A method for increasing the content of DHA and the proportion of sn-2 DHA in schizochytrium oil fermentation liquid, schizochytrium algae powder and application thereof

Abstract

The present invention belongs to the fields of bioengineering technology and food engineering technology, and discloses a method for regulating and controlling the content of DHA and the proportion of sn-2 DHA in the fermentation liquid of Schizochytrium oil, Schizochytrium algae powder and application, wherein Schizochytrium is subjected to batch feeding fermentation culture, and the pH of the fermentation liquid in the fermentation process is controlled by supplementing acid or alkali in stages during the fermentation culture process. The method of the present invention regulates the pH of the fermentation liquid in stages during the fermentation culture of Schizochytrium, and by regulating the pH of the fermentation process, Schizochytrium obtains a faster division and reproduction speed, a longer oil accumulation period, and a shorter oil consumption terminal period. The obtained Schizochytrium oil not only has a significantly higher oil yield than the traditional fermentation strategy, but also has a relatively significant increase in the content of DHA and the proportion of sn-2 DHA in the oil.

Description

Technical Field

[0001] The present invention belongs to the fields of bioengineering technology and food engineering technology, and in particular to a method for regulating and controlling the content of DHA and the proportion of sn-2 DHA in a schizochytrium oil fermentation liquid, schizochytrium algae powder and application thereof. Background Art

[0002] Docosahexaenoic acid (DHA, 22:6, n-3) is an important polyunsaturated fatty acid that has the functions of promoting the brain and nerve development of infants and young children, improving lipid metabolism disorders, and enhancing immunity. It is known as the "brain gold". In the past, most people obtained DHA mainly by consuming foods rich in α-linolenic acid (ALA, 18:3, n-3), such as nuts, meat, egg products, etc., and then using the body's elongation / desaturation enzyme pathway to convert ALA into DHA for use. However, the DHA obtained in this way is very limited. In recent years, deep-sea fish have become the main source of DHA for humans.

[0003] Moreover, the effective absorption rate of DHA by humans is closely related to the storage form of DHA. The high-purity DHA commonly sold on the market is mainly ethyl ester (EE) DHA obtained by processing fish oil, and the effective absorption rate of EE-DHA by the human body is only 21%. In addition, EE-DHA may cause adverse reactions in children and some people with physical diseases because it decomposes to form ethanol during the utilization process. Natural algae oil DHA mainly exists in the form of triglycerides (TAG). The comprehensive effective absorption rate of TAG-DHA by the human body is 57%, while the effective absorption rate of TAG sn-2 DHA is close to 100%. Schizochytrium is a heterotrophic and oil-rich marine organism with huge potential for the production of oil compounds. Schizochytrium can accumulate oil that accounts for nearly 70% of its own biomass and is considered to be the most suitable microorganism for industrial mass production of DHA oil. However, according to previous studies, fatty acids in Schizochytrium are mainly stored in the form of triglycerides. Although DHA accounts for nearly 50% of the total fatty acids, the proportion of sn-2 DHA is generally lower than 50%, and the quality of DHA oil needs to be further improved.

[0004] At present, there are many reports on the production of DHA oil by Schizochytrium, but the production of DHA oil by Schizochytrium has problems such as low DHA ratio, low sn-2 DHA ratio, lack of application, etc. For example, Chinese patent CN101979623A can promote the synthesis of DHA by Schizochytrium by adding one or more combinations of exogenous regulatory factors such as acetic acid, citric acid and simvastatin, so that the final DHA yield in Schizochytrium is increased from the initial 35.51% to 45%. Chinese patent CN117946875A obtains Schizochytrium TKD-2212 strains by screening, mutagenesis and breeding from the natural environment. The DHA content of algae oil produced by batch feeding fermentation is 40-65%, of which the proportion of DHA at the sn-2 position is only 35-50%. Chinese patent CN114703238A solves the problem of low production efficiency of glucose and glycerol co-utilization by Schizochytrium by controlling the timing of carbon source glycerol addition, and the DHA content in oil reaches 46.28%. Chinese patent CN118185850A determined the optimal addition amount of clove extract, resveratrol, and epigallocatechin gallate (EGCG) through single factor experiments, and determined the optimal ratio of composite FAS inhibitors through orthogonal experiments, and the final DHA ratio at the sn-2 position was close to 60%. Chinese patent CN117844646A provides a high-DHA-producing Schizochytrium HSc-01, which was fermented in a fermenter and the DHA ratio in the fermentation broth was as high as 60.2%, but the oil yield was only 42g / L, and it did not deeply test the edible properties of Schizochytrium algae powder.

[0005] Therefore, it is urgent to obtain a fermentation method that is more efficient in producing high-quality DHA algae oil (high DHA ratio and high sn-2 DHA ratio) and Schizochytrium algae powder with excellent edible properties. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for increasing the DHA content and the proportion of sn-2 DHA in Schizochytrium oil fermentation broth, Schizochytrium algae powder and application.

[0007] The technical solution adopted by the present invention to solve its technical problem is:

[0008] A method for increasing the DHA content and sn-2 DHA ratio in a Schizochytrium oil fermentation broth, the method comprising: subjecting the Schizochytrium to fed-batch fermentation culture, and controlling the pH of the fermentation broth in the fermentation process by supplementing acid or alkali in stages during the fermentation culture, wherein the acid is at least one of lactic acid, citric acid, malic acid, and fumaric acid, and the alkali is at least one of sodium hydroxide, potassium hydroxide, and sodium carbonate;

[0009] The method of controlling the pH of the fermentation liquid during the fermentation process by supplementing acid or alkali comprises the following steps:

[0010] 1) At 0h of fermentation, the fermentation liquid was made weakly alkaline by adding alkali, with a pH of 7.2 ± 0.1, and maintained until the nitrogen source was exhausted to accelerate the fission and reproduction period of Schizochytrium;

[0011] 2) During the oil accumulation period, i.e., 15h, 18h or 21h of fermentation, acid was added to make the fermentation liquid weakly acidic, with a pH of 6.8±0.1, and the pH was maintained until the growth of Schizochytrium slowed down, thereby extending the oil accumulation period of Schizochytrium and enhancing the accumulation of oil and DHA;

[0012] 3) In the late fermentation stage, i.e. 95h, 100h or 105h of fermentation, the pH of the fermentation liquid is adjusted to neutral, pH 7.0 ± 0.1, by adding alkali and acid, and maintained until the end of fermentation, i.e. 120h of fermentation, to reduce the consumption of oil in the late fermentation stage.

[0013] Furthermore, the concentration of the acid for adjusting the pH of the fermentation liquid is between 20 g / L and 250 g / L, and the concentration of the alkali for adjusting the pH of the fermentation liquid is between 40 g / L and 400 g / L. The concentration refers to the final concentration of the acid and alkali regulating liquid during the fermentation process.

[0014] Furthermore, the method specifically includes the following steps:

[0015] (1) Streak the Schizochytrium plate from the cryotube, incubate at 28°C for 72 hours, and pick a single colony;

[0016] (2) A single colony was inoculated into the seed medium of the seed solution, and the pH was not adjusted at 28°C and 230 rpm for 48 h;

[0017] (3) Inoculate the seed medium with 1% inoculum in the seed solution, without adjusting the pH, and culture at 28°C and 170 rpm for 24 h. If OD600>3 at 24 h and no bacteria are found under microscopic examination, the next generation can be infected.

[0018] (4) Inoculate the seed medium with 1% inoculum into the seed solution, without adjusting the pH, and culture at 28°C and 170 rpm for 24 h. If OD600>5 at 24 h and no bacteria are found under microscopic examination, the next generation will be infected.

[0019] (5) Inoculate the seed tank containing seed medium with seed solution at a 2% inoculum, culture at 28°C, 150 rpm, 20 L / min for 24 h. After 20 h, OD600>8 and no bacteria are found under microscopic examination, and the next generation is infected.

[0020] (6) Inoculate the fermenter containing fermentation medium at a rate of 2% at 28°C, 100 rpm, 15 m3 / h to start fermentation and adjust pH during fermentation;

[0021] Wherein, the method for controlling pH by fermentation comprises the following steps:

[0022] 1) At 0h of fermentation, the fermentation liquid was made weakly alkaline by adding alkali, with a pH of 7.2 ± 0.1, and maintained until the nitrogen source was exhausted to accelerate the fission and reproduction period of Schizochytrium;

[0023] 2) During the oil accumulation period, i.e., 15h, 18h or 21h of fermentation, acid was added to make the fermentation liquid weakly acidic, with a pH of 6.8±0.1, and the pH was maintained until the growth of Schizochytrium slowed down, thereby extending the oil accumulation period of Schizochytrium and enhancing the accumulation of oil and DHA;

[0024] 3) In the late fermentation stage, i.e. 95h, 100h or 105h of fermentation, the pH of the fermentation liquid is adjusted to neutral, pH 7.0 ± 0.1, by adding alkali and acid, and maintained until the end of fermentation, i.e. 120h of fermentation, to reduce the consumption of oil in the late fermentation stage.

