Schizochytrium fermenting method, fermentation product and application thereof

Abstract

The application provides a schizochytrium fermenting method, which comprises the following steps: fermenting schizochytrium, and adding ammonia water into the fermentation system 20-30 hours before the end of fermentation, so as to maintain the ammonium ion concentration in the fermentation system at 10-20 mmol / L. By adding ammonia water to supplement nitrogen in the later stage of fermentation, the application stimulates the thickening of the cell wall of schizochytrium, so that the schizochytrium cells are not easy to break and the oil is not easy to seep out in the subsequent spray drying process; and the stability of the algal powder product is high.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, and in particular to a fermentation method for Schizochytrium sp., fermentation products, and applications. Background Technology

[0002] Schizochytrium is a marine microalga, also known as schizochytrium. It is characterized by its rapid growth rate, high DHA content, safety, and ease of cultivation, and is widely used in scientific research and commercial production.

[0003] Algal cell walls are primarily composed of polysaccharides and glycoproteins. In industrial algal production, the accumulation of fermentation products often leads to thinner cell walls and larger cell volumes. In subsequent processing, especially in algal cell drying processes such as spray drying, the cells are easily compressed and ruptured, causing oil leakage. This results in algal powder sticking to the cell walls or clogging the outlet during drying, leading to low product yields and significantly hindering the production of algal powder products. Furthermore, the leaked polyunsaturated fatty acids are easily lost at high temperatures, and the resulting algal powder product has a high oil content on its surface, which is detrimental to product stability.

[0004] Existing technologies typically address this problem by adding excipients to dilute the algae powder, such as:

[0005] Patent CN101999522B describes a process where the fermentation broth is first concentrated, then an antioxidant is added, followed by the addition of the broth to an aqueous phase containing starch and emulsifier. The mixture is then spray-dried, and finally the powder is coated with resin to obtain microalgae powder.

[0006] Patent CN114041601A describes a method that uses multiple enzymes to hydrolyze the cell walls of microalgae to obtain an enzymatic hydrolysate, centrifuges it to obtain an emulsifiable oil, and then mixes it with excipients to prepare microcapsule powder.

[0007] There are also methods to change the drying process, such as:

[0008] Patent CN1344514A discloses a processing method for spirulina powder, which uses freeze drying instead of spray drying to prepare spirulina powder, thus avoiding the yield problems caused by spray drying.

[0009] While adding excipients can alleviate the problem of wall adhesion during spray drying to some extent, the addition of emulsifiers, wall materials, and other components increases costs, and cost control is paramount in the animal nutrition sector where algae powder is marketed. Freeze-drying also suffers from high costs, low throughput, and limited industrial application. Therefore, neither of these approaches is suitable for the preparation of products in the animal nutrition field. Summary of the Invention

[0010] To address the problems existing in the prior art, this invention provides a fermentation method for *Schizochytridica*, its fermentation products, and their applications. By controlling the fermentation of *Schizochytridica*, the cell walls of the plant can be effectively thickened, solving the problem of cell wall rupture and oil seepage during spray drying, thereby increasing the yield of algal powder products, reducing surface oil content, and improving the stability of the algal powder products.

[0011] This invention provides a fermentation method for Schizochytrium, comprising the following steps:

[0012] Fermentation culture of *Schizochytrium* was carried out. 20-30 hours before the end of fermentation, ammonia water was added to the fermentation system to maintain the ammonium ion concentration in the fermentation system at 10-20 mmol / L.

[0013] Preferably, the pH of the fermentation system is maintained at ≤8.0 for 20-30 hours before the end of fermentation. As is well known to those skilled in the art, the pH can be maintained by supplementing with conventional pH adjusters such as citric acid and phosphoric acid to stabilize the pH of the system.

[0014] Preferably, the fermentation ends after 110-130 hours of fermentation.

[0015] Preferably, the phosphorus content in the fermentation system is maintained at ≥20 ppm for 20-30 hours before the end of fermentation. Maintaining a phosphorus content of ≥20 ppm aims to further preserve the integrity of the cell morphology. This maintenance is primarily achieved through phosphorus supplementation, preferably including: adjusting the pH using phosphoric acid; or supplementing with phosphates such as dipotassium hydrogen phosphate or potassium dihydrogen phosphate. In a specific embodiment of this invention, the phosphorus content is maintained at 20-50 ppm.

