose technology, specifically relating to a method for preparing a supplementary nitrogen source for 2'-fucosylated lactose fermentation. Background Technology

[0002] Human milk oligosaccharides (HMOs) are the third most abundant component of breast milk after lactose and fat, and are essential for protecting the health of newborns. 2'-fucosyllactose (2'-FL) has the highest content among HMOs, reaching up to 31%, and is also the simplest oligosaccharide in structure. Furthermore, it possesses most of the functions of HMOs. Therefore, exploring the safe and efficient synthesis of 2'-FL is of great significance for promoting its industrialization.

[0003] The whole-cell microbial synthesis of 2'-FL is currently the most commonly used method due to its mild conditions and low cost. Existing fermentation cycles for 2'-FL using Bacillus subtilis as the fermentation strain are typically 68–74 hours, and the synthesis efficiency is highly correlated with the cell mass in the early stages of fermentation. A scheme involving the addition of 5 g / L / h yeast extract after 9 hours of fermentation promotes strain growth. Compared to batches without added yeast extract, this method shortens the time to reach a 2'-FL product concentration of 65 g / L by 15 hours (experimental data: the final product concentration in the 5 g / L / h yeast extract group was 79.02 g / L, with an OD600 peak of 222, reached in 62 hours). However, the additional yeast extract presents two major problems: firstly, it significantly increases fermentation costs; secondly, it leads to excessive accumulation of proteins, peptides, and amino acids in the fermenter, causing severe foaming problems. Therefore, we propose a method for preparing a supplementary nitrogen source for 2'-fucosylated lactose fermentation.

[0004] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a supplemental nitrogen source for 2'-fucosylated lactose fermentation.

[0006] To achieve the above objectives, the present invention provides the following technical solution: 8. A method for preparing a fed-batch nitrogen source for 2'-fucosylated lactose fermentation, comprising the following steps: S1: Prepare fermentation medium: Prepare fermentation medium suitable for a 50L fermenter; S2: Preparation of folic acid solution: Dissolve folic acid in 0.01 mol / L phosphate buffer and bring the volume up to a solution containing 25 mg of folic acid; S3: Calibrate fermenter parameters; S4: Inoculate the strain and carry out fermentation treatment: Inoculate the activated Bacillus subtilis seed liquid into the fermenter calibrated in step S3 at an inoculation amount of 5% (v / v), then add the sterile folic acid solution prepared in step S2, stir evenly, so that the final concentration of folic acid in the fermentation system reaches 1.0 mg / L. S5: Parameter control of the fermentation process in step S4; S6: Fermentation complete and products collected: Total fermentation time is 74-80 hours.

[0007] Preferably, the components of the culture medium include 24 g / L corn steep liquor powder, 24 g / L yeast extract powder, and K2HP. 3H2O 13.07g / L, KH2PO4 3g / L, urea 6.67g / L, anhydrous MgSO4 3g / L, KIO3 3.5g / L, glucose 10g / L, lactose 30g / L.

[0008] Preferably, in step S2, the solution is filtered and sterilized using a 0.22μm filter membrane, and the sterilized folic acid solution is sealed and stored away from light.

[0009] Preferably, in step S3, the internal temperature of the fermenter is 37°C, the initial fermentation volume is set to 25L, the aeration rate is adjusted to 25L / min, the initial rotation speed is 100rpm, and the pH value is adjusted to 6.90 to complete parameter calibration.

[0010] Preferably, the temperature inside the fermenter in step S5 is 37°C.

[0011] Preferably, the dissolved oxygen concentration during fermentation in step S5 is between 40% and 50%.

[0012] Preferably, in step S6, if the 2'-FL concentration does not continue to increase after 8 hours, fermentation is terminated, and the fermentation broth is extracted, purified, and the 2'-FL product is collected.

[0013] Compared with the prior art, the present invention has the following beneficial effects: This invention addresses the shortcomings of "high cost and excessive foaming when adding yeast powder" in 2'-FL fermentation by using "ammonia water + folic acid" as a nitrogen source replacement solution. This eliminates the need for additional yeast powder, reduces raw material costs, and decreases foaming caused by protein accumulation and the amount of defoaming agent required. Detailed Implementation

[0014] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0015] In the description of this invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer" and other terms indicating orientation or positional relationships are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on the invention.

[0016] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "joining" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0017] A method for preparing a fed-batch nitrogen source for 2'-fucosylated lactose fermentation includes the following steps: S1: Prepare fermentation medium: Prepare fermentation medium suitable for a 50L fermenter; S2: Preparation of folic acid solution: Dissolve folic acid in 0.01 mol / L phosphate buffer and bring the volume up to a solution containing 25 mg of folic acid; S3: Calibrate fermenter parameters; S4: Inoculate the strain and carry out fermentation treatment: Inoculate the activated Bacillus subtilis seed liquid into the fermenter calibrated in step S3 at an inoculation amount of 5% (v / v), then add the sterile folic acid solution prepared in step S2, stir evenly, so that the final concentration of folic acid in the fermentation system reaches 1.0 mg / L. S5: Parameter control of the fermentation process in step S4; S6: Fermentation complete and products collected: Total fermentation time is 74-80 hours.