[0025] Further, the pH value of the Schizochytrium plate culture medium is 6.0-6.5, and comprises: 40-60 g / L of glucose, 4-6 g / L of yeast extract, 5-8 g / L of sodium sulfate, 2-4 g / L of magnesium sulfate, 4-8 g / L of ammonium sulfate, 1-2 g / L of potassium chloride, 0.5-1 g / L of potassium sulfate, 0.5-2 g / L of potassium dihydrogen phosphate, 8-12 g / L of sodium glutamate, 1-5 mg / L of zinc sulfate heptahydrate, 0.01-0.1 mg / L of cobalt chloride hexahydrate, 2-6 mg / L of copper sulfate pentahydrate, 1-2 mg / L of nickel sulfate hexahydrate, 8-15 mg / L of iron sulfate heptahydrate, 3-5 mg / L of manganese chloride tetrahydrate, 0.04 mg / L of sodium molybdate dihydrate, 10-30 g / L of agar powder, and the solvent is water;

[0026] The seed culture medium of the seed solution has a pH value of 6.0-6.5 and comprises: 40-60 g / L of glucose, 4-6 g / L of yeast extract, 5-8 g / L of sodium sulfate, 2-4 g / L of magnesium sulfate, 4-8 g / L of ammonium sulfate, 1-2 g / L of potassium chloride, 0.5-1 g / L of potassium sulfate, 0.5-2 g / L of potassium dihydrogen phosphate, 8-12 g / L of sodium glutamate, 1-5 mg / L of zinc sulfate heptahydrate, 0.01-0.1 mg / L of cobalt chloride hexahydrate, 2-6 mg / L of copper sulfate pentahydrate, 1-2 mg / L of nickel sulfate hexahydrate, 8-15 mg / L of iron sulfate heptahydrate, 3-5 mg / L of manganese chloride tetrahydrate, 0.04 mg / L of sodium molybdate dihydrate, and the solvent is water;

[0027] The pH value of the fermentation medium is 6.0-6.5, and the fermentation medium comprises: 60-100 g / L of glucose, 5-15 g / L of yeast extract, 5-12 g / L of sodium sulfate, 2-4 g / L of magnesium sulfate, 4-8 g / L of ammonium sulfate, 1-2 g / L of potassium chloride, 0.5-1 g / L of potassium sulfate, 0.5-2 g / L of potassium dihydrogen phosphate, 15-20 g / L of sodium glutamate, 1-5 mg / L of zinc sulfate heptahydrate, 0.01-0.1 mg / L of cobalt chloride hexahydrate, 2-6 mg / L of copper sulfate pentahydrate, 1-2 mg / L of nickel sulfate hexahydrate, 8-15 mg / L of iron sulfate heptahydrate, 3-5 mg / L of manganese chloride tetrahydrate, 0.04 mg / L of sodium molybdate dihydrate, and the solvent is water;

[0028] Alternatively, the regulator acid is citric acid, and the final concentration of the citric acid is 150 g / L; the regulator base is sodium hydroxide, and the final concentration of the sodium hydroxide is 250 g / L;

[0029] Alternatively, when the pH is adjusted during fermentation, the pH is adjusted to 7.2 at 0 h of fermentation, the pH is adjusted to 6.8 at 18 h of fermentation during the oil accumulation period, and the pH is adjusted to 7.0 at 100 h of fermentation in the late fermentation period.

[0030] Further, the pH of the Schizochytrium plate culture medium is 6.1, and includes: 50 g / L glucose, 5 g / L yeast extract, 8 g / L sodium sulfate, 4 g / L magnesium sulfate, 8 g / L ammonium sulfate, 2 g / L potassium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 10 g / L sodium glutamate, 2 mg / L zinc sulfate heptahydrate, 0.04 mg / L cobalt chloride hexahydrate, 4 mg / L copper sulfate pentahydrate, 2 mg / L nickel sulfate hexahydrate, 10 mg / L iron sulfate heptahydrate, 4 mg / L manganese chloride tetrahydrate, 0.04 mg / L sodium molybdate dihydrate, 20 g / L agar powder, and the solvent is water.

[0031] The seed culture medium of the seed solution has a pH of 6.1 and comprises: 50 g / L glucose, 5 g / L yeast extract, 8 g / L sodium sulfate, 4 g / L magnesium sulfate, 8 g / L ammonium sulfate, 2 g / L potassium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 10 g / L sodium glutamate, 2 mg / L zinc sulfate heptahydrate, 0.04 mg / L cobalt chloride hexahydrate, 4 mg / L copper sulfate pentahydrate, 2 mg / L nickel sulfate hexahydrate, 10 mg / L iron sulfate heptahydrate, 4 mg / L manganese chloride tetrahydrate, 0.04 mg / L sodium molybdate dihydrate, and the solvent is water;

[0032] The pH value of the fermentation medium is 6.1, and the fermentation medium comprises: 80 g / L glucose, 5 g / L yeast extract, 8 g / L sodium sulfate, 4 g / L magnesium sulfate, 8 g / L ammonium sulfate, 2 g / L potassium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 10 g / L sodium glutamate, 2 mg / L zinc sulfate heptahydrate, 0.04 mg / L cobalt chloride hexahydrate, 4 mg / L copper sulfate pentahydrate, 2 mg / L nickel sulfate hexahydrate, 10 mg / L iron sulfate heptahydrate, 4 mg / L manganese chloride tetrahydrate, 0.04 mg / L sodium molybdate dihydrate, and the solvent is water.

[0033] Furthermore, the Schizochytrium is Schizochytrium sp. HX-308 or Schizochytrium sp. ATCC 20888.

[0034] A Schizochytrium algae powder comprising the Schizochytrium oil fermentation liquid obtained by the regulation method as described above.

[0035] The method for preparing the Schizochytrium algae powder as described above comprises the following steps:

[0036] 100 mg / L of tea polyphenol extract and 20 mg / L of sesamol were added to the Schizochytrium oil fermentation liquid, and then spray-dried to make powder. During drying, the inlet temperature was 180° C. and the outlet temperature was 80° C., so as to obtain Schizochytrium algae powder.

[0037] Use of the Schizochytrium algae powder as described above in native milk and / or aquatic feed.

[0038] The use of Schizochytrium algae powder as described above in feed.

[0039] The advantages and positive effects achieved by the present invention are:

[0040] 1. The method of the present invention regulates the pH of the fermentation liquid in stages during the fermentation and cultivation of Schizochytrium. By regulating the pH of the fermentation process, Schizochytrium can obtain a faster division and reproduction speed, a longer oil accumulation period, and a shorter oil consumption terminal period. The obtained Schizochytrium oil not only has a significantly higher oil yield than the traditional fermentation strategy, but also has a relatively significant increase in the DHA content and sn-2 DHA ratio in the oil. Furthermore, the regulation method is applied to the preparation of Schizochytrium algae powder or Schizochytrium oil, which can make the Schizochytrium algae powder or Schizochytrium oil rich in DHA and sn-2 DHA, which is more conducive to its application in the field of native milk and aquatic feed.

[0041] 2. The present invention can increase the DHA content and the proportion of sn-2 DHA in Schizochytrium oil and / or Schizochytrium algae powder. The method can promote the synthesis of DHA and the selective storage of sn-2 DHA during the growth process of Schizochytrium, and improve the quality of DHA in Schizochytrium oil and algae powder.

[0042] 3. In the experiment of feeding dairy cows with the Schizochytrium algae powder provided by the present invention, the DHA in the cow's milk accumulates more rapidly and has a higher DHA content, which will make the quality of DHA native milk higher and more easily meet people's demand for DHA intake.

[0043] 4. In the experiment of feeding rainbow trout with the Schizochytrium algae powder provided by the present invention, the DHA in the fish oil accumulated more rapidly, the DHA content was better, and the quality of rainbow trout was closer to that of native marine salmon.