[0016] Preferably, the fermentation medium for *Schizochytrium* comprises carbon sources, nitrogen sources, and inorganic salts known to those skilled in the art. Carbon sources may be selected from glucose, corn syrup, etc.; nitrogen sources may include natural nitrogen sources, inorganic nitrogen sources, or amino acids, such as yeast extract, yeast powder, ammonium sulfate, monosodium glutamate, etc.; inorganic salts include common sodium chloride, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, sodium bicarbonate, sodium sulfate, ammonium sulfate, and potassium chloride, etc. In some specific embodiments, vitamins or other trace metal elements may also be included.

[0017] Preferably, the fermentation culture temperature is 25-33℃, the aeration ratio is 1-2vvm, and the rotation speed is 200-400rpm.

[0018] This invention provides the fermentation product obtained by the aforementioned Schizochytrium fermentation method.

[0019] This invention provides the fermentation method of Schizochytrium or the application of the fermentation product in the preparation of DHA Schizochytrium powder.

[0020] This invention provides a DHA-containing Schizochytrium powder, which is obtained by fermenting Schizochytrium using the aforementioned method and then drying it, or by directly drying the fermentation product.

[0021] Preferably, the drying is spray drying, wherein the inlet air temperature of the spray drying is 180-220℃, the feed rate is 50-100mL / h, and the outlet air temperature is 80-100℃.

[0022] Beneficial effects:

[0023] This invention provides a fermentation method for *Schizochytridica*, in which *Schizochytridica* is fermented and cultured. 20-30 hours before the end of fermentation, ammonia is added to the fermentation system to maintain an ammonium ion concentration of 10-20 mmol / L. This invention addresses the technical problems of low yield and high surface oil content in algal powder during spray drying. By adding ammonia to supplement nitrogen in the later stages of fermentation, the cell walls of *Schizochytridica* are stimulated to thicken. This makes it less likely for the oil from *Schizochytridica* to seep out during spray drying, thus increasing the algal powder yield. The resulting algal powder product has low surface oil content and high stability.

[0024] Because nitrogen supplementation plays a crucial role in the growth of microorganisms, existing technologies often supplement nitrogen with ammonia water during the early growth stages of fermentation bacteria to promote biomass growth. For example, the method provided by patent CN107208119B continuously introduces ammonia water into the culture medium. However, in this patented technology, nitrogen supplementation is mainly carried out during the growth and logarithmic phases, and is discontinued after 50 hours of fermentation. The purpose of this invention, which supplements nitrogen with ammonia water in the later stages of fermentation, is to stimulate cell wall thickening, thereby preventing cell wall rupture and oil exudation, and improving algal powder yield and quality. This invention differs from existing conventional technologies in its technical concept.

[0025] Patent CN101812484B describes a technical solution for using ammonia water to regulate the pH of the fermentation system throughout the entire process. However, the ammonia water used there is only for pH regulation, and its dosage is completely different from the amount of ammonia water used for nitrogen supplementation in this invention. This invention requires the addition of acid while supplementing nitrogen to control the fermentation pH to ≤8.0. Combined with the scheme of Comparative Example 2 of this invention, it can be seen that a small amount of ammonia water supplementation has little effect on cell wall regulation.

[0026] Patent CN101519678B states that additional ammonia can affect lipid productivity, reducing DHA content from 18-22% to 9-12.6%. In this invention, the ammonia is added during the later stages of fermentation, avoiding the peak period of product synthesis; therefore, the DHA content does not show a significant decrease. Attached Figure Description

[0027] Figure 1 This is a microscopic image of *Schizochytridica* after fermentation in Example 1 of the present invention.

