Fermentation method of schizochytrium limacinum with low surface oil and high DHA

Abstract

The invention provides a method for fermenting schizochytrium limacinum with low surface oil and high DHA (docosahexaenoic acid), which comprises the following steps: performing multiplication culture on an activated schizochytrium limacinum strain, performing fermentation culture on an obtained seed solution, and maintaining the pH value of a fermentation system to be 5.0-7.0 by using a first composite buffer system consisting of ammonia water and citric acid in the early stage of fermentation; maintaining the pH value of the fermentation system at 5.0-7.0 by using a second composite buffer system at least composed of calcium carbonate and citric acid in the middle and later periods of fermentation; the residual sugar concentration of the fermentation system is maintained at 10-50 g / L through material supplementation, and finally a fermentation product is obtained. According to the method disclosed by the invention, the surface oil is obviously reduced while the dry cell weight and the DHA yield are improved through multiple regulation and control effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial fermentation, and provides a Schizochytrium sp. fermentation method with low surface oil and high DHA, which involves specific fermentation medium formula and segmented fermentation control strategy to realize high-density culture of Schizochytrium sp., significantly reduce surface oil content and increase DHA yield. BACKGROUND

[0002] Docosahexaenoic acid (DHA), as a long-chain omega-3 polyunsaturated fatty acid with six cis double bonds, plays an important role in physiology and nutritional health. DHA is a key structural lipid that constitutes the phospholipid bilayer of specific biomembranes such as the synaptic membrane of cerebral cortex neurons, the outer segment disc membrane of retinal photoreceptor cells, and the head of sperm. In these highly specialized membrane structures, DHA greatly increases the fluidity and flexibility of the membrane through its long and polyunsaturated carbon chain, which is important for maintaining the normal conformation and function of membrane proteins (such as ion channels and G protein-coupled receptors) and ensuring efficient transmission of neural signals. At the same time, DHA is also an important precursor of bioactive molecules, which can be metabolized to produce lipid mediators such as resolvin and protectin with strong anti-inflammatory and pro-inflammatory resolution effects, and is involved in the regulation of immune response, neuroprotection, and tissue repair processes. Therefore, DHA has extremely wide and in-depth applications in the fields of infant formula, nutritional supplements for pregnant and lactating women, functional health foods, special medical use formula foods, and high-end aquaculture feed.

[0003] The commercial production of DHA mainly comes from traditional fish oil extraction and microbial fermentation. The oil of deep-sea fish (such as tuna and salmon) is the main source of DHA. However, fish oil production of DHA has problems such as periodic fluctuations in fishery resources, risk of heavy metal bioaccumulation due to marine environmental pollution, difficulty in removing fishy smell, and high cost due to complex decolorization and deodorization purification process. Moreover, the DHA content in fish oil is usually between 12% and 30%, and it often coexists with eicosapentaenoic acid (EPA), making it difficult to separate and purify high-purity DHA products. In addition, the ecological sustainability concerns caused by overfishing also prompt the industry to seek more green and controllable alternatives. In contrast, microbial fermentation, especially the marine thraustochytrid microorganisms represented by Schizochytrium sp. and Aurantiochytrium sp., has advantages such as high efficiency and environmental protection. Its fermentation period is short, growth is not limited by climate and region, can utilize various cheap carbon sources (such as glucose, glycerol, and lignocellulose hydrolysate), oil content is extremely high (can account for more than 50% of cell dry weight), and DHA accounts for a high proportion of total fatty acids (can reach 40-60%), making it an ideal "cell factory" for realizing sustainable industrial production of DHA.

[0004] In order to achieve the economic feasibility of the Schizochytrium DHA fermentation, the high cell density fermentation process optimization of Schizochytrium has gradually matured. By optimizing the culture conditions, the biomass of the cell (in terms of dry cell weight) is maximized, so as to obtain the highest total DHA yield (g DHA / L) in the unit fermentation volume. At present, through fed-batch fermentation, optimization of nutrients, precise control of dissolved oxygen and pH and other strategies, the dry cell weight of Schizochytrium can reach 150~200 g / L or even higher in the laboratory and pilot scale, and the DHA yield can reach 40~80 g / L, showing strong industrialization potential.