[0044] 5. The fermentation pH of the present invention is regulated in three stages. Specifically, in the early stage of Schizochytrium fermentation, the pH of the fermentation liquid is adjusted to weak alkalinity, thereby accelerating the division and reproduction speed of Schizochytrium; in the oil accumulation stage, the pH of the fermentation liquid is adjusted to weak acidity, so that Schizochytrium obtains a longer oil accumulation period, and the accumulation of oil and DHA is significantly enhanced; in the late stage of fermentation, the pH of the fermentation liquid is adjusted to neutrality, so that the oil consumption period of Schizochytrium is shortened. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a pH change diagram of Schizochytrium in the present invention after unregulated pH fed-batch fermentation, normal pH regulated fed-batch fermentation and this optimized fermentation;

[0046] Figure 2 This is a graph showing changes in the sodium glutamate content in the fermentation broth of Schizochytrium sp. HX-308 after unregulated pH fed-batch fermentation, normal pH-regulated fed-batch fermentation, and this optimized pH-regulated fermentation;

[0047] Figure 3 This is a graph showing the biomass and oil yield changes of Schizochytrium HX-308 in the present invention after unregulated pH fed-batch fermentation, normal pH regulated fed-batch fermentation, and this optimized pH regulated fermentation;

[0048] Figure 4 This is a graph showing the changes in biomass and oil yield of Schizochytrium ATCC 20888 in the present invention after unregulated pH fed-batch fermentation, normal pH regulated fed-batch fermentation, and this optimized pH regulated fermentation. DETAILED DESCRIPTION

[0049] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0050] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0051] A method for increasing the DHA content and sn-2 DHA ratio in a Schizochytrium oil fermentation broth, the method comprising: subjecting the Schizochytrium to fed-batch fermentation culture, and controlling the pH of the fermentation broth in the fermentation process by supplementing acid or alkali in stages during the fermentation culture, wherein the acid is at least one of lactic acid, citric acid, malic acid, and fumaric acid, and the alkali is at least one of sodium hydroxide, potassium hydroxide, and sodium carbonate;

[0052] The method of controlling the pH of the fermentation liquid during the fermentation process by supplementing acid or alkali comprises the following steps:

[0053] 1) At 0h of fermentation, the fermentation liquid was made weakly alkaline by adding alkali, with a pH of 7.2 ± 0.1, and maintained until the nitrogen source was exhausted to accelerate the fission and reproduction period of Schizochytrium;

[0054] 2) During the oil accumulation period, i.e., 15h, 18h or 21h of fermentation, acid was added to make the fermentation liquid weakly acidic, with a pH of 6.8±0.1, and the pH was maintained until the growth of Schizochytrium slowed down, thereby extending the oil accumulation period of Schizochytrium and enhancing the accumulation of oil and DHA;

[0055] 3) In the late fermentation stage, i.e. 95h, 100h or 105h of fermentation, the pH of the fermentation liquid is adjusted to neutral, pH 7.0 ± 0.1, by adding alkali and acid, and maintained until the end of fermentation, i.e. 120h of fermentation, to reduce the consumption of oil in the late fermentation stage.

[0056] Preferably, the concentration of the acid for adjusting the pH of the fermentation broth is between 20 g / L and 250 g / L, and the concentration of the base for adjusting the pH of the fermentation broth is between 40 g / L and 400 g / L.

[0057] Preferably, the method specifically comprises the following steps:

[0058] (1) Streak the Schizochytrium plate from the cryotube, incubate at 28°C for 72 hours, and pick a single colony;

[0059] (2) A single colony was inoculated into the seed medium of the seed solution, and the pH was not adjusted at 28°C and 230 rpm for 48 h;

[0060] (3) Inoculate the seed medium with 1% inoculum into the seed solution, without adjusting the pH, and culture at 28°C and 170 rpm for 24 h. If OD600>3 at 24 h and no bacteria are found under microscopic examination, the next generation can be infected.

[0061] (4) Inoculate the seed medium with 1% inoculum into the seed solution, without adjusting the pH, and culture at 28°C and 170 rpm for 24 h. If OD600>5 at 24 h and no bacteria are found under microscopic examination, the next generation will be infected.

[0062] (5) Inoculate the seed tank containing seed medium with seed solution at a 2% inoculum, culture at 28°C, 150 rpm, 20 L / min for 24 h. After 20 h, OD600>8 and no bacteria are found under microscopic examination, and the next generation is infected.

[0063] (6) Inoculate the fermenter containing fermentation medium at a rate of 2% at 28°C, 100 rpm, 15 m 3 / h to start fermentation and adjust pH during fermentation;

[0064] Wherein, the method for controlling pH by fermentation comprises the following steps:

[0065] 1) At 0h of fermentation, the fermentation liquid was made weakly alkaline by adding alkali, with a pH of 7.2 ± 0.1, and maintained until the nitrogen source was exhausted to accelerate the fission and reproduction period of Schizochytrium;

[0066] 2) During the oil accumulation period, i.e., 15h, 18h or 21h of fermentation, acid was added to make the fermentation liquid weakly acidic, with a pH of 6.8±0.1, and the pH was maintained until the growth of Schizochytrium slowed down, thereby extending the oil accumulation period of Schizochytrium and enhancing the accumulation of oil and DHA;

[0067] 3) In the late fermentation stage, i.e. 95h, 100h or 105h of fermentation, the pH of the fermentation liquid is adjusted to neutral, pH 7.0 ± 0.1, by adding alkali and acid, and maintained until the end of fermentation, i.e. 120h of fermentation, to reduce the consumption of oil in the late fermentation stage.

[0068] Preferably, the pH value of the Schizochytrium plate culture medium is 6.0-6.5, and comprises: 40-60 g / L glucose, 4-6 g / L yeast extract, 5-8 g / L sodium sulfate, 2-4 g / L magnesium sulfate, 4-8 g / L ammonium sulfate, 1-2 g / L potassium chloride, 0.5-1 g / L potassium sulfate, 0.5-2 g / L potassium dihydrogen phosphate, 8-12 g / L sodium glutamate, 1-5 mg / L zinc sulfate heptahydrate, 0.01-0.1 mg / L cobalt chloride hexahydrate, 2-6 mg / L copper sulfate pentahydrate, 1-2 mg / L nickel sulfate hexahydrate, 8-15 mg / L iron sulfate heptahydrate, 3-5 mg / L manganese chloride tetrahydrate, 0.04 mg / L sodium molybdate dihydrate, 10-30 g / L agar powder, and the solvent is water;

[0069] The seed culture medium of the seed solution has a pH value of 6.0-6.5 and comprises: 40-60 g / L of glucose, 4-6 g / L of yeast extract, 5-8 g / L of sodium sulfate, 2-4 g / L of magnesium sulfate, 4-8 g / L of ammonium sulfate, 1-2 g / L of potassium chloride, 0.5-1 g / L of potassium sulfate, 0.5-2 g / L of potassium dihydrogen phosphate, 8-12 g / L of sodium glutamate, 1-5 mg / L of zinc sulfate heptahydrate, 0.01-0.1 mg / L of cobalt chloride hexahydrate, 2-6 mg / L of copper sulfate pentahydrate, 1-2 mg / L of nickel sulfate hexahydrate, 8-15 mg / L of iron sulfate heptahydrate, 3-5 mg / L of manganese chloride tetrahydrate, 0.04 mg / L of sodium molybdate dihydrate, and the solvent is water;

[0070] The pH value of the fermentation medium is 6.0-6.5, and the fermentation medium comprises: 60-100 g / L of glucose, 5-15 g / L of yeast extract, 5-12 g / L of sodium sulfate, 2-4 g / L of magnesium sulfate, 4-8 g / L of ammonium sulfate, 1-2 g / L of potassium chloride, 0.5-1 g / L of potassium sulfate, 0.5-2 g / L of potassium dihydrogen phosphate, 15-20 g / L of sodium glutamate, 1-5 mg / L of zinc sulfate heptahydrate, 0.01-0.1 mg / L of cobalt chloride hexahydrate, 2-6 mg / L of copper sulfate pentahydrate, 1-2 mg / L of nickel sulfate hexahydrate, 8-15 mg / L of iron sulfate heptahydrate, 3-5 mg / L of manganese chloride tetrahydrate, 0.04 mg / L of sodium molybdate dihydrate, and the solvent is water;

[0071] Alternatively, the acid is citric acid with a concentration of 150 g / L; the alkali is sodium hydroxide with a concentration of 250 g / L;

[0072] Alternatively, when the pH is adjusted during fermentation, the pH is adjusted to 7.2 at 0 h of fermentation, the pH is adjusted to 6.8 at 18 h of fermentation during the oil accumulation period, and the pH is adjusted to 7.0 at 100 h of fermentation in the late fermentation period.