[0028] Figure 2 This is a microscopic image of *Schizochytridica* after fermentation in Comparative Example 1 of this invention. Detailed Implementation

[0029] The present invention provides a fermentation method for Schizochytrium, comprising the following steps: fermenting Schizochytrium, and adding ammonia water to the fermentation system 20-30 hours before the end of fermentation to maintain the ammonium ion concentration in the fermentation system at 10-20 mmol / L.

[0030] This invention involves the fermentation and cultivation of *Schizochytridica*. The specific type and source of the *Schizochytridica* are not particularly limited; any commonly used industrial strains in the art are acceptable. In a preferred embodiment of this invention, the *Schizochytridica* preferably includes one or more of the following: *Schizochytridica* CABIO-A-2-II strain (biological preservation number: CCTCC M 2019990), *Schizochytridica* CABIO-A-2-III strain (biological preservation number: CCTCC NO: M 2021564), and *Schizochytridica* CABIO-A-2-IV strain (biological preservation number: CCTCC NO: M 2021565).

[0031] In a preferred embodiment of the present invention, the fissile chytrids is first activated and expanded in culture, and then inoculated into a fermentation medium for fermentation culture.

[0032] This invention does not impose any particular limitation on the formulation of the culture medium for activation and expansion cultures; commercially available culture media commonly used in the art for the activation and expansion cultures of industrial strains are acceptable. In a more preferred embodiment of this invention, the formulation of both the activation and expansion culture media is: 4% glucose, 3% monosodium glutamate, 0.5% yeast extract, 2% sodium chloride, 0.2% potassium dihydrogen phosphate, 0.5% magnesium sulfate, and 0.02% calcium chloride (mass percentage concentration, dispersion system is water). In this invention, the preferred culture temperature for activation and expansion cultures is 25-33℃, more preferably 28℃; the preferred rotation speed for activation and expansion cultures is 200-250 rpm, more preferably 220 rpm; the preferred activation culture time is 35-45 h, more preferably 40 h. The preferred expansion culture time is 25-35 h, more preferably 30 h.

[0033] The present invention does not impose any particular limitation on the access method, and any conventional access method in the art is acceptable.

[0034] In this invention, the fermentation medium preferably comprises an organic carbon source, an organic nitrogen source, and inorganic salts; the organic carbon source preferably comprises glucose; the organic nitrogen source preferably comprises yeast extract and monosodium glutamate; and the inorganic salts preferably comprise sodium chloride, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, sodium bicarbonate, sodium sulfate, ammonium sulfate, and potassium chloride. In a more preferred embodiment of this invention, the fermentation medium is formulated as follows: glucose 40 g / L, yeast extract 4 g / L, monosodium glutamate 30 g / L, sodium chloride 5 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 5 g / L, calcium chloride 0.5 g / L, sodium bicarbonate 0.5 g / L, sodium sulfate 8 g / L, ammonium sulfate 6 g / L, and potassium chloride 0.5 g / L (dispersion system is water), with a natural pH. In this invention, the fermentation temperature is preferably 25-33℃, more preferably 28℃; the aeration ratio is preferably 1-2 vvm, and the rotation speed is preferably 200-400 rpm.

[0035] In this invention, ammonia water is added to the fermentation system 20-30 hours before the end of fermentation to maintain the ammonium ion concentration in the fermentation system at 10-20 mmol / L.

[0036] In this invention, the fermentation process is preferably completed after 110-130 hours of fermentation; more preferably after 120 hours. The addition of ammonia water is performed 20-30 hours before the end of fermentation, preferably 30 hours before the end of fermentation. After the addition of ammonia water, the ammonium ion concentration in the fermentation system is 10-20 mmol / L, preferably 10-15 mmol / L. This invention preferably involves adding acid to the fermentation system to maintain a pH value ≤ 8.0; the timing of acid addition is preferably the same as the timing of ammonia water addition; the acid is preferably citric acid and / or phosphoric acid. Using phosphoric acid can effectively maintain the phosphorus content in the fermentation system.

[0037] In this invention, phosphorus is preferably added to the fermentation system 20-30 hours before the end of fermentation to maintain a phosphorus content of ≥20 ppm. Based on the fermentation experience of the strain used in this application, adjusting the pH with phosphate is sufficient to maintain the phosphorus content above 20 ppm (as phosphorus). Of course, in other optional methods, citric acid can be used to adjust the pH, and then phosphates such as dipotassium hydrogen phosphate or potassium dihydrogen phosphate can be added to adjust the phosphorus content.

[0038] The cell walls of *Schizochytridica* contain a large amount of protein. The large amount of nitrogen from the subsequent ammonia supplementation directly stimulates the synthesis of *Schizochytridica* cell walls, achieving cell wall thickening and solving the problems of cell wall rupture and oil leakage during spray drying. Simultaneously, controlling the timing of ammonia supplementation has minimal impact on DHA accumulation in *Schizochytridica*. The fermentation broth prepared by the method described in this invention, after spray drying, exhibits high direct yield, low surface oil content, and high stability.

[0039] This invention provides a fermentation product obtained by the aforementioned Schizochytridium fermentation method, and the application of the fermentation method and fermentation product in the preparation of DHA-containing Schizochytridium powder. The Schizochytridium in the fermentation product of this invention has a thicker cell wall and can be used to prepare high-DHA-content Schizochytridium powder.

[0040] This invention also provides a DHA-containing Schizochytrium powder, obtained by drying the fermentation product. In this invention, the drying is preferably performed by spray drying; the inlet air temperature of the spray drying is preferably 180-220℃, more preferably 200℃; the feed rate is preferably 50-100mL / h, more preferably 80mL / h; and the outlet air temperature is preferably 80-100℃, more preferably 90℃.

[0041] The DHA-containing Schizochytrium powder provided by this invention is rich in DHA and has a simple preparation method that does not involve the addition of emulsifiers or wall materials, nor does it involve freeze-drying processes, resulting in low product production costs.

[0042] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are all conventional methods; the materials and reagents used are all commercially available.

[0043] Example 1

[0044] (1) The *Cyclotrum schistosum* CABIO-A-2-II was inoculated into the primary activation medium and cultured at 28℃ with a shaking speed of 220 r / min for 40 h. The primary activation solution was then inoculated into the secondary expansion medium at an inoculation rate of 2.5%, and cultured at 28℃ with a shaking speed of 220 r / min for 30 h. The formulations of the primary and secondary culture media (mass percentage concentration, dispersion system: water) were as follows: 4% glucose, 3% monosodium glutamate, 0.5% yeast extract, 2% sodium chloride, 0.2% potassium dihydrogen phosphate, 0.5% magnesium sulfate, and 0.02% calcium chloride.

[0045] (2) The secondary expansion inoculation was carried out onto the fermentation medium at an inoculation rate of 5% and the culture temperature was 28℃. The fermentation medium formula (dispersion system is water) was as follows: glucose 40g / L, yeast extract 4g / L, monosodium glutamate 30g / L, sodium chloride 5g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 5g / L, calcium chloride 0.5g / L, sodium bicarbonate 0.5g / L, sodium sulfate 8g / L, ammonium sulfate 6g / L, potassium chloride 0.5g / L, pH natural.

[0046] (3) Ammonia-controlled fermentation process for *Schizochytrium* with thickened cell walls in a 50L tank: Temperature 28℃, aeration ratio 1-2 vvm, rotation speed 300 rpm. Dissolved oxygen was controlled above 5%, and pH was maintained at 7.8 using 30% citric acid. Sugar was added based on residual sugar; when residual sugar was less than 10 g / L, 5% was added at once. After 90 h of fermentation, ammonia was added continuously to maintain the ammonium ion concentration in the fermentation broth at 12 ± 2 mmol / L. At the same time, phosphoric acid and citric acid were added according to the pH to maintain the pH of the fermentation broth between 6.8 and 7.2 and the phosphorus content in the fermentation broth at 25 ± 5 ppm. Fermentation was completed after 120 h, and the fermentation broth was collected.

[0047] (4) Process for preparing DHA Schizochytrium powder by spray drying of fermentation broth: The mixed product is centrifuged and dehydrated until the solid content accounts for 50% of the total mass, and then spray dried. The inlet air temperature is 200℃, the feed rate is 80mL / h, the outlet air temperature reaches 90℃, the water content of algal oil powder is less than 5%, and the direct yield is 95.5%.

[0048] (5) The DHA content in the algae powder product is 30.1% (based on dry bacterial cells, the same below), the surface oil is 3.5%, and the odor is normal and has no rancid smell after 10 days of accelerated testing at 60℃.

[0049] Microscopic examination of schistosacchari cells sampled from the 120-hour fermentation broth yielded the following results: Figure 1 As shown. Figure 1 The results showed that the cell walls of *Schizochytridica* were intact and relatively thick after being regulated with ammonia.

[0050] Example 2

[0051] (1) *Cyclotrum schistosum* CABIO-A-2-IV was inoculated into primary activation medium and cultured at 28℃ with a shaking speed of 220 r / min for 40 h. The primary activation solution was then inoculated into secondary expansion medium at an inoculation rate of 2.5%, and cultured at 28℃ with a shaking speed of 220 r / min for 30 h. The formulations of the primary and secondary culture media (mass percentage concentration, dispersion system: water) were as follows: 4% glucose, 3% monosodium glutamate, 0.5% yeast extract, 2% sodium chloride, 0.2% potassium dihydrogen phosphate, 0.5% magnesium sulfate, and 0.02% calcium chloride.

[0052] (2) The secondary expansion inoculation was carried out onto the fermentation medium at an inoculation rate of 5% and the culture temperature was 28℃. The fermentation medium formula (dispersion system is water) was as follows: glucose 40g / L, yeast extract 4g / L, monosodium glutamate 30g / L, sodium chloride 5g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 5g / L, calcium chloride 0.5g / L, sodium bicarbonate 0.5g / L, sodium sulfate 8g / L, ammonium sulfate 6g / L, potassium chloride 0.5g / L, pH natural.

[0053] (3) Ammonia-controlled fermentation process for *Schizochytrium* with thickened cell walls in a 50L tank: Temperature 28℃, aeration ratio 1-2 vvm, rotation speed 350 rpm. Dissolved oxygen was controlled above 5%, and pH was maintained at 7.8 using 30% citric acid. Sugar was added based on residual sugar; when residual sugar was less than 10 g / L, 5% was added at once. After 95 h of fermentation, ammonia was added continuously to maintain the ammonium ion concentration in the fermentation broth at 15 ± 2 mmol / L. At the same time, phosphoric acid and citric acid were added according to the pH to maintain the pH of the fermentation broth between 6.8 and 7.2 and the phosphorus content in the fermentation broth at 25 ± 5 ppm. Fermentation was completed after 120 h, and the fermentation broth was collected.

[0054] (4) Process for preparing high DHA-containing Schizochytrium powder by spray drying of fermentation broth: The mixed product is centrifuged and dehydrated until the solids account for 50% of the total mass, and then spray dried. The inlet air temperature is 200℃, the feed rate is 80mL / h, the outlet air temperature reaches 90℃, the water content of algal oil powder is less than 5%, and the direct yield is 96.6%.

[0055] (5) The DHA content in the algae powder product is 25.7% (dry bacterial cells), the surface oil is 2.7%, and the odor is normal and has no rancid smell after 10 days of accelerated testing at 60℃.

[0056] Example 3

[0057] The method was followed as described in Example 1. The only difference from Example 1 was that in step (3), citric acid was added according to the pH to maintain the pH of the fermentation broth between 6.8 and 7.2. The phosphorus content was not adjusted, and the phosphorus content in the fermentation broth was measured to be 5 ppm when the product was placed in the tank. After spray drying, the moisture content of the algal oil powder was less than 5%, and the direct yield was 92.2%. The DHA content in the algal powder product was 25.7% (dry cells), and the surface oil content was 4.9%. After 10 days of accelerated testing at 60°C, the odor was normal and there was no rancid smell.

[0058] Comparative Example 1

[0059] The difference from Example 1 is that no additional ammonia was added for regulation during the later stage of fermentation.

[0060] The direct yield of algal powder was 79.6%, with the remainder collected manually. The algal powder product contained 26.6% DHA (dry cells) and 15.8% surface oil. During a 2-day accelerated drying test at 60℃, the algal powder developed a rancid smell. The Schizochytrium cells, which were not treated with ammonia, were prone to rupture during the drying process.

[0061] Microscopic examination of schistosacchari cells sampled from the 120-hour fermentation broth yielded the following results: Figure 2 As shown, some cell edges are blurred (not due to viewing distance).

[0062] Comparative Example 2

[0063] The difference from Example 1 is that the concentration of ammonium ions regulated by ammonia water is controlled at 1-5 mmol / L.

[0064] The direct yield of algal powder was 85.7%, and its effect on thickening the cell walls of schizophyllum was not obvious. The DHA content in the algal powder product was 27.5% (dry cells), and the surface oil content was 8.7%. After 2 days of accelerated testing at 60℃, the algal powder developed a rancid smell.

[0065] Comparative Example 3

[0066] The difference from Example 1 is that the concentration of ammonium ions regulated by ammonia water is controlled between 25-30 mmol / L.

[0067] The direct yield of algae powder was 90.7%, but the salt concentration in the later stage affected the accumulation of DHA. The DHA content in the algae powder product was 10.3% (dry bacterial cells), and the surface oil was 4.5%. After 10 days of accelerated testing at 60℃, the algae powder had a normal odor.

[0068] Comparative Example 4

[0069] The difference from Example 1 is that the ammonia water control start time is advanced. Ammonia water is added after 64 hours of fermentation to maintain the ammonia water concentration in the fermentation broth at 10-15 mmol / L. At the same time, citric acid is added according to the pH to maintain the pH of the fermentation broth between 6.8 and 7.2. Fermentation ends after 120 hours.

[0070] The direct yield of algae powder was 91.5%, the DHA content in the algae powder product was 9.6% (dry cells), the surface oil content was 4.7%, and the algae powder had a normal odor after 10 days of accelerated testing at 60℃.

[0071] Comparative Example 5

[0072] The difference from Example 1 is that the start time of ammonia water control is delayed. Ammonia water is added after 110 hours of fermentation to maintain the ammonia water concentration in the fermentation broth at 10-15 mmol / L. At the same time, citric acid is added according to the pH to maintain the pH of the fermentation broth between 6.8 and 7.2. Fermentation ends after 120 hours.

[0073] The direct yield of algae powder was 83.7%. The DHA content in the algae powder product was 23.0% (dry bacterial cells), and the surface oil content was 7.4%. After 5 days of accelerated testing at 60℃, the algae powder developed a rancid smell.

[0074] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fermentation method for Schizochytrium, characterized in that, The process includes the following steps: fermenting and culturing *Schizochytrium*, adding ammonia water to the fermentation system 20-30 hours before the end of fermentation to maintain the ammonium ion concentration in the fermentation system at 10-20 mmol / L; For 20-30 hours before the end of the fermentation, maintain the pH value of the fermentation system ≤ 8.0; The fermentation process ends after 110-130 hours of fermentation.

2. The fermentation method for Schizochytrium according to claim 1, characterized in that, For 20-30 hours before the end of the fermentation, maintain the phosphorus content in the fermentation system at ≥20ppm.

3. The fermentation method for Schizochytrium according to claim 1 or 2, characterized in that, The fermentation culture temperature is 25-33℃, the aeration ratio is 1-2vvm, and the rotation speed is 200-400rpm.

4. The fermentation product obtained by the fermentation method of Schizochytrium according to any one of claims 1-3.

5. The fermentation method of Schizochytrium according to any one of claims 1-3 or the fermentation product according to claim 4 in the preparation of DHA Schizochytrium powder.

6. DHA-containing Schizochytrium powder, characterized in that, The DHA-containing Schizochytrium powder is obtained by fermenting Schizochytrium using the method described in any one of claims 1-3 and then drying it, or by drying the fermentation product described in claim 4.

7. The DHA-containing Schizochytrium powder according to claim 6, characterized in that, The drying process is spray drying, with an inlet air temperature of 180-220℃, a feed rate of 50-100mL / h, and an outlet air temperature of 80-100℃.

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