[0018] In step S1, the fermentation medium suitable for a 50L fermenter is precisely prepared according to the following formula: Components include 24g / L corn steep liquor powder, 24g / L yeast extract powder, and K2HP. The following ingredients were added: 13.07 g / L H₂O, 3 g / L KH₂PO₄, 6.67 g / L urea, 3 g / L anhydrous MgSO₄, 3.5 g / L KIO₃, 10 g / L glucose, and 30 g / L lactose. A polyether-modified silicone defoamer (0.15 mL / L) was added to the above components, and the remainder was made up with deionized water. The mixture was stirred until all components were completely dissolved to form a homogeneous fermentation medium. The prepared medium was then transferred to a 50 L fermenter to complete the pre-fermentation nutrient system setup. In step S2, folic acid was dissolved in 0.01 mol / L phosphate buffer (PBS, pH 7.0-7.4) and the solution was brought to a final volume of 25 mg folic acid. The solution was then filtered through a 0.22 μm membrane for sterilization (to avoid high-temperature sterilization which could destroy the vitamin activity of folic acid and ensure that it could participate normally in the one-carbon unit metabolism of the strain during fermentation). The sterilized folic acid solution was then sealed, protected from light, and stored for later use. In step S3, the 50L fermenter containing the culture medium prepared in step 1 is placed in a constant temperature environment to maintain the temperature inside the tank at 37°C; the initial fermentation volume is set to 25L, the aeration rate is adjusted to 25L / min, the initial rotation speed is 100rpm, and the maximum dissolved oxygen value of the fermentation system is corrected; the pH value of the culture medium is adjusted to 6.90 using ammonia water to complete the calibration of temperature, dissolved oxygen, and pH parameters before fermentation, ensuring that the environment meets the growth requirements of Bacillus subtilis at the time of inoculation.

[0019] In step S4, the activated Bacillus subtilis seed culture is inoculated into the fermenter calibrated in step 3 at an inoculation rate of 5% (v / v). After the inoculation is completed, the sterile folic acid solution prepared in step 2 is immediately added to the fermentation system and stirred evenly to make the final concentration of folic acid in the fermentation system reach 1.0 mg / L. This step ensures that the strain can use folic acid to participate in metabolism from the initial stage of growth, replacing the nitrogen source function of traditional yeast powder.

[0020] Parameter control during fermentation: Temperature control: The fermenter temperature is maintained at 37℃ throughout the process using a temperature control system, with a deviation of no more than ±0.5℃; pH control: Ammonia is added to the fermentation system in real time via an automatic dripping device to stabilize the pH value at 7.25±0.05 (no additional ammonia flow rate setting is required, with pH stability as the core control indicator), while ammonia is used to supplement the basic nitrogen source; Dissolved oxygen control: By adjusting the rotation speed and aeration rate, the dissolved oxygen concentration of the fermentation system is maintained at 40%~50% throughout the process to ensure the respiratory and metabolic needs of the strain. In step S6, the total fermentation time for human milk oligosaccharides is 74 to 80 hours. Fermentation is terminated when the 2'-FL concentration has not continued to rise for nearly 8 hours. The fermentation broth is then extracted, purified, and the 2'-FL product is collected.

[0021] Based on the premise of adding folic acid simultaneously with vaccination, the concentration of folic acid was adjusted in a gradient manner to screen the optimal concentration.

[0022] Table 1: Effects of different supplemental nitrogen sources on 2'-FL concentration and cell count

[0023] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for making a feed nitrogen source for the fermentation of 2'-fucosyllactose, characterized in that, The method comprises the following steps: S1: preparing a fermentation medium: preparing a fermentation medium suitable for a 50L fermenter; S2: preparing a folic acid solution: dissolving folic acid in 0.01mol / L phosphate buffer and diluting to a solution containing 25mg of folic acid; S3: calibrating the parameters of the fermenter; S4: inoculating the strain and performing fermentation: inoculating the activated Bacillus subtilis seed liquid into the fermenter calibrated in step S3 at a 5% (v / v) inoculation amount, then adding the sterile folic acid solution prepared in step S2, and stirring uniformly to make the final concentration of folic acid in the fermentation system reach 1.0mg / L; S5: controlling the parameters of the fermentation process in step S4; S6: completing fermentation and collecting the product: the total fermentation time is 74-80h.

2. The method for preparing the fed-batch nitrogen source for 2'-fucosylated lactose fermentation according to claim 1, characterized in that, The components of the medium include corn steep liquor dry powder 24 g / L, yeast extract powder 24 g / L, K2HP 3H2O 13.07 g / L, KH2PO4 3 g / L, urea 6.67 g / L, anhydrous MgSO4 3 g / L, KIO3 3.5 g / L, glucose 10 g / L, lactose 30 g / L.

3. The method for preparing the fed-batch nitrogen source for 2'-fucosylated lactose fermentation according to claim 1, characterized in that, The solution is filtered with a 0.22μm filter to remove bacteria in step S2, and the bacteria-free folic acid solution is sealed and stored in the dark.

4. The method for preparing the fed-batch nitrogen source for 2'-fucosylated lactose fermentation according to claim 1, characterized in that, The temperature in the fermenter in step S3 is 37℃, the initial volume is set to 25L, the aeration amount is adjusted to 25L / min, the initial rotation speed is 100rpm, the pH value is adjusted to 6.90, and the parameter calibration is completed.

5. The method for preparing the fed-batch nitrogen source for 2'-fucosylated lactose fermentation according to claim 1, characterized in that, The temperature in the fermenter in step S5 is 37℃.

6. The method for preparing the fed-batch nitrogen source for 2'-fucosylated lactose fermentation according to claim 1, characterized in that, The dissolved oxygen concentration in the fermentation in step S5 is 40%-50%.

7. The method for preparing the fed-batch nitrogen source for 2'-fucosylated lactose fermentation according to claim 1, characterized in that, The concentration of 2'-FL does not continue to increase after 8h in step S6, the fermentation is terminated, and the 2'-FL product is collected after extraction and purification of the fermentation broth.

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