[0073] Preferably, the pH of the Schizochytrium plate culture medium is 6.1, and comprises: 50 g / L glucose, 5 g / L yeast extract, 8 g / L sodium sulfate, 4 g / L magnesium sulfate, 8 g / L ammonium sulfate, 2 g / L potassium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 10 g / L sodium glutamate, 2 mg / L zinc sulfate heptahydrate, 0.04 mg / L cobalt chloride hexahydrate, 4 mg / L copper sulfate pentahydrate, 2 mg / L nickel sulfate hexahydrate, 10 mg / L iron sulfate heptahydrate, 4 mg / L manganese chloride tetrahydrate, 0.04 mg / L sodium molybdate dihydrate, 20 g / L agar powder, and the solvent is water.

[0074] The seed culture medium of the seed solution has a pH of 6.1 and comprises: 50 g / L glucose, 5 g / L yeast extract, 8 g / L sodium sulfate, 4 g / L magnesium sulfate, 8 g / L ammonium sulfate, 2 g / L potassium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 10 g / L sodium glutamate, 2 mg / L zinc sulfate heptahydrate, 0.04 mg / L cobalt chloride hexahydrate, 4 mg / L copper sulfate pentahydrate, 2 mg / L nickel sulfate hexahydrate, 10 mg / L iron sulfate heptahydrate, 4 mg / L manganese chloride tetrahydrate, 0.04 mg / L sodium molybdate dihydrate, and the solvent is water;

[0075] The pH value of the fermentation medium is 6.1, and the fermentation medium comprises: 80 g / L glucose, 5 g / L yeast extract, 8 g / L sodium sulfate, 4 g / L magnesium sulfate, 8 g / L ammonium sulfate, 2 g / L potassium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 10 g / L sodium glutamate, 2 mg / L zinc sulfate heptahydrate, 0.04 mg / L cobalt chloride hexahydrate, 4 mg / L copper sulfate pentahydrate, 2 mg / L nickel sulfate hexahydrate, 10 mg / L iron sulfate heptahydrate, 4 mg / L manganese chloride tetrahydrate, 0.04 mg / L sodium molybdate dihydrate, and the solvent is water.

[0076] Preferably, the Schizochytrium sp. is Schizochytrium sp. HX-308 or Schizochytrium sp. ATCC 20888.

[0077] A Schizochytrium algae powder comprising the Schizochytrium oil fermentation liquid obtained by the regulation method as described above.

[0078] The method for preparing the Schizochytrium algae powder as described above comprises the following steps:

[0079] 100 mg / L of tea polyphenol extract and 20 mg / L of sesamol were added to the Schizochytrium oil fermentation liquid, and then spray-dried to make powder. During drying, the inlet temperature was 180° C. and the outlet temperature was 80° C., so as to obtain Schizochytrium algae powder.

[0080] Use of the Schizochytrium algae powder as described above in native milk and / or aquatic feed.

[0081] The use of Schizochytrium algae powder as described above in feed.

[0082] Specifically, the relevant preparation and detection are as follows:

[0083] In the following examples, Schizochytrium sp. HX-308 is currently deposited in China Center for Type Culture Collection (CCTCC) with the deposit number CCTCC No.M209059, which has been disclosed in patent application CN101575584A;

[0084] Schizochytrium sp. ATCC 20888 was purchased from the American Type Culture Collection with the number ATCC 20888.

[0085] Unless otherwise specified, room temperature refers to 25±5℃.

[0086] The method for determining biomass is:

[0087] Take 1 mL of the fermentation liquid that has just been fermented, centrifuge it at 4000 r / min for 5 min, discard the supernatant, add 1 mL of 2% NaCl solution to wash once, then wash once with 1 mL of distilled water, dry it at 105°C to constant weight, and finally weigh and calculate.

[0088] The culture medium used in the following examples is as follows:

[0089] The pH of the plate culture medium is 6.1, and includes: 50 g / L glucose, 5 g / L yeast extract, 8 g / L sodium sulfate, 4 g / L magnesium sulfate, 8 g / L ammonium sulfate, 2 g / L potassium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 10 g / L sodium glutamate, 2 mg / L zinc sulfate heptahydrate, 0.04 mg / L cobalt chloride hexahydrate, 4 mg / L copper sulfate pentahydrate, 2 mg / L nickel sulfate hexahydrate, 10 mg / L iron sulfate heptahydrate, 4 mg / L manganese chloride tetrahydrate, 0.04 mg / L sodium molybdate dihydrate, 20 g / L agar powder, and the solvent is water.

[0090] The pH of the seed culture medium of the seed solution is 6.1, and includes: 50 g / L glucose, 5 g / L yeast extract, 8 g / L sodium sulfate, 4 g / L magnesium sulfate, 8 g / L ammonium sulfate, 2 g / L potassium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 10 g / L sodium glutamate, 2 mg / L zinc sulfate heptahydrate, 0.04 mg / L cobalt chloride hexahydrate, 4 mg / L copper sulfate pentahydrate, 2 mg / L nickel sulfate hexahydrate, 10 mg / L iron sulfate heptahydrate, 4 mg / L manganese chloride tetrahydrate, 0.04 mg / L sodium molybdate dihydrate, and the solvent is water.

[0091] The pH value of the fermentation medium is 6.1, and it includes: 80 g / L glucose, 5 g / L yeast extract, 8 g / L sodium sulfate, 4 g / L magnesium sulfate, 8 g / L ammonium sulfate, 2 g / L potassium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 10 g / L sodium glutamate, 2 mg / L zinc sulfate heptahydrate, 0.04 mg / L cobalt chloride hexahydrate, 4 mg / L copper sulfate pentahydrate, 2 mg / L nickel sulfate hexahydrate, 10 mg / L iron sulfate heptahydrate, 4 mg / L manganese chloride tetrahydrate, 0.04 mg / L sodium molybdate dihydrate, and the solvent is water.

[0092] The regulator acid is citric acid, and the final concentration of the citric acid is 150 g / L. The regulator base is sodium hydroxide, and the final concentration of the sodium hydroxide is 250 g / L.

[0093] The DHA content in the microbial oil prepared by the method provided in the above preferred embodiment is higher.

[0094] Example 1: Control of Schizochytrium HX-308 fermentation by three-stage pH control mode

[0095] 1. The specific fermentation steps of Schizochytrium HX-308 are:

[0096] (1) Streak the Schizochytrium plate culture medium from the cryotube and incubate it in a 28°C incubator for 72 hours before use. Pick a single colony.

[0097] (2) A single colony was inoculated into a test tube (test tube generation 1) (seed medium with 5 mL of seed solution, without pH adjustment) and cultured at 28°C and 230 rpm for 48 h;

[0098] (3) Inoculate 250 mL baffled shake flask (seed medium with 50 mL seed solution, no pH adjustment) at 1% inoculum, and culture at 28°C and 170 rpm for 24 h (2nd generation in shake flask). If OD600>3 at 24 h and no bacteria are found under microscopic examination, the next generation can be infected;

[0099] (4) Inoculate 1% of the inoculum into a 1000 mL baffled shake flask (seed medium with 200 mL of seed solution, without adjusting pH), and culture at 28°C and 170 rpm for 24 h (3rd generation of shake flask). If OD600>5 at 24 h and no bacteria are found under microscopic examination, the next generation can be infected.

[0100] (5) Inoculate 2% of the inoculum into a 50L seed tank (a total of 40L of seed liquid seed medium), culture at 28°C, 150rpm, 20L / min for 24h (1st generation of the tank). If OD600>8 at 20h and no bacteria are found under microscopic examination, the next generation can be infected.

[0101] (6) Inoculate 2% of the inoculum into a 5000L fermenter (a total of 2000L of fermentation medium) at 28°C, 100 rpm, 15 m 3 / h to start fermentation and perform different pH adjustments.

[0102] The acid for adjusting pH is citric acid with a concentration of 150 g / L; the alkali for adjusting pH is sodium hydroxide with a concentration of 250 g / L.

[0103] The different pH controls in step (6) above specifically include fermentation pH control (i.e., this optimized pH control fermentation), fermentation without pH control, and fermentation with normal pH control, specifically:

[0104] The method for controlling pH by fermentation comprises the following steps:

[0105] (1) At 0h of fermentation, the fermentation liquid was made weakly alkaline by adding alkali, with a pH of 7.2 ± 0.1, and maintained until the nitrogen source was basically exhausted, thereby accelerating the fission and reproduction period of Schizochytrium;

[0106] (2) During the oil accumulation period, i.e., fermentation for 15 h, 18 h or 21 h, preferably 18 h, acid is added to make the fermentation liquid weakly acidic, with a pH of 6.8 ± 0.1, and the pH is maintained until the growth of Schizochytrium slows down, thereby extending the oil accumulation period of Schizochytrium and enhancing the accumulation of oil and DHA;

[0107] (3) In the late fermentation stage, i.e., 95 h, 100 h or 105 h, preferably 100 h, the pH of the fermentation liquid is adjusted to neutral, i.e., 7.0 ± 0.1, by adding alkali and acid, and maintained until the end of fermentation, i.e., 120 h, to reduce the consumption of oil in the late fermentation stage.

[0108] (4) Glucose and monosodium glutamate detection: Take 1 mL of fermentation broth at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, and 32h, centrifuge at 12000 rpm for 1 min, and take the supernatant for gradient dilution to 100 times, centrifuge the dilution, and detect it with the biosensor SBA-40ES. Glucose and monosodium glutamate were determined using different enzyme membranes and calibrated with standard solutions in advance.

[0109] The method of fermentation without pH control comprises the following steps:

[0110] During the fermentation process, the fermentation is carried out without adding acid or alkali, and the pH is naturally regulated until the end of the fermentation, that is, the fermentation takes 120 hours.

[0111] Glucose and sodium glutamate detection: Take 1 mL of fermentation broth at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, and 32h, centrifuge at 12000 rpm for 1min, take the supernatant and dilute it to 100 times, centrifuge the dilution, and detect it with biosensor SBA-40ES. Glucose and sodium glutamate were determined with different enzyme membranes and calibrated with standard solutions in advance.

[0112] The method for normal pH control of fermentation comprises the following steps:

[0113] (1) At 0 h of fermentation, the pH of the fermentation liquid was not regulated by adding alkali, and was maintained until the nitrogen source was basically exhausted;

[0114] (2) After the nitrogen source is exhausted, i.e., 22 h after fermentation, the fermentation liquid is made weakly acidic by adding acid, with a pH of 6.8 ± 0.1, and maintained until the end of the Schizochytrium fermentation, i.e., 120 h after fermentation;

[0115] (3) Glucose and monosodium glutamate detection: Take 1 mL of fermentation broth at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, and 32h, centrifuge at 12000 rpm for 1 min, take the supernatant and dilute it to 100 times, centrifuge the dilution, and detect it with the biosensor SBA-40ES. Glucose and monosodium glutamate were determined using different enzyme membranes and calibrated with standard solutions in advance.

[0116] 2. Collecting the cells to extract oil, including the following steps:

[0117] (1) After the fermentation culture is completed, NaOH solution is added to the fermentation broth to adjust the pH to 10-13, and then 0.01-0.2% of the fermentation broth weight of the cell wall breaking enzyme is added, and the mixture is shaken at 40-60° C. and 100-200 r / min for 5-15 hours;

[0118] (2) Cool to room temperature and add an equal volume of anhydrous ethanol to the fermentation broth to inactivate the cell wall-breaking enzyme;

[0119] (3) Add an equal volume of n-hexane to the fermentation liquid to extract the oil:

[0120] The extracted organic phase was allowed to stand for 5 hours, and after the upper and lower layers were clearly separated, the upper organic phase was collected. The upper organic phase was taken out and placed in a rotary evaporation bottle, and the organic phase was rotary evaporated in a 45°C water bath at 120 r / min. After the organic phase no longer evaporated, the rotary evaporation bottle was removed and dried in a 60°C oven until the weight no longer changed, and the oil was weighed;

[0121] (4) Gas phase detection and analysis of fatty acids. The specific procedure is as follows:

[0122] Take 20 μL of the oil obtained in step (3) and add it to an EP tube containing 1 ml of 1M potassium hydroxide-methanol solution. Oscillate at 20°C and 1000 r / min for 6 h. Add 50 μL of concentrated sulfuric acid to terminate the reaction. Add 1 ml of n-hexane and oscillate at 20°C and 1000 r / min for 0.5 h to extract the oil.

[0123] The extract was placed in a liquid phase vial for gas phase detection. The analysis was performed using a GC-2010 (Shimadzu, Japan) gas phase system equipped with a DB-23 capillary column (60 m * 0.22 mm) and a flame ionization detector (FID). Nitrogen was used as the carrier gas. The injection volume was 1 μL and the injection temperature was 250 °C. The column temperature was increased from 100 °C to 200 °C at a rate of 25 °C / min, and then increased to 230 °C at a rate of 4 °C / min and maintained for 9 min. The FID detector temperature was 280 °C. The different fatty acid compositions were identified by comparison with relevant external standards (Sigma, USA). The content of individual fatty acids was calculated from the peak area on the chromatogram using non-endogenous fatty acids (C19:0) as internal standards.

[0124] 3. Determine the sn-2 DHA content in oils and fats, including the following steps:

[0125] (1) Removal of phospholipids:

[0126] ① Place about 10 mL of the oil obtained in step (3) in a sealed bottle, heat it in a water bath to about 90°C, filter / suction filter, remove insoluble matter, and obtain a sample.

[0127] ② Use a clean beaker to weigh 2 g of the sample obtained in the previous step, heat it to 80°C, add 2.0 mL of ultrapure water, and stir thoroughly to hydrate it.

[0128] ③ Centrifuge the centrifuge tube at 12000rpm for 5 minutes. The upper layer is glycerolipids, the middle layer is phospholipids, and the lower layer is the aqueous phase. Try to collect the glycerides in the upper layer.

[0129] (2) Preparation of 2-monoglyceride

[0130] ① Add 1 g of the upper glyceride obtained in the previous step and 4 g of anhydrous ethanol into the reactor, and then add 0.03 g (3%, w / w, mass concentration) of lipase to start the alcoholysis reaction. The reaction temperature was 23°C and stirred continuously at 350 r / min for 4 h.

[0131] ② Centrifuge the reaction mixture at 12000 rpm for 5 min and then filter through an organic filter to remove lipase.

[0132] ③ Dissolve the product in 15 mL of n-hexane, then add 6 mL of 75% ethanol-water solution, transfer the mixed solution to a separation funnel and let stand to separate the layers.

[0133] ④ After two layers are formed, the upper layer containing esters and unreacted triglyceride residues is removed, and the extraction layer containing 2-MAG, i.e., the lower organic phase, is retained.

[0134] ⑤ Add 10 mL of a mixed solution of V (ethanol): V (n-hexane) = 90:10 (volume ratio) to the lower organic phase, centrifuge at 3500 r / min for 1 min, aspirate the upper n-hexane phase containing 2-MAG, perform gas phase detection, and analyze the DHA and sn-2 DHA content.

[0135] (3) Methylation

[0136] The oil obtained in step 2 and the n-hexane phase obtained in step 3 (2) are subjected to methyl esterification: ① add 1 ml of potassium hydroxide methanol solution to a 1.5 ml centrifuge tube, add 20 μl of the oil / n-hexane phase thereto and mix well, then add it to a 20 ml volumetric flask, then add 2 ml of potassium hydroxide-methanol solution to the volumetric flask (rinse the 1.5 ml centrifuge tube and aspirate the oil into the volumetric flask as much as possible), mix well, place in a 65°C water bath for 17 min, and cool to room temperature; ② add 2 ml of boron trifluoride ether (boron trifluoride: ether = 3:7, volume ratio), mix well, place in a 65°C water bath for 7 min; ③ add 2 ml of saturated potassium chloride and shake well, add 3 ml of n-hexane (chromatographic grade), and let stand to separate; ④ pour into a small centrifuge tube, take the upper n-hexane phase and pass it through a microporous membrane to remove impurities; ⑤ seal and store before applying to gas chromatography.

[0137] The fermentation results of different pH fermentation modes are as follows Figure 1As shown. When fermenting without controlling pH, the pH of Schizochytrium gradually increases as the fermentation proceeds. However, after 24 hours of fermentation, the pH of the fermentation broth exceeds 8, and the pH of the fermentation broth then slowly increases. When the pH is regulated in normal fermentation, when the sodium glutamate in the fermentation broth is exhausted, that is, 22 hours of fermentation, the pH of the fermentation broth is maintained at 6.8 ± 0.1 by adding acid. In the three-stage pH-regulated fermentation, that is, fermentation regulation, at the beginning of the fermentation, that is, 0 hours of fermentation, the fermentation broth is weakly alkaline by adding alkali solution, and the pH is 7.2 ± 0.1, which is maintained until the nitrogen source is basically exhausted; in the oil accumulation period, that is, 18 hours of fermentation, the fermentation broth is weakly acidic by adding acid solution, and the pH is 6.8 ± 0.1, which is maintained until the growth of Schizochytrium slows down. In the late fermentation period, that is, 100 hours of fermentation, the pH of the fermentation broth is adjusted to neutral by adding alkali solution and acid solution, and the pH is 7.0 ± 0.1, which is maintained until the end of fermentation. The three-stage fermentation optimization timely adjusted the optimal fermentation pH during different fermentation time periods of Schizochytrium to facilitate the synthesis of DHA and lipid accumulation of Schizochytrium.

[0138] Figure 2 The changes of sodium glutamate content in the fermentation broth of Schizochytrium HX-308 after unregulated pH fed-batch fermentation, normal pH-regulated fed-batch fermentation and this optimized pH-regulated fermentation. Figure 2 As shown, when Schizochytrium HX-308 is fed-fermented without pH control, it takes about 32 hours to consume 20g / L sodium glutamate; when Schizochytrium HX-308 is fed-fermented with normal pH control, it takes about 28 hours to consume 20g / L sodium glutamate; when Schizochytrium HX-308 is fed-fermented under the three-stage pH control mode, it only takes 22 hours to consume 20g / L sodium glutamate. When Schizochytrium uses nitrogen sources for reproduction and division, carbon sources are used to consume energy instead of producing oil. Therefore, the three-stage pH control mode accelerates the utilization of nitrogen sources and increases the oil accumulation period of Schizochytrium.

[0139] Figure 3 The biomass and oil yield changes of Schizochytrium HX-308 after unregulated pH fed-batch fermentation, normal pH-regulated fed-batch fermentation and this optimized pH-regulated fermentation. Figure 3As shown, when Schizochytrium HX-308 was fermented without pH control, the biomass and oil content grew slowly, and the final biomass and oil content were 47.2 g / L and 25.9 g / L, respectively. When Schizochytrium HX-308 was fermented under normal pH control, the biomass and oil content grew normally, and the final biomass and oil content were 117.5 g / L and 61.5 g / L, respectively. When Schizochytrium HX-308 was fermented under the three-stage pH control mode, the biomass and oil content grew rapidly, and the final biomass and oil content were 133.2 g / L and 82.5 g / L, respectively. Therefore, the present invention uses a three-stage pH control mode to ferment Schizochytrium, and its oil yield is significantly higher than other reported fermentation modes.

[0140] Finally, the fermentation properties of Schizochytrium HX-308 under three pH control modes are shown in Table 1. When Schizochytrium HX-308 was fed-batch fermented without pH control, DHA and DHA / DPA were 50.3% and 3.0, respectively, and the final DHA was 13.0 g / L, and the sn-2 DHA ratio was 42.2%. When Schizochytrium HX-308 was fed-batch fermented under normal pH control, DHA and DHA / DPA were 46.8% and 3.1, respectively, and the final DHA was 28.8 g / L, and the sn-2 DHA ratio was 38.3%. When Schizochytrium HX-308 was fed-batch fermented under three-stage pH control mode, DHA and DHA / DPA were 55.7% and 3.8, respectively, and the final DHA was 46.0 g / L, and the sn-2 DHA ratio was 60.4%. Therefore, the present invention uses a three-stage pH control mode (i.e., this optimized pH control fermentation) to ferment Schizochytrium, and its DHA ratio is as high as 55.7%, DHA oil production is as high as 46.0 g / L, oil production is as high as 82.5 g / L, and sn-2 DHA ratio is as high as 60.4%. Both the single fermentation results and the comprehensive fermentation results are far better than the reported levels.

[0141] In addition, the production of DHA oil by microbial method is the most effective way to solve the problems of unstable supply chain and fishy taste of traditional fish DHA oil. However, the biggest cost of industrialization of microbial fermentation is time cost. This three-stage control strategy can produce DHA oil more efficiently without increasing production time, laying a solid foundation for the sustainable development of DHA oil industry chain.

[0142] At the same time, it can also be seen that the pH control of the fermentation in the method of the present invention (i.e., the optimized pH control fermentation) in step (1) at 0h of fermentation, by adding alkali solution to make the fermentation liquid weakly alkaline, the pH is 7.2±0.1 and the pH control of the fermentation liquid in step (2) at the oil accumulation period, i.e., 15h, 18h or 21h of fermentation, preferably 18h of fermentation, by adding acid solution to make the fermentation liquid weakly acidic, the pH is 6.8±0.1, have a synergistic effect, and can synergistically improve the DHA ratio, DHA oil yield, oil yield, and sn-2 DHA ratio of the prepared product.

[0143]

[0144] The pH change time of the three-stage adjustment was further optimized, and the results are shown in Table 2. Except for the differences in the pH fermentation at different time points of the three-stage optimization, the remaining steps are the same as the relevant steps of "1. Specific fermentation steps of Schizochytrium HX-308 in the three-stage pH control mode in Example 1".

[0145] When the regulation mode A was changed, the DHA content and sn-2 DHA ratio were lower than those of the fermentation mode regulated by mode B. And the oil content of the former was only 60.2g / L, and the DHA oil yield was only 30.9g / L. Compared with mode B, mode A entered the oil production period early before the Schizochytrium entered the end of complete division and reproduction, resulting in insufficient biomass accumulation and failure to accumulate more oil. When the regulation mode C was changed, the DHA content and sn-2 DHA ratio were also lower than those of the fermentation mode regulated by mode B. And the oil content of mode C was only 55.9g / L, and the DHA oil yield was only 29.8g / L. Compared with mode B, mode C did not enter the oil production mode in time when the Schizochytrium entered the end of complete division and reproduction, missing the golden time for accumulating oil. Therefore, although the oil biomass of Schizochytrium was as high as 150.5g / L in the fermentation mode of mode C, the oil yield and DHA accumulation were less than those of mode B. A large amount of carbon source was used to accumulate biomass, resulting in insufficient oil accumulation.

[0146]

[0147] In addition, this data is far higher than the 35% standard index stipulated in the current national standard (GB26400-2011 Food Additive Docosahexaenoic Acid Oil (Fermentation Method)), and is expected to meet the requirements for DHA content in DHA algae oil in the fields of medicine and special medical food.

[0148] At the same time, it can also be seen that the fermentation pH control in the method of the present invention (i.e., the optimized pH control fermentation) in step (2) during the oil accumulation period, i.e., 18 hours of fermentation, by adding acid to make the fermentation liquid weakly acidic, the pH is 6.8 ± 0.1 and the step (3) in the late fermentation period, i.e., 100 hours of fermentation, by adding alkali and acid to adjust the pH of the fermentation liquid to neutral, the pH is 7.0 ± 0.1, have a synergistic effect, and can synergistically improve the DHA ratio, DHA oil yield, oil yield, and sn-2 DHA ratio of the prepared product.

[0149] Example 2: Three-stage pH control mode to control the fermentation of Schizochytrium ATCC 20888

[0150] The fermentation of Schizochytrium was carried out according to the method of Example 1 to obtain microbial lipids, except that Schizochytrium HX-308 was replaced by Schizochytrium ATCC 20888.

[0151] After 120 hours of batch fed-batch fermentation, the biomass and oil yield of Schizochytrium ATCC 20888 in the uncontrolled pH fed-batch fermentation, the normal pH controlled pH fed-batch fermentation and the optimized pH controlled fermentation were as follows: Figure 4 As shown. When Schizochytrium ATCC 20888 was fed-fermented without pH control, the biomass and oil content increased slowly, and the final biomass and oil content were 40.2 g / L and 20.9 g / L, respectively. When Schizochytrium ATCC 20888 was fed-fermented with normal pH control, the biomass and oil content increased normally, and the final biomass and oil content were 102.5 g / L and 51.5 g / L, respectively. When Schizochytrium ATCC 20888 was fed-fermented under the three-stage pH control mode, the biomass and oil content increased rapidly, and the final biomass and oil content were 113.2 g / L and 71.5 g / L, respectively.

[0152] Finally, the fermentation properties of Schizochytrium ATCC 20888 under three pH control modes are shown in Table 3. When Schizochytrium ATCC 20888 was fed-batch fermented without pH control, DHA and DHA / DPA were 48.8% and 2.8, respectively, and the final DHA was 10.2 g / L, and the sn-2 DHA ratio was 37.2%. When Schizochytrium ATCC 20888 was fed-batch fermented under normal pH control, DHA and DHA / DPA were 46.9% and 2.8, respectively, and the final DHA was 24.1 g / L, and the sn-2 DHA ratio was 36.3%. When Schizochytrium ATCC 20888 was fed-batch fermented under three-stage pH control mode, DHA and DHA / DPA were 54.2% and 3.5, respectively, and the final DHA was 38.8 g / L, and the sn-2 DHA ratio was 55.3%. For Schizochytrium ATCC 20888, the three-stage mode was used to regulate the fermentation, and the DHA oil production was increased by 3.8 times compared with the unregulated fermentation, the sn-2 DHA ratio was increased by 48.7%, and the oil and biomass were increased by 3.4 times and 2.8 times, respectively. Therefore, through the three-stage mode of fermentation regulation, the oil and DHA production levels of Schizochytrium ATCC 20888 can also reach the leading level in the market. In addition, this optimized fermentation strategy not only does not increase the fermentation cost, but also does not add inedible substances during the fermentation process, and the usability of the fermented oil is guaranteed. On the other hand, this regulation strategy has strong applicability and has shown positive applicability for different Schizochytrium, resulting in a significant increase in the DHA oil production capacity per unit time, greatly shortening the time production cost of DHA oil.

[0153] At the same time, it can also be seen that the pH control of the fermentation in the method of the present invention (i.e., the optimized pH control fermentation) in step (1) at 0h of fermentation, by adding alkali solution to make the fermentation liquid weakly alkaline, the pH is 7.2±0.1 and the pH control of the fermentation liquid in step (2) at the oil accumulation period, i.e., 15h, 18h or 21h of fermentation, preferably 18h of fermentation, by adding acid solution to make the fermentation liquid weakly acidic, the pH is 6.8±0.1, have a synergistic effect, and can synergistically improve the DHA ratio, DHA oil yield, oil yield, and sn-2 DHA ratio of the prepared product.

[0154]

[0155] Example 3 Preparation of Schizochytrium algae powder

[0156] The Schizochytrium HX-308 was fermented and cultured using the above-mentioned regulation method (i.e., "the three-stage pH regulation mode in Example 1 regulates the fermentation of Schizochytrium HX-308 1, the specific fermentation steps of Schizochytrium HX-308, wherein the pH regulation in step (6) is the fermentation regulation pH (i.e., this optimized regulation pH fermentation), and the specific parameters are: 0h adjusts the pH to 7.2, 18h adjusts the pH to 6.8, and 100h adjusts the pH to 7.0") to obtain a fermentation broth; 100 mg / L of tea polyphenol extract and 20 mg / L of sesamol were added to the fermentation broth, and the fermentation broth was dried and powdered, and the drying temperature was 180°C at the inlet temperature and 80°C at the outlet temperature.

[0157] The algae powder obtained according to the preparation method of the algae powder (i.e., the algae powder obtained by spray drying under the condition of adding natural antioxidants, i.e., tea polyphenol extract and sesamol) was compared with the algae powder obtained by normal direct spray drying (i.e., the algae powder obtained by spray drying under the condition of not adding natural antioxidants, i.e., tea polyphenol extract and sesamol). The acid value and peroxide value of the oil in the algae powder were detected using a kit according to the instructions after being stored for one year at room temperature and 40°C.

[0158] The results are shown in Table 4. The oil in the algae powder obtained by spray drying with the addition of natural antioxidants is more stable than the oil in the algae powder obtained by normal spray drying under the same storage environment. Both the acid value and the peroxide value are much lower than those of the normal fermentation group, and the quality performance is more outstanding. Under 4℃, the acid value of the oil in the algae powder under normal spray drying conditions is 0.9 mg (KOH) / g, and increases to 1.35 mg (KOH) / g after 30 days; while the acid value of the oil in the algae powder obtained by spray drying with the addition of natural antioxidants is 0.75 mg (KOH) / g, and only increases to 0.9 mg (KOH) / g after 30 days, respectively. The initial and final acid values ​​are lower. When stored at 40℃ for 30 days, 60 days, 90 days, 180 days and 360 days, the acid value of the oil in the algae powder under normal spray drying conditions is 0.3, 0.45, 0.6, 0.65 and 0.65 mg (KOH) / g higher than that in the algae powder obtained by spray drying with the addition of natural antioxidants. In terms of peroxide value, the oil in the algae powder under normal spray drying conditions began to peroxidize in about 15 days, while the oil in the algae powder obtained by spray drying with the addition of natural antioxidants did not begin to peroxidize until about 90 days. Therefore, the oil obtained by this spraying strategy has a more reliable shelf life, better quality, and more reliable edible stability. The main reason is the addition of natural edible antioxidants (i.e., tea polyphenol extract and sesamol), which prevent the oil in the algae powder from being oxidized, extend the shelf life of the algae powder, and improve the quality of the algae powder.

[0159] Table 4 Comparison of storage index parameters of oil in algae powder obtained by spray drying of Schizochytrium fermentation liquid under normal spray drying conditions and adding natural antioxidants under different environments

[0160]

[0161] Example 4 Feeding Cow Experiment

[0162] The Schizochytrium fermentation liquid obtained by normal pH control and three-stage pH control mode (wherein the pH control in step (6) is fermentation pH control (i.e., this optimized pH control fermentation), with specific parameters: pH adjusted to 7.2 at 0h, pH adjusted to 6.8 at 18h, and pH adjusted to 7.0 at 100h) corresponding to Example 1 was spray-dried to obtain algae powder under the conditions of adding natural antioxidants in Example 3. The two algae powders were mixed with dairy cow feed at a ratio of 6g algae powder / kg feed, and the dairy cows were continuously fed and milk was collected. The milk produced by dairy cows continuously fed with normal feed was used as a comparison to detect the DHA content in the milk. The results are shown in Table 5.

[0163] Table 5 Comparison of the changes in DHA content in milk of cows fed with Schizochytrium algae powder obtained by normal pH control and three-stage pH control mode

[0164]

[0165] The results in Table 4 show that when the algae powder feed obtained by spray drying in Example 3 containing the Schizochytrium fermentation liquid containing the three-stage pH-controlled fermentation corresponding to Example 1 is continuously fed to dairy cows for 30 days, the DHA content in the milk reaches and stably maintains around 300 mg / kg, and exceeds 350 mg / kg after feeding for 50 days. Therefore, dairy cows fed with Schizochytrium algae powder obtained in the three-stage pH control mode are more likely to enrich DHA in milk, and have more advantages in producing DHA native milk.

[0166] Example 5 Feeding freshwater rainbow trout experiment

[0167] The Schizochytrium fermentation liquid obtained by normal pH control and three-stage pH control mode (wherein the pH control in step (6) is fermentation pH control (i.e., this optimized pH control fermentation), with specific parameters: pH adjusted to 7.2 at 0h, pH adjusted to 6.8 at 18h, and pH adjusted to 7.0 at 100h) corresponding to Example 1 was spray-dried to obtain algae powder under the conditions of adding natural antioxidants in Example 3. The two algae powders were mixed with rainbow trout feed at a ratio of 100g algae powder / kg feed, and 30 rainbow trout fry of the same age, size, and developmental status were selected and divided into three groups on average. The same amount of normal feed, feed with algae powder obtained by normal pH control fermentation, and feed with algae powder obtained by three-stage pH control fermentation were fed respectively, and the feeding was continued for 60 days. Each time the DHA content in fish oil was tested, 2 rainbow trout were randomly picked from each test group for testing and the average value was taken. The results are shown in Table 6.

[0168] Table 6 Comparison of the changes in DHA content in fish oil of rainbow trout fed with Schizochytrium algae powder obtained by normal pH control and three-stage pH control mode

[0169]

[0170] The results in Table 6 show that when rainbow trout were fed with algae powder feed obtained by spray drying in Example 3 containing the Schizochytrium fermentation broth with three-stage pH-controlled fermentation corresponding to Example 1 for continuous feeding for 45 days, the DHA content in the fish oil exceeded 10%, and exceeded 12% after 60 days of feeding.

[0171] Among them, the method for determining the composition of polyunsaturated fatty acids in samples such as microbial oils / DHA algae powder / milk / eggs in the above embodiment is as follows: 50 mg milk / 100 g is taken for fatty acid methyl esterification, added to an EP tube containing 1 mL of 1 M potassium hydroxide-methanol solution, and shaken for 6 h at 20 ° C and 1000 rpm, 50 μL of concentrated sulfuric acid is added to terminate the reaction, and 1 mL of n-hexane is added to shake for 0.5 h at 20 ° C and 1000 rpm to extract oil; the extract is placed in a liquid phase vial, and gas phase detection is performed, and the gas phase system is analyzed using a GC-2010 (Shimadzu, Japan) equipped with a DB-23 capillary column (60 m * 0.22mm) and a flame ionization detector (FID). Nitrogen was used as the carrier gas with an injection volume of 1 μL and an injection temperature of 250°C. The column temperature was increased from 100°C to 200°C at a rate of 25°C / min, and then to 230°C at a rate of 4°C / min and maintained for 9 min. The FID detector temperature was 280°C. The different polyunsaturated fatty acid compositions were identified by comparison with relevant external standards (Sigma, USA). The content of individual polyunsaturated fatty acids was calculated from the peak area on the chromatogram using non-endogenous fatty acids (C19:0) as the internal standard.

[0172] The glucose concentration and sodium glutamate concentration during the fermentation process were detected using a sugar meter (SBA-40ES).

[0173] The present invention regulates the fermentation of Schizochytrium HX-308 by a three-stage pH fermentation strategy, and obtains 82.5 g / L of oil within 120 hours, and the DHA and DHA / DPA are 55.7% and 3.8 respectively, the final DHA is 46.0g / L, and the sn-2 DHA ratio is 60.4%. The oil quality is significantly higher than the reported patent level, and the strategy has also achieved significant fermentation effects in Schizochytrium ATCC 20888. Furthermore, the present invention provides a Schizochytrium algae powder spray drying strategy, and the algae powder obtained by this strategy has a better guarantee of the oil quality in the storage process. Finally, the Schizochytrium algae powder containing the high-quality algae oil was subjected to feeding experiments on dairy cows and rainbow trout. For dairy cow feeding, after continuous feeding of dairy cows for 30 days, the DHA content in milk reached and stably maintained at around 300 mg / kg, and exceeded 350 mg / kg after 50 days of feeding. For rainbow trout feeding, after continuous feeding of rainbow trout for 45 days, the DHA content in fish oil exceeded 10%, and exceeded 12% after 60 days of feeding. In summary, the present invention provides a method and application for increasing the DHA content and sn-2 DHA ratio in Schizochytrium oil and / or Schizochytrium algae powder, which can promote the synthesis of DHA and the selective storage of sn-2 DHA during the growth of Schizochytrium, and improve the quality of DHA in Schizochytrium oil and algae powder. A method for preparing Schizochytrium algae powder is also provided, which includes better protection of the quality of algae powder, and confirms the application of high-quality Schizochytrium algae powder in the field of native milk and aquatic feed.

[0174] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for increasing the DHA content and sn-2 DHA ratio in Schizochytrium oil fermentation broth, characterized in that: The method comprises the following steps: subjecting Schizochytrium to fed-batch fermentation culture, and controlling the pH of the fermentation liquid in the fermentation process by supplementing acid or alkali in stages during the fermentation culture process; The specific steps include: (1) Streak the Schizochytrium plate from the cryotube, incubate at 28°C for 72 hours, and pick a single colony; (2) A single colony was inoculated into the seed medium of the seed solution, and the pH was not adjusted at 28°C and 230 rpm for 48 h; (3) Inoculate the seed medium with 1% inoculum into the seed solution, without adjusting the pH, and culture at 28°C and 170 rpm for 24 h. If OD600>3 at 24 h and no bacteria are found under microscopic examination, the next generation can be infected. (4) Inoculate the seed medium with 1% inoculum into the seed solution, without adjusting the pH, and culture at 28°C and 170 rpm for 24 h. If OD600>5 at 24 h and no bacteria are found under microscopic examination, the next generation will be infected. (5) Inoculate the seed tank containing seed medium with seed solution at a 2% inoculum, culture at 28°C, 150 rpm, 20 L / min for 24 h. After 20 h, OD600>8 and no bacteria are found under microscopic examination, and the next generation is infected. (6) Inoculate the fermenter containing fermentation medium at a rate of 2% at 28°C, 100 rpm, 15 m 3 / h to start fermentation and adjust pH during fermentation; Wherein, the method for controlling pH by fermentation comprises the following steps: 1) At 0h of fermentation, the fermentation liquid was made weakly alkaline by adding alkali, with a pH of 7.2 ± 0.1, and maintained until the nitrogen source was exhausted to accelerate the fission and reproduction period of Schizochytrium; 2) During the oil accumulation period, i.e., 18 hours of fermentation, acid was added to make the fermentation liquid weakly acidic, with a pH of 6.8 ± 0.1, and this was maintained until the growth of Schizochytrium slowed down, thereby extending the oil accumulation period of Schizochytrium and enhancing the accumulation of oil and DHA; 3) In the late fermentation stage, i.e., 100 hours of fermentation, the pH of the fermentation liquid is adjusted to neutral, pH 7.0 ± 0.1, by adding alkali and acid, and maintained until the end of fermentation, i.e., 120 hours of fermentation, to reduce the consumption of oil in the late fermentation stage; The pH value of the Schizochytrium plate culture medium is 6.0-6.5, and the medium comprises: 40-60 g / L of glucose, 4-6 g / L of yeast extract, 5-8 g / L of sodium sulfate, 2-4 g / L of magnesium sulfate, 4-8 g / L of ammonium sulfate, 1-2 g / L of potassium chloride, 0.5-1 g / L of potassium sulfate, 0.5-2 g / L of potassium dihydrogen phosphate, 8-12 g / L of sodium glutamate, 1-5 mg / L of zinc sulfate heptahydrate, 0.01-0.1 mg / L of cobalt chloride hexahydrate, 2-6 mg / L of copper sulfate pentahydrate, 1-2 mg / L of nickel sulfate hexahydrate, 8-15 mg / L of iron sulfate heptahydrate, 3-5 mg / L of manganese chloride tetrahydrate, 0.04 mg / L of sodium molybdate dihydrate, 10-30 g / L of agar powder, and the solvent is water; The seed culture medium of the seed solution has a pH value of 6.0-6.5 and comprises: 40-60 g / L glucose, 4-6 g / L yeast extract, 5-8 g / L sodium sulfate, 2-4 g / L magnesium sulfate, 4-8 g / L ammonium sulfate, 1-2 g / L potassium chloride, 0.5-1 g / L potassium sulfate, 0.5-2 g / L potassium dihydrogen phosphate, 8-12 g / L sodium glutamate, 1-5 mg / L zinc sulfate heptahydrate, 0.01-0.1 mg / L cobalt chloride hexahydrate, 2-6 mg / L copper sulfate pentahydrate, 1-2 mg / L nickel sulfate hexahydrate, 8-15 mg / L iron sulfate heptahydrate, 3-5 mg / L manganese chloride tetrahydrate, 0.04 mg / L sodium molybdate dihydrate, and the solvent is water; The pH value of the fermentation medium is 6.0-6.5, and the fermentation medium comprises: 60-100 g / L of glucose, 5-15 g / L of yeast extract, 5-12 g / L of sodium sulfate, 2-4 g / L of magnesium sulfate, 4-8 g / L of ammonium sulfate, 1-2 g / L of potassium chloride, 0.5-1 g / L of potassium sulfate, 0.5-2 g / L of potassium dihydrogen phosphate, 15-20 g / L of sodium glutamate, 1-5 mg / L of zinc sulfate heptahydrate, 0.01-0.1 mg / L of cobalt chloride hexahydrate, 2-6 mg / L of copper sulfate pentahydrate, 1-2 mg / L of nickel sulfate hexahydrate, 8-15 mg / L of iron sulfate heptahydrate, 3-5 mg / L of manganese chloride tetrahydrate, 0.04 mg / L of sodium molybdate dihydrate, and the solvent is water; The acid is citric acid with a concentration of 150 g / L; the alkali is sodium hydroxide with a concentration of 250 g / L; The Schizochytrium Schizochytrium sp. HX-308 or Schizochytrium Schizochytrium sp. ATCC 20888.

2. The control method according to claim 1, characterized in that: The pH of the Schizochytrium plate culture medium is 6.1, and includes: 50 g / L glucose, 5 g / L yeast extract, 8 g / L sodium sulfate, 4 g / L magnesium sulfate, 8 g / L ammonium sulfate, 2 g / L potassium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 10 g / L sodium glutamate, 2 mg / L zinc sulfate heptahydrate, 0.04 mg / L cobalt chloride hexahydrate, 4 mg / L copper sulfate pentahydrate, 2 mg / L nickel sulfate hexahydrate, 10 mg / L iron sulfate heptahydrate, 4 mg / L manganese chloride tetrahydrate, 0.04 mg / L sodium molybdate dihydrate, 20 g / L agar powder, and the solvent is water; The seed culture medium of the seed solution has a pH of 6.1 and comprises: 50 g / L glucose, 5 g / L yeast extract, 8 g / L sodium sulfate, 4 g / L magnesium sulfate, 8 g / L ammonium sulfate, 2 g / L potassium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 10 g / L sodium glutamate, 2 mg / L zinc sulfate heptahydrate, 0.04 mg / L cobalt chloride hexahydrate, 4 mg / L copper sulfate pentahydrate, 2 mg / L nickel sulfate hexahydrate, 10 mg / L iron sulfate heptahydrate, 4 mg / L manganese chloride tetrahydrate, 0.04 mg / L sodium molybdate dihydrate, and the solvent is water; The pH value of the fermentation medium is 6.1, and the fermentation medium comprises: 80 g / L glucose, 5 g / L yeast extract, 8 g / L sodium sulfate, 4 g / L magnesium sulfate, 8 g / L ammonