A low-surface oil, high-dha fragilaria sp. fermentation method

Abstract

The application provides a Schizochytrium fermenting method with low surface oil and high DHA, wherein activated Schizochytrium sp. is cultured after amplification, and then seed liquid obtained is subjected to fermentation culture; in the early fermentation stage, a first composite buffer system composed of ammonia and citric acid is used to maintain the pH value of the fermentation system at 5.0-7.0; in the middle and late fermentation stage, a second composite buffer system composed of at least calcium carbonate and citric acid is used to maintain the pH value of the fermentation system at 5.0-7.0; the residual sugar concentration of the fermentation system is maintained at 10-50 g / L through feeding, and finally the fermentation product is obtained. Through the multiple regulation, the method can improve the cell dry weight and DHA yield, and significantly reduce the surface oil.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology and provides a fermentation method for Schizochytrium algae with low surface oil and high DHA content. The method involves achieving high-density cultivation of Schizochytrium algae through a specific fermentation medium formulation and a segmented fermentation control strategy, while significantly reducing surface oil content and increasing DHA yield. Background Technology

[0002] Docosahexaenoic acid (DHA), a long-chain ω-3 polyunsaturated fatty acid with six cis double bonds, plays a vital role in physiological and nutritional health. DHA is a key structural lipid constituting the phospholipid bilayer of specific biological membranes, such as those in cerebral cortex neuronal synapses, extracellular disc membranes of retinal photoreceptors, and sperm heads. In these highly specialized membrane structures, DHA, through its long and polyunsaturated carbon chains, significantly increases membrane fluidity and flexibility, playing a crucial role in maintaining the normal conformation and function of membrane proteins (such as ion channels and G protein-coupled receptors) and ensuring efficient nerve signal transmission. Simultaneously, DHA is also an important precursor to bioactive molecules, capable of metabolizing into lipid mediators with potent anti-inflammatory and pro-inflammatory-reduction effects, such as reductase and proton pump inhibitors, participating in the regulation of immune responses, neuroprotection, and tissue repair. Therefore, DHA has extremely wide and in-depth applications in infant formula, nutritional supplements for pregnant and lactating women, functional health foods, special medical purpose formula foods, and high-end aquaculture feeds.

[0003] Commercial DHA production primarily utilizes two pathways: traditional fish oil extraction and microbial fermentation. Oils from deep-sea fish (such as tuna and salmon) are the main source of DHA. However, DHA production from fish oil faces challenges including cyclical fluctuations in fishery resources, the risk of heavy metal bioaccumulation due to marine pollution, difficulty in removing fishy odor, and high costs resulting from complex decolorization, deodorization, and purification processes. Furthermore, the DHA content in fish oil typically ranges from 12% to 30%, and it often coexists with eicosapentaenoic acid (EPA), making the separation and purification of high-purity DHA products technically challenging. In addition, concerns about ecological sustainability caused by overfishing are prompting the industry to seek greener and more controllable alternatives. In contrast, microbial fermentation, especially using marine schizochytrium microorganisms such as Schizochytrium sp. and Aurantiochytrium sp., has advantages such as high efficiency and environmental friendliness. Its fermentation cycle is short, its growth is not limited by climate and region, it can utilize a variety of inexpensive carbon sources (such as glucose, glycerol, and lignocellulose hydrolysate), and its oil content is extremely high (accounting for more than 50% of the cell dry weight) and DHA accounts for a high proportion of total fatty acids (up to 40-60%). It is an ideal "cell factory" for realizing the sustainable industrial production of DHA.

[0004] To achieve the economic feasibility of DHA fermentation from *Schizochytrium*, the optimization of high-cell-density fermentation processes has gradually matured. By optimizing culture conditions, the cell biomass (based on cell dry weight) is maximized, thereby achieving the highest total DHA yield (g DHA / L) per unit fermentation volume. Currently, through strategies such as fed-batch fermentation, nutrient optimization, and precise control of dissolved oxygen and pH, the cell dry weight of *Schizochytrium* can reach 150-200 g / L or even higher in laboratory and pilot-scale operations, with DHA yields reaching 40-80 g / L, demonstrating strong industrialization potential.

[0005] However, in high-cell-density fermentation processes, a significant technical bottleneck affecting yield, cost, and product quality is becoming increasingly prominent—the formation of "surface oil" or "extracellular oil." Surface oil is not the ideal form for the target product, triglycerides (TAG). It mainly refers to the free oil released from the cell into the fermentation broth during the later stages of fermentation due to cell autolysis, loss of membrane integrity, or active / passive secretion. This oil floats on the surface of the fermentation broth or emulsifies within the system, forming a significant oil layer. In traditional or unoptimized high-density fermentation processes, surface oil can account for a staggering 30-60% of the total lipid yield. The surface oil, free in the extracellular fermentation broth, is lost through centrifugation, leading to a significant decrease in total oil recovery. Recovering emulsified or dissolved oil from the fermentation supernatant requires additional demulsification and extraction steps, greatly increasing production costs. Furthermore, the presence of surface oil increases the viscosity and complexity of the fermentation broth, interfering with cell aggregation and sedimentation, thus reducing solid-liquid separation efficiency. Meanwhile, during high-cell-density fermentation, a large amount of surface oil coats the microbial cells, affecting the mass transfer efficiency of oxygen and nutrients, potentially exacerbating anaerobic stress and cell death, creating a vicious cycle. Oxidation products of the oils may also inhibit microbial activity. Free oils present in the fermentation broth over a long period are more prone to oxidative rancidity, producing undesirable flavor compounds that affect the color, odor, and oxidative stability of the Schizochytrium powder or algal oil, thus reducing its commercial value.

[0006] Therefore, developing a fermentation method for Schizochytrium with low surface oil and high DHA has become a key technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0007] To address the problems mentioned in the background section, this invention provides a fermentation method for Schizochytrium algae with low surface oil content and high DHA yield. This invention enables the simultaneous achievement of low surface oil content and high DHA production in Schizochytrium algae fermentation under high-density fermentation conditions, thereby enhancing the competitiveness of microalgae DHA products in high-end food, pharmaceutical, and other fields.

[0008] In one aspect, a fermentation method for *Schizochytridica* with low surface oil and high DHA is provided, comprising: amplifying and culturing activated *Schizochytridica* strains; fermenting the resulting seed culture; adjusting the pH of the fermentation system to 5.0-7.0 using a first composite buffer system composed of ammonia and citric acid within 0-20 h of fermentation; adjusting the pH of the fermentation system to 5.0-7.0 using a second composite buffer system composed of at least calcium carbonate and citric acid within 65-75 h of fermentation; and maintaining the residual sugar concentration of the fermentation system at 10-50 g / L by feeding.

[0009] In some embodiments, the conditions for fermentation culture of the seed culture are: 20-30°C, dissolved oxygen level maintained at 20%-70%, and total fermentation culture time of 85-120 hours. In some embodiments, the conditions for fermentation culture of the seed culture are: 20-30°C, dissolved oxygen level maintained at 20%-40%, and total fermentation culture time of 110-120 hours. In some embodiments, during fermentation culture, glucose with a mass concentration of 50%-70% is added to the seed culture, and the sugar content of the fermentation broth is controlled at 15-45 g / L after 65-75 hours.

[0010] In some embodiments, the culture medium for fermenting the seed liquid comprises 30-60 g / L yeast extract, 0.2-0.5 g / L calcium chloride, 0.5-1.5 g / L potassium chloride, 1-3 g / L ammonium sulfate, 1-3 g / L potassium dihydrogen phosphate, 6-10 g / L magnesium sulfate, 20-45 g / L sodium sulfate, 1-2 g / L potassium sulfate, 10-15 g / L monosodium glutamate, 2-5 g / L yeast powder, and vitamin B12.

[0011] In some instances, the vitamin B12 content in the culture medium is 0.1–1.0 mg / L.

[0012] In some embodiments, the culture medium for fermenting the seed liquid consists of 40 g / L yeast extract, 0.2 g / L calcium chloride, 1.5 g / L potassium chloride, 1 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 6 g / L magnesium sulfate, 45 g / L sodium sulfate, 1 g / L potassium sulfate, 15 g / L monosodium glutamate, 2 g / L yeast powder, and 0.5 mg / L vitamin B12.

[0013] In some embodiments, the mass concentration of the ammonia water in the first composite buffer system ranges from 5% to 25%.

[0014] In some embodiments, the concentration of calcium carbonate in the second composite buffer system ranges from 5 to 25 g / L.

[0015] In some instances, the concentration of citric acid in the first and second composite buffer systems ranges from 1 to 3 g / L.

[0016] In some embodiments, malic acid may be added to the second composite buffer system, wherein the concentration of malic acid is in the range of 1 to 3 g / L.

[0017] In some embodiments, the amplification culture process is as follows:

[0018] S1. The activated Schizochytrium strain was inoculated into the seed culture and cultured at 20-30℃ and 180-220 r / min for 24 h at pH 5.0-7.0. When the residual sugar concentration of the fermentation system was less than 20 g / L, it was transferred to the secondary seed tank for large-scale culture.

[0019] S2. Inoculate the culture medium of S1 into the expansion medium at an inoculation rate of 10-20% and culture at a temperature of 20-30℃, a shaking speed of 180-220 r / min, a culture time of 24h, and a pH of 5.0-7.0. When the residual sugar concentration of the fermentation system is less than 20g / L, it is transferred to a one-ton fermenter.

[0020] S3. Inoculate the expanded seed culture solution from S2 into a one-ton fermentation tank containing one ton of fermentation medium at an inoculation rate of 5-15% for fermentation culture. Maintain the fermentation temperature at 20-30℃, pH at 5.0-7.0, and aeration rate of 10-30 m³ / h. 3 / h, with a rotation speed of 35~50Hz, and the residual sugar concentration of the fermentation system is less than 20g / L when it is connected to an eight-ton fermenter;

[0021] S4. Inoculate the expanded seed culture from S3 into an 8-ton fermentation tank containing 8 tons of fermentation medium at an inoculation rate of 10-25%. Fermentation should be carried out at a temperature of 20-30℃, pH maintained at 5.0-7.0, and an aeration rate of 50-80 m³ / h. 3 Seed culture was obtained by incubating at a speed of 35-50 Hz for 10-15 hours.

[0022] In some embodiments, during the amplification culture, the pH of the fermentation system is adjusted by adding a pH adjuster selected from at least one of ammonia, sodium hydroxide, sodium carbonate, calcium carbonate, sodium acetate, malic acid, citric acid, acetic acid, hydrochloric acid, phosphoric acid, and sulfuric acid. In some examples, the pH is adjusted using ammonia / sodium acetate / calcium carbonate and malic acid / citric acid. In some examples, the pH is adjusted using ammonia / calcium carbonate and malic acid / citric acid. In some examples, the pH is adjusted using ammonia / calcium carbonate and citric acid. In some examples, the pH is adjusted using ammonia / calcium carbonate and phosphoric acid. In some examples, the pH is adjusted using ammonia and malic acid / citric acid.

[0023] In some embodiments, the seed culture medium, expansion culture medium, one-ton fermentation medium, and eight-ton fermentation medium include at least one of a carbon source, a nitrogen source, an inorganic salt, and a vitamin. In some examples, the carbon source includes at least one of glucose, corn steep liquor powder, glycerol, sucrose, lactose, and maltose. In some examples, the carbon source concentration is 40-80 g / L. In some examples, the nitrogen source of the culture medium is selected from at least one of yeast extract, corn steep liquor powder, soybean meal, ammonium sulfate, and soybean protein powder. In some examples, the nitrogen source concentration is 1-30 g / L. In some examples, the inorganic salt is selected from at least one of calcium chloride, potassium chloride, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate, sodium sulfate, and potassium sulfate. In some instances, the inorganic salt is selected from at least one of the following: calcium chloride 0.2-0.5 g / L, potassium chloride 0.5-1.5 g / L, ammonium sulfate 1-3 g / L, potassium dihydrogen phosphate 1-3 g / L, magnesium sulfate 6-10 g / L, sodium sulfate 20-45 g / L, and potassium sulfate 1-2 g / L.

[0024] In some embodiments, the seed culture medium comprises 40-70 g / L glucose, 5-15 g / L yeast extract, 10-25 g / L sodium sulfate, 3-6 g / L magnesium sulfate, 0.5-1.5 g / L potassium sulfate, 0.5-1.5 g / L potassium dihydrogen phosphate, 0.5-1.5 g / L ammonium sulfate, 0.1-0.4 g / L calcium chloride, and 1-3 g / L calcium carbonate.

[0025] In some embodiments, the expanded culture medium comprises 40-70 g / L glucose, 5-15 g / L yeast extract, 10-25 g / L sodium sulfate, 3-6 g / L magnesium sulfate, 0.5-1.5 g / L potassium sulfate, 0.5-1.5 g / L potassium dihydrogen phosphate, 0.5-1.5 g / L ammonium sulfate, 0.1-0.4 g / L calcium chloride, 1-3 g / L calcium carbonate, and 0.1-1.0 mg / L vitamin B6.

[0026] In some embodiments, the one ton fermentation medium comprises: calcium chloride 0.2~0.5 g / L, potassium chloride 0.5~1.5 g / L, ammonium sulfate 1~3 g / L, potassium dihydrogen phosphate 1~3 g / L, magnesium sulfate 6~10 g / L, sodium sulfate 20~45 g / L, potassium sulfate 1~2 g / L, monosodium glutamate 10~15 g / L, and yeast powder 2~5 g / L.

[0027] In some embodiments, the eight-ton fermentation culture medium comprises: yeast extract 30-60 g / L, calcium chloride 0.2-0.5 g / L, potassium chloride 0.5-1.5 g / L, ammonium sulfate 1-3 g / L, potassium dihydrogen phosphate 1-3 g / L, magnesium sulfate 6-10 g / L, sodium sulfate 20-45 g / L, potassium sulfate 1-2 g / L, monosodium glutamate 10-15 g / L, yeast powder 2-5 g / L, and vitamin B12 0.1-1.0 mg / L.

[0028] In some embodiments, during the fermentation process, an appropriate defoamer may be added to eliminate foam; conventional defoamers in the art can be used.

[0029] Compared with the prior art, one of the above technical solutions has the following advantages or beneficial effects:

[0030] This invention provides a fermentation method for Schizochytrium algae with low surface oil and high DHA content, which can significantly reduce the surface oil ratio while increasing cell dry weight and DHA yield. Specifically, it utilizes a high cell density fermentation medium for Schizochytrium algae with added vitamin B. 12 It provides cofactors for methionine synthase and methylmalonyl-CoA mutase, ensuring the smooth operation of one-carbon metabolism and propionic acid-assisted oxidation pathways, maintaining cell methylation reactions (promoting membrane phospholipid synthesis and repair) and reducing propionic acid accumulation (avoiding toxicity and metabolic disorders), thereby delaying cell autolysis in the later stages of fermentation. In the early stages of fermentation, ammonia / citric acid is added. Ammonia provides a nitrogen source and prevents a sudden drop in pH, while citric acid replenishes intermediates in the tricarboxylic acid cycle, jointly promoting rapid cell growth. In the middle and later stages of fermentation, calcium carbonate / citric acid is added. Calcium carbonate acts as a solid buffer to continuously stabilize the pH, alleviating metabolic acid stress, and the released Ca2+... 2+ It forms ion bridges with cell membrane phospholipids, enhancing the mechanical strength of the cell membrane while Ca... 2+ It combines with leaked free fatty acids to form calcium soaps, reducing their emulsifying and destructive effects. Through multiple regulatory mechanisms, a significant reduction in surface oil is ultimately achieved.

[0031] This invention obtains a culture by high-density fermentation of *Schizochytrium*, and further extracts *Schizochytrium* oil. The extracted *Schizochytrium* powder exhibits increased DHA content while maintaining extremely low surface oil content. This invention, while ensuring the same raw materials, increases DHA content and reduces DHA loss caused by high surface oil levels during algal powder extraction, thereby lowering production costs and improving production efficiency.

[0032] Terminology Explanation

[0033] Certain embodiments of the invention will now be described in detail, examples of which are illustrated by the accompanying structural and chemical formulas. The invention is intended to encompass all alternatives, modifications, and equivalents, all of which are included within its scope. Those skilled in the art will recognize that many similar or equivalent methods and materials can be used to practice the invention. The invention is by no means limited to the methods and materials described herein. In the event that one or more of the incorporated documents, patents, and similar materials differ from or contradict this application (including, but not limited to, defined terminology, application of terminology, described techniques, etc.), this application shall prevail.

[0034] It should be further appreciated that certain features of the invention, for clarity, have been described in multiple independent embodiments, but may also be provided in combination in a single embodiment. Conversely, various features of the invention, for brevity, have been described in a single embodiment, but may also be provided individually or in any suitable sub-combination.

[0035] Unless otherwise stated, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. All patents and publications related to this invention are incorporated herein by reference in their entirety.

[0036] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0037] In the following content, all numbers disclosed herein, whether or not they use words such as "approximately" or "about," are approximate values. The value of each number may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20%. Whenever a number with a value of N is disclosed, any numbers with values ​​of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15%, or N+ / -20% will be explicitly disclosed, where "+ / -" indicates addition or subtraction. Attached Figure Description

[0038] Figure 1This is a microscopic image of cells fermented for 120 hours under the conditions of Example 6-0.

[0039] Figure 2 The images are microscopic images of cells fermented for 120 hours under the conditions of Examples 6-9.

[0040] Figure 3 The images are microscopic images of cells fermented for 120 hours under the conditions of Examples 6-10. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0042] All reagents used in this invention can be purchased commercially or prepared by the methods described in this invention.

[0043] In this invention, the DHA content was detected using the internal standard method in GB 5009.168-2016.

[0044] The *Schizochytrium* used in this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 40902. It has been disclosed in the following patent: Specifically, patent application number CN202311728644.8, patent title: "A *Schizochytrium* strain rich in n-3 fatty acids and its screening and cultivation," publication number CN117701403A, publication date 2024-03-15.

[0045] The method for detecting surface oil in the fermentation broth of *Schizochytrium* in this invention includes: accurately weighing 1-2g of sample, and sequentially performing gradient extraction with 10ml of ethanol, 20ml of diethyl ether, and 20ml of petroleum ether, mixing each solvent by shaking. After the extract is allowed to stand and separate into layers for 30-60 minutes, accurately measure 20ml of the supernatant, evaporate it to dryness in a water bath below 60℃, and then dry it to constant weight in an oven at 103±2℃. Calculate the surface oil concentration according to the formula. The specific formula is as follows: X = (m1-m0) × (v1-v2) / m × 20 × (1-a) × 100. Where: X: fat content in the sample (on a dry basis), wt%, retained to one decimal place; a: water content in the sample, wt%; m1 is the weight of the supernatant in the glass dish; m0 is the weight of the empty glass dish; m is the weight of the supernatant after drying to constant weight in the glass dish; v1 is the total volume of the extract after standing and separating into layers; v2 is the volume of the lower layer of the extract after standing and separating into layers.

[0046] General Example 1

[0047] Seed culture activation: Laboratory-preserved *Schizochytrium* strains were inoculated into seed culture medium and cultured at 20–30℃ and 180–220 rpm for 24 h at pH 5.0–7.0. The seed culture medium consisted of 40–70 g / L glucose, 5–15 g / L yeast extract, 10–25 g / L sodium sulfate, 3–6 g / L magnesium sulfate, 0.5–1.5 g / L potassium sulfate, 0.5–1.5 g / L potassium dihydrogen phosphate, 0.5–1.5 g / L ammonium sulfate, 0.1–0.4 g / L calcium chloride, and 1–3 g / L calcium carbonate. When the residual sugar concentration in the fermentation system was below 20 g / L, the culture was transferred to a secondary seed tank for further expansion. The specific seed culture medium is shown in Table 1 below.

[0048] Table 1 Seed Culture Medium Information

[0049]

[0050] General Example 2

[0051] Seed culture expansion: The culture solution from Example 1-1 was inoculated into the expansion medium at an inoculum rate of 10-20% and cultured at a temperature of 20-30℃, a shaking speed of 180-220 r / min, for 24 hours, with a pH of 5.0-7.0. The expansion medium consisted of 40-70 g / L glucose, 5-15 g / L yeast extract, 10-25 g / L sodium sulfate, 3-6 g / L magnesium sulfate, 0.5-1.5 g / L potassium sulfate, 0.5-1.5 g / L potassium dihydrogen phosphate, 0.5-1.5 g / L ammonium sulfate, 0.1-0.4 g / L calcium chloride, 1-3 g / L calcium carbonate, and 0.1-1.0 mg / L vitamin B6. When the residual sugar concentration of the fermentation system was below 20 g / L, it was inoculated into a one-ton fermenter. Specific culture conditions are shown in Table 2 below.

[0052] Table 2 Cultivation Status

[0053]

[0054] General Example 3

[0055] One-ton tank culture: The expanded seed culture broth from Example 2 was inoculated into a one-ton tank at an inoculation rate of 5-15% for fermentation culture. The culture temperature was 20-30℃, the pH was maintained at 5.0-7.0, and the aeration rate was 10-30 m³ / h. 3The fermentation medium is prepared at a speed of 35-50 Hz. One ton of fermentation medium includes: calcium chloride 0.2-0.5 g / L, potassium chloride 0.5-1.5 g / L, ammonium sulfate 1-3 g / L, potassium dihydrogen phosphate 1-3 g / L, magnesium sulfate 6-10 g / L, sodium sulfate 20-45 g / L, potassium sulfate 1-2 g / L, monosodium glutamate 10-15 g / L, and yeast powder 2-5 g / L. When the residual sugar concentration of the fermentation system is below 20 g / L, it is introduced into an eight-ton fermenter. Specific cultivation conditions are shown in Table 3 below.

[0056] Table 3 Cultivation Status

[0057]

[0058] General Example 4

[0059] Eight-ton tank culture: One ton of seed culture solution from Example 3 was inoculated into an eight-ton tank at an inoculation rate of 10-25% for fermentation culture. The culture temperature was 20-30℃, the pH was maintained at 5.0-7.0, and the aeration rate was 50-80 m³ / h. 3 The fermentation medium was 35-50 Hz, with a rotation speed of 1 h. The eight-ton fermentation medium consisted of: yeast extract 30-60 g / L, calcium chloride 0.2-0.5 g / L, potassium chloride 0.5-1.5 g / L, ammonium sulfate 1-3 g / L, potassium dihydrogen phosphate 1-3 g / L, magnesium sulfate 6-10 g / L, sodium sulfate 20-45 g / L, potassium sulfate 1-2 g / L, monosodium glutamate 10-15 g / L, yeast powder 2-5 g / L, and vitamin B12 0.1-1.0 mg / L. After 10-15 hours of cultivation, the medium was transferred to an eighty-ton fermenter. Specific cultivation conditions are shown in Table 4 below.

[0060] Table 4 Cultivation Status

[0061]

[0062] General Example 5

[0063] 80-ton tank culture: The 8 tons of seed culture solution from Example 4 was inoculated into an 80-ton tank at an inoculation rate of 10-25% for culture. The culture temperature was 20-30℃, and the pH was maintained at 5.0-7.0. The pH was adjusted throughout the process using sodium acetate and malic acid, with the addition amounts being 3 g / L malic acid and 12 g / L sodium acetate. The aeration rate was 500-1000 m³ / h. 3The fermentation speed is 40-50 Hz. During fermentation, 60% glucose is added, and the sugar content of the fermentation broth is controlled at 15-45 g / L after 65-75 hours. The 80-ton fermentation medium includes: yeast extract 30-60 g / L, calcium chloride 0.2-0.5 g / L, potassium chloride 0.5-1.5 g / L, ammonium sulfate 1-3 g / L, potassium dihydrogen phosphate 1-3 g / L, magnesium sulfate 6-10 g / L, sodium sulfate 20-45 g / L, potassium sulfate 1-2 g / L, monosodium glutamate 10-15 g / L, and yeast powder 2-5 g / L. Specific cultivation conditions are shown in Table 5 below.

[0064] Table 5 Cultivation Status

[0065]

[0066] The culture time was 120 hours, and the results of the products are shown in Table 6 below.

[0067] Table 6 Results of the products

[0068]

[0069] As can be seen from Example 5, during the fermentation culture in the 80-ton tank, changes in fermentation conditions (Examples 5-1, 5-2, 5-3, 5-6, and 5-7) had little impact on the product; however, adjustments to the components in the fermentation medium, especially changes in the types of vitamins, had a significant impact on the surface oil. Changes in surface oil in Examples 5-1, 5-4, and 5-5 show that the introduction of vitamin B12 can effectively control key metabolic pathways and has a certain impact on cell autolysis.

[0070] General Example 6

[0071] The difference from Examples 5-5 lies in the pH adjustment process used during the 80-ton tank culture, the dissolved oxygen level maintained at 20-40%, and the addition of 50-70% glucose during fermentation to maintain the residual sugar concentration in the fermentation broth at 10-50 g / L, as detailed in Table 7 below.

[0072] Table 7 Changes in relevant parameters

[0073]

[0074] The results of the obtained products are shown in Table 8 below.

[0075] Table 8 Results of the products

[0076]

[0077] As can be seen from the general example 6 during the 80-ton tank fermentation process:

[0078] Comparing Examples 6-2 and 6-3, the introduction of vitamin B12 into the culture medium, in conjunction with pH regulation during the culture process, not only effectively ensures the healthy growth of Schizochytrium cells, but also effectively controls key metabolic pathways and has a certain impact on cell autolysis.

[0079] Comparing Examples 6-1 and 6-3, pH adjustment alone in the early stage of cultivation cannot effectively control the production of surface oil.

[0080] Compared with Examples 6-4 to 6-8, different pH reagents were used to regulate the pH of the system during the culture process. The introduction of vitamin B12 in the culture medium can effectively ensure healthy cell production and maintain cell membrane integrity, so that the surface oil of the obtained product can be effectively controlled. In particular, in Examples 6-7 and 6-8, the surface oil content of the obtained product was reduced to below 5%.

[0081] Compared with Examples 6-1 to 6-8, Examples 6-9 and 6-10 adopted a step-by-step control of the pH of the fermentation environment. In the early stage (0-20h), ammonia / citric acid was added, and in the later stage (65-75h), calcium carbonate / citric acid was added to regulate the fermentation environment in stages to maintain healthy cell growth, thereby inhibiting the formation of surface oil at the source.

[0082] like Figure 1 , Figure 2 and Figure 3 The comparison shows that Example 6-0 did not optimize the pH control of the fermentation process, nor did it improve the culture medium. After 120 hours of fermentation, the resulting cell membranes were thin and ruptured in the later stage of fermentation, with oil seeping into the fermentation broth. In contrast, Examples 6-9 and 6-10 optimized the pH control process and improved the culture medium, resulting in thicker cell membranes that showed almost no rupture in the later stage of 120 hours of fermentation.

[0083] In summary, the fermentation method of this invention aims to promote rapid cell growth by supplementing ammonia / citric acid in the early stage of fermentation; in the middle and later stages, it switches to a calcium carbonate / citric acid buffer system, whose Ca... 2+ It can stabilize pH and enhance cell membrane integrity; throughout the fermentation process, it is combined with vitamin B. 12 By regulating key metabolic pathways, the cells can be de-saturated and their autolysis can be delayed. This stable physicochemical environment not only promotes the growth of Schizochytrium cells, but also facilitates the efficient synthesis of its metabolites (especially lipids and DHA), and inhibits the surface oil caused by cell membrane breakage, thereby achieving a simultaneous increase in biomass yield, total lipid yield, and DHA content and quality in the lipids.

[0084] General Example 7

[0085] The fermentation broths of *Schizochytrium* obtained in Examples 6-7, 6-9, and 6-10 were centrifuged to collect the bacterial cells. Using the oil produced by 76.88% of the bacterial cells as raw material, 20% sodium octenyl succinate starch, 2.5% sodium ascorbate, 0.01% sodium citrate, and 0.01% maltodextrin were added as excipients. The mixture was sheared at 10000 r / min for 15 min and homogenized twice at 800 bar. Then, it was spray-dried at an inlet air temperature of 170±5℃ and an outlet air temperature of 70±5℃. After sieving, it was mixed with 0.6% tricalcium phosphate to obtain microcapsule products 7-1, 7-2, and 7-3.

[0086] Surface oil detection showed that the surface oil content of microcapsule products 7-1, 7-2, and 7-3 was below the method detection limit and could be counted as 0%. This indicates that after the surface oil in the fermentation broth is reduced, the subsequent physical encapsulation process can eliminate the surface oil in the fermentation broth.

[0087] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention.

Claims

1. A fermentation method for Schizochytrium algae with low surface oil and high DHA, characterized in that, The steps include the following: After the activated *Schizochytrium* strain was expanded and cultured, the resulting seed culture was fermented. During the first 0-20 hours of fermentation, the pH of the fermentation system was maintained at 5.0-7.0 using a first complex buffer system composed of ammonia and citric acid. During the second 65-75 hours of fermentation, the pH of the fermentation system was maintained at 5.0-7.0 using a second complex buffer system composed of at least calcium carbonate and citric acid. The residual sugar concentration of the fermentation system was maintained at 10-50 g / L by feeding. The culture medium for fermenting the seed liquid consists of yeast extract 30-60 g / L, calcium chloride 0.2-0.5 g / L, potassium chloride 0.5-1.5 g / L, ammonium sulfate 1-3 g / L, potassium dihydrogen phosphate 1-3 g / L, magnesium sulfate 6-10 g / L, sodium sulfate 20-45 g / L, potassium sulfate 1-2 g / L, monosodium glutamate 10-15 g / L, yeast powder 2-5 g / L, and vitamin B12 0.1-1.0 mg / L.

2. The fermentation method according to claim 1, characterized in that, The conditions for fermentation culture of the seed liquid are: 20~30℃, dissolved oxygen level maintained at 20%~70%, and total fermentation culture time of 110~120 hours.

3. The fermentation method according to claim 1, characterized in that, During the fermentation culture of the seed liquid, glucose with a mass concentration of 50% to 70% is added, and the sugar content of the fermentation liquid is controlled at 15 to 45 g / L for 65 to 75 hours.

4. The fermentation method according to claim 1, characterized in that, The culture medium for fermenting the seed liquid consisted of 40 g / L yeast extract, 0.2 g / L calcium chloride, 1.5 g / L potassium chloride, 1 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 6 g / L magnesium sulfate, 45 g / L sodium sulfate, 1 g / L potassium sulfate, 15 g / L monosodium glutamate, 2 g / L yeast powder, and 0.5 mg / L vitamin B12.

5. The fermentation method according to claim 1, characterized in that, In the first composite buffer system, the mass concentration of the ammonia solution ranges from 5% to 25%; in the second composite buffer system, the concentration of the calcium carbonate ranges from 5 to 25 g / L. In the first and second composite buffer systems, the concentration of citric acid ranges from 1 to 3 g / L.

6. The fermentation method according to claim 1, characterized in that, In the second composite buffer system, malic acid may also be added, with the concentration of malic acid ranging from 1 to 3 g / L.

7. The fermentation method according to claim 1, characterized in that, The amplification and culture process is as follows: S1. The activated Schizochytrium strain was inoculated into the seed culture and cultured at 20-30℃ and 180-220 r / min for 24 h at pH 5.0-7.

0. When the residual sugar concentration of the fermentation system was less than 20 g / L, it was transferred to the secondary seed tank for large-scale culture. S2. Inoculate the culture medium of S1 into the expansion medium at an inoculation rate of 10-20% and culture at a temperature of 20-30℃, a shaking speed of 180-220 r / min, a culture time of 24h, and a pH of 5.0-7.

0. When the residual sugar concentration of the fermentation system is less than 20g / L, it is transferred to a one-ton fermenter. S3. Inoculate the expanded seed culture solution from S2 into a one-ton fermentation tank containing one ton of fermentation medium at an inoculation rate of 5-15% and ferment for 24 hours at a temperature of 20-30℃, pH maintained at 5.0-7.0, and aeration rate of 10-30 m³ / h. 3 / h, with a rotation speed of 35~50Hz, and the residual sugar concentration of the fermentation system is less than 20g / L when it is connected to an eight-ton fermenter; S4. Inoculate the expanded seed culture from S3 into an 8-ton fermentation tank containing 8 tons of fermentation medium at an inoculation rate of 10-25%. Fermentation should be carried out at a temperature of 20-30℃, pH maintained at 5.0-7.0, and an aeration rate of 50-80 m³ / h. 3 Seed culture was obtained by incubating at a speed of 35-50 Hz for 10-15 hours.

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