[0005] However, in the process of high cell density fermentation, a serious technical bottleneck affecting yield, cost and product quality is increasingly prominent - the formation of "surface oil" or "extracellular oil". Surface oil is not the ideal form of the target product triglyceride (TAG), which mainly refers to a large amount of oil released from the cell in the late fermentation due to cell autolysis, loss of membrane integrity or active / passive secretion. These oils float on the surface of the fermentation broth or emulsify in the system, forming a significant oil layer. In the traditional or unoptimized high-density fermentation process, the proportion of surface oil can surprisingly account for 30~60% of the total lipid yield. The surface oil is free in the extracellular fermentation broth, and the method of collecting the cell by centrifugation will lose the oil of the surface oil, resulting in a significant decrease in the total oil recovery rate. And if the emulsified or dissolved oil is recovered from the fermentation supernatant, additional demulsification and extraction steps are required, which greatly increases the production cost. And the presence of surface oil increases the viscosity and complexity of the fermentation broth, interfering with the coagulation and sedimentation of the cell, reducing the efficiency of solid-liquid separation. At the same time, in the process of high cell density fermentation, a large amount of surface oil will wrap the cell, affecting the mass transfer efficiency of oxygen and nutrients, and may exacerbate the anoxic stress and death of the cell, forming a vicious cycle. The oxidation products of oil can also have an inhibitory effect on the activity of the cell. The long-term presence of free oil in the fermentation broth is more prone to oxidation and spoilage, producing undesirable flavor substances, affecting the color, odor and oxidation stability of Schizochytrium powder or algal oil, and reducing its commercial value.

[0006] Therefore, developing a low-surface-oil, high-DHA Schizochytrium fermentation method has become a key technical problem to be solved in the field. SUMMARY

[0007] In view of the problems in the above background art, the present application provides a low-surface-oil, high-DHA Schizochytrium fermentation method. The present application can realize low-surface-oil and high-DHA Schizochytrium fermentation process under high-density fermentation conditions, and promote the competitiveness of microalgae DHA products in high-end food, medicine and other fields.

[0008] In a first aspect, a fermentation method of low surface oil and high DHA Schizochytrium sp. is provided, comprising After the activated Schizochytrium sp. is expanded and cultured, the obtained seed liquid is subjected to fermentation culture, and within 0-20 h of fermentation, a first composite buffer system composed of ammonia and citric acid is used to adjust the pH value of the fermentation system to 5.0-7.0; within 65-75 h of fermentation, a second composite buffer system composed of at least calcium carbonate and citric acid is used to adjust the pH value of the fermentation system to 5.0-7.0; and the residual sugar concentration of the fermentation system is maintained at 10-50 g / L by feeding.

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

[0010] In some embodiments, the culture medium for the fermentation culture of the seed liquid is 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.

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

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

[0013] In some embodiments, the mass concentration of ammonia in the first composite buffer system is in the range of 5%-25%.

[0014] In some embodiments, the concentration of calcium carbonate in the second composite buffer system is in the range of 5-25 g / L.

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

[0016] In some embodiments, malic acid is also added to the second complex buffer system, and the concentration of the malic acid ranges from 1 to 3 g / L.

[0017] In some embodiments, the process of the amplification culture is as follows: S1, the activated Schizochytrium sp. is inoculated into a seed culture, and the seed culture is cultured at 20-30°C and 180-220 r / min for 24 h, and the pH is 5.0-7.0. When the residual sugar concentration of the fermentation system is less than 20 g / L, the second seed tank is inoculated for expansion culture; S2, the culture solution of S1 is inoculated into the expansion culture medium at an inoculation amount of 10-20% for culture, the culture temperature is 20-30°C, the shaking speed is 180-220 r / min, the culture time is 24 h, the pH is 5.0-7.0, and when the residual sugar concentration of the fermentation system is less than 20 g / L, a one-ton fermentation tank is inoculated; S3, the expansion seed culture solution of S2 is inoculated into a one-ton tank of the one-ton fermentation medium at an inoculation amount of 5-15% for fermentation culture, the culture temperature is 20-30°C, the pH is maintained at pH 5.0-7.0, the aeration amount is 10-30 m 3 / h, the speed is 35-50 Hz, and when the residual sugar concentration of the fermentation system is less than 20 g / L, an eight-ton fermentation tank is inoculated; S4, the expansion seed culture solution of S3 is inoculated into an eight-ton tank of the eight-ton fermentation medium at an inoculation amount of 10-25% for fermentation culture, the culture temperature is 20-30°C, the pH is maintained at pH 5.0-7.0, the aeration amount is 50-80 m 3 / h, the speed is 35-50 Hz, and the culture is performed for 10-15 h to obtain the seed solution.

[0018] In some embodiments, in the process of the expansion culture, the pH of the fermentation system is regulated by adding a pH regulator, and the pH regulator is at least one selected from the group consisting 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 regulated by ammonia / sodium acetate / calcium carbonate and malic acid / citric acid. In some examples, the pH is regulated by ammonia / calcium carbonate and malic acid / citric acid. In some examples, the pH is regulated by ammonia / calcium carbonate and citric acid. In some examples, the pH is regulated by ammonia / calcium carbonate and phosphoric acid. In some examples, the pH is regulated by ammonia and malic acid / citric acid.

[0019] In some embodiments, the seed medium, the expansion medium, the one-ton fermentation medium, and the eight-ton fermentation medium comprise at least one of a carbon source, a nitrogen source, inorganic salts, and vitamins. In some examples, the carbon source comprises at least one of glucose, corn steep powder, glycerol, sucrose, lactose, maltose. In some examples, the carbon source concentration is 40-80 g / L. In some examples, the medium nitrogen source is selected from at least one of yeast powder, corn steep powder, soybean meal, ammonium sulfate, soybean protein powder. In some examples, the medium nitrogen source concentration is 1-30 g / L. In some examples, the inorganic salts are selected from at least one of calcium chloride, potassium chloride, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate, sodium sulfate, potassium sulfate. In some examples, the inorganic salts are selected from at least one of 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.

[0020] In some embodiments, the seed medium is glucose 40-70 g / L, yeast powder 5-15 g / L, sodium sulfate 10-25 g / L, magnesium sulfate 3-6 g / L, potassium sulfate 0.5-1.5 g / L, potassium dihydrogen phosphate 0.5-1.5 g / L, ammonium sulfate 0.5-1.5 g / L, calcium chloride 0.1-0.4 g / L, calcium carbonate 1-3 g / L.

[0021] In some embodiments, the expansion medium is glucose 40-70 g / L, yeast powder 5-15 g / L, sodium sulfate 10-25 g / L, magnesium sulfate 3-6 g / L, potassium sulfate 0.5-1.5 g / L, potassium dihydrogen phosphate 0.5-1.5 g / L, ammonium sulfate 0.5-1.5 g / L, calcium chloride 0.1-0.4 g / L, calcium carbonate 1-3 g / L, vitamin B6 0.1-1.0 mg / L.

[0022] 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, yeast powder 2-5 g / L.

[0023] In some embodiments, the eight tons of fermentation 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, vitamin B12 0.1-1.0 mg / L.

[0024] In some embodiments, during the fermentation culture process, a defoaming agent can be appropriately added to eliminate foam, and a conventional defoaming agent in the art can be used.

[0025] Compared with the prior art, one of the above technical solutions has the following advantages or beneficial effects: The application provides a low-surface-oil and high-DHA Schizochytrium fermenting method, which can significantly reduce the surface oil ratio while improving the cell dry weight and DHA yield. 12 , provides the cofactor of methionine synthetase and methylmalonyl-CoA mutase, ensures the smoothness of one-carbon metabolism and propionic acid auxiliary oxidation pathway, maintains the cell methylation reaction (promotes membrane phospholipid synthesis and repair) and reduces propionic acid accumulation (avoids toxicity and metabolic disorder), thereby delaying the autolysis phenomenon of cells in the late fermentation period. Ammonia water / ammonia water is supplemented in the early fermentation period, ammonia water provides nitrogen source and avoids pH sudden drop, and citric acid supplements intermediates in the tricarboxylic acid cycle, which together promotes rapid growth of the bacteria; calcium carbonate / citric acid is supplemented in the middle and late fermentation period, calcium carbonate is used as a solid buffer to continuously stabilize pH, and the released Ca 2+ forms an ionic bridge with the cell membrane phospholipid, enhances the mechanical strength of the cell membrane, and Ca 2+ binds with the leaked free fatty acid to form calcium soap, reduces the emulsification damage.

[0026] The application obtains a culture by high-cell-density fermentation of Schizochytrium, and further extracts Schizochytrium oil, the DHA content of the extracted Schizochytrium powder is improved, and the surface oil content is extremely low. The application improves the DHA content under the condition of ensuring the same raw material, reduces the DHA loss caused by high surface oil in the process of extracting algal powder, reduces the production cost, and improves the production efficiency.

[0027] Term explanation 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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

[0032] Figure 1Microscopic image of cells fermented for 120h under the conditions of Example 6-0.

[0033] Figure 2 Microscopic image of cells fermented for 120h under the conditions of Example 6-9.

[0034] Figure 3 Microscopic image of cells fermented for 120h under the conditions of Example 6-10. DETAILED DESCRIPTION

[0035] In order to make the objectives, technical solutions and advantages of the present application clearer, the following further describes the present application in combination with examples. The specific examples described herein are only used to explain the present application and do not constitute any limitation on the present application. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present disclosure. Such structures and technologies are also described in many publications.

[0036] The reagents used in the present application can be purchased from the market or can be prepared by the methods described in the present application.

[0037] In the present application, the DHA content is detected by the internal standard method in GB 5009.168-2016.

[0038] The Schizochytrium used in the present application is deposited in the China General Microbiological Culture Collection Center (CGMCC) with the accession number CGMCC No.40902. It has been disclosed in the following patent. The specific patent application number is CN202311728644.8, the patent name is: Schizochytrium strain rich in n-3 fatty acids and its screening and culture, the publication number is CN117701403A, and the publication date is March 15, 2024.

[0039] The surface oil detection method of the Schizochytrium fermentation broth in the present application includes: accurately weighing 1-2g of the sample, and sequentially extracting with 10ml of ethanol, 20ml of diethyl ether and 20ml of petroleum ether, and oscillating mixing each solvent. After the extraction liquid is stratified for 30-60min, 20ml of supernatant is accurately taken, dried in a water bath below 60℃, and then placed in a 103±2℃ oven for drying to constant weight. The surface oil concentration is calculated according to the formula. The specific formula is as follows: X=(m1-m0) ×(v1-v2) / m×20×(1-a) ×100. In the formula: X: the content of fat in the sample (calculated on a dry basis), wt%, retained to one decimal place; a: the content of moisture 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 in the glass dish after drying to constant weight; v1 is the total volume after the extraction liquid is stratified; v2 is the lower layer volume after the extraction liquid is stratified.

[0040] Total Example 1 Seed activation culture: The laboratory preserved Schizochytrium sp. seed was inoculated into seed culture medium and cultured at 20-30°C, 180-220 r / min for 24 h, pH 5.0-7.0. The seed culture medium was glucose 40-70 g / L, yeast powder 5-15 g / L, sodium sulfate 10-25 g / L, magnesium sulfate 3-6 g / L, potassium sulfate 0.5-1.5 g / L, potassium dihydrogen phosphate 0.5-1.5 g / L, ammonium sulfate 0.5-1.5 g / L, calcium chloride 0.1-0.4 g / L, calcium carbonate 1-3 g / L. When the residual sugar concentration of the fermentation system was less than 20 g / L, the second seed tank was inoculated for expansion culture. The specific seed culture medium is shown in Table 1 below: Table 1 Seed culture medium

[0041] Total Example 2 Seed expansion culture: The culture solution of Example 1-1 was inoculated into the expansion culture medium at an inoculation amount of 10-20% for culture, the culture temperature was 20-30°C, the shaking speed was 180-220 r / min, the culture time was 24 h, and the pH was 5.0-7.0. The expansion culture medium was glucose 40-70 g / L, yeast powder 5-15 g / L, sodium sulfate 10-25 g / L, magnesium sulfate 3-6 g / L, potassium sulfate 0.5-1.5 g / L, potassium dihydrogen phosphate 0.5-1.5 g / L, ammonium sulfate 0.5-1.5 g / L, calcium chloride 0.1-0.4 g / L, calcium carbonate 1-3 g / L, vitamin B6 0.1-1.0 mg / L. When the residual sugar concentration of the fermentation system was less than 20 g / L, a ton of fermentation tank was inoculated. The specific culture conditions are shown in Table 2 below: Table 2 Culture conditions

[0042] Total Example 3 Ton tank culture: The expanded seed culture solution of Total Example 2 was inoculated into a ton tank for fermentation culture at an inoculation amount of 5-15%, the culture temperature was 20-30°C, the pH was maintained at 5.0-7.0, the aeration amount was 10-30 m 3 / h, and the rotation speed was 35-50 Hz. The ton fermentation medium included: 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. When the residual sugar concentration of the fermentation system was less than 20 g / L, an eight-ton fermentation tank was inoculated. The specific culture conditions are shown in Table 3 below: Table 3 Cultivation conditions

[0043] Total Example 4 Eight-ton tank cultivation: the eight-ton seed culture solution cultivated in total example 3 was inoculated into an eight-ton tank at an inoculation amount of 10-25% for fermentation cultivation, the cultivation temperature was 20-30°C, the pH was maintained at pH 5.0-7.0, the aeration amount was 50-80 m 3 / h, and the rotation speed was 35-50 Hz. The eight-ton fermentation medium included: 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 cultivation for 10-15 h, the eight-ton seed culture solution was inoculated into an eighty-ton fermentation tank. The specific cultivation conditions are shown in Table 4 below: Table 4 Cultivation conditions

[0044] Total Example 5 Eighty-ton tank cultivation: the eighty-ton seed culture solution cultivated in total example 4 was inoculated into an eighty-ton tank at an inoculation amount of 10-25% for cultivation, the cultivation temperature was 20-30°C, the pH was maintained at pH 5.0-7.0, the pH was adjusted with sodium acetate and malic acid throughout the whole process, the addition amount was malic acid 3 g / L and sodium acetate 12 g / L, the aeration amount was 500-1000 m 3 / h, and the rotation speed was 40-50 Hz. During the fermentation process, glucose with a mass concentration of 60% was supplemented, and the sugar content in the fermentation solution was controlled at 15-45 g / L at 65-75 h. The eighty-ton fermentation medium included: 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. The specific cultivation conditions are shown in Table 5 below: Table 5 Cultivation conditions

[0045] The cultivation time was 120 h, and the results of the product are shown in Table 6 below.

[0046] Table 6 Results of the product

[0047] From the total example 5, it can be seen that the change of fermentation conditions (example 5-1, 5-2, 5-3, 5-6 and 5-7) has little effect on the product during the fermentation culture process of the 80-ton tank; the change of the components in the fermentation medium, especially the change of the type of vitamins, has a greater effect on the surface oil. From the change of the surface oil in example 5-1, 5-4 and 5-5, it can be seen that the introduction of vitamin B12 can effectively control the key metabolic pathway and has a certain effect on cell autolysis.

[0048] Total example 6 The difference from example 5-5 is that the process of using pH adjuster during the 80-ton tank culture process is different, the dissolved oxygen is maintained at 20-40%, and the mass concentration of glucose is supplemented during the fermentation process to maintain the residual sugar concentration in the fermentation broth at 10-50 g / L, as shown in the following table 7: Table 7 Change of related parameters

[0049] The results of the obtained product are shown in the following table 8.

[0050] Table 8 Results of the product

[0051] From the total example 6, it can be seen that during the fermentation culture process of the 80-ton tank: Comparing example 6-2 and example 6-3, the introduction of vitamin B12 in the culture medium cooperates with the pH regulation during the culture process, which not only effectively ensures the healthy growth of the Schizochytrium sp. cell, but also effectively controls the key metabolic pathway and has a certain effect on cell autolysis.

[0052] Comparing example 6-1 and example 6-3, only the pH regulation in the early culture period cannot effectively control the surface oil production.

[0053] Comparing example 6-4 to example 6-8, different pH reagents are used to regulate the pH of the culture system during the culture process, and the introduction of vitamin B12 in the culture medium can effectively ensure the healthy production of cells and maintain the integrity of the cell membrane, so that the surface oil of the obtained product can be effectively controlled. Especially in example 6-7 and example 6-8, the surface oil content of the obtained product is reduced to less than 5%.

[0054] Comparing example 6-1 to example 6-8, example 6-9 and example 6-10 use step-by-step control of the pH of the fermentation environment. In the early stage (0-20h), ammonia / citric acid is supplemented, and in the later stage (65-75h), calcium carbonate / citric acid is supplemented to maintain the healthy growth of cells, thereby inhibiting the generation of surface oil from the source.

[0055] As 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.

[0056] 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.

[0057] General Example 7 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.

[0058] 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.

[0059] 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 method for fermenting low surface oil, high DHA Schizochytrium sp., characterized in that, The method comprises the following steps: The activated Schizochytrium sp. is expanded and cultured, and then the obtained seed liquid is fermented, and during 0-20 hours of the fermentation, 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; during 65-75 hours of the fermentation, 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; and the residual sugar concentration of the fermentation system is maintained at 10-50 g / L through feeding.

2. The fermentation process of claim 1, wherein, The seed liquid is fermented under the conditions of 20-30 ℃ and a dissolved oxygen level of 20%-70%, and the total fermentation time is 110-120 hours.

3. The fermentation process of claim 1, wherein, During the fermentation of the seed liquid, glucose with a mass concentration of 50%-70% is fed, and the sugar content of the fermentation liquid is controlled at 15-45 g / L at 65-75 h.

4. The fermentation process of claim 1, wherein, The fermentation medium for the seed liquid is 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.

5. The fermentation process of claim 4, wherein, The content of vitamin B12 in the medium is 0.1-1.0 mg / L.

6. The fermentation process of claim 1, wherein, The fermentation medium for the seed liquid is yeast extract 40 g / L, calcium chloride 0.2 g / L, potassium chloride 1.5 g / L, ammonium sulfate 1 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate 6 g / L, sodium sulfate 45 g / L, potassium sulfate 1 g / L, monosodium glutamate 15 g / L, yeast powder 2 g / L, and vitamin B12 0.5 mg / L.

7. The fermentation process of claim 1, wherein, In the first composite buffer system, the mass concentration of the ammonia is 5%-25%; and in the second composite buffer system, the concentration of the calcium carbonate is 5-25 g / L. In the first composite buffer system and the second composite buffer system, the concentration of the citric acid is 1-3 g / L.

8. The fermentation method according to claim 1, characterized in that, In the second composite buffer system, malic acid can also be added, and the concentration of the malic acid is 1-3 g / L.

9. The fermentation process of claim 1, wherein, The expansion and culture process is as follows: S1, the activated Schizochytrium sp. is inoculated into a seed culture medium and cultured at 20-30 ℃ and 180-220 r / min for 24 h, and when the residual sugar concentration of the fermentation system is lower than 20 g / L, the culture liquid is inoculated into a secondary seed tank for expansion and culture; S2, the culture liquid of S1 is inoculated into an expansion medium at an inoculation amount of 10-20% for culture, the culture temperature is 20-30 ℃, the shaking speed is 180-220 r / min, the culture time is 24 h, the pH value is 5.0-7.0, and when the residual sugar concentration of the fermentation system is lower than 20 g / L, the culture liquid is inoculated into a one-ton fermentation tank. S3, inoculate the expanded seed culture liquid of S2 into a one-ton fermenter according to a 5-15% inoculation amount, and carry out fermentation culture for 24 hours, the culture temperature is 20-30°C, the pH is kept at pH 5.0-7.0, the aeration amount is 10-30 m3 / h, the rotating speed is 35-50 Hz, and when the residual sugar concentration of the fermentation system is lower than 20 g / L, inoculate into an eight-ton fermenter; 3 ; S4, inoculate the expanded seed culture liquid of S3 into eight-ton fermentor according to 10~25% inoculation amount, and carry out fermentation culture, the culture temperature is 20~30℃, the pH is kept at pH 5.0~7.0, the aeration amount is 50~80m / h, the rotating speed is 35~50Hz, and the culture is carried out for 10~15h to obtain the seed liquid. 3 / h, the rotating speed is 35~50Hz, and the culture is carried out for 10~15h to obtain the seed liquid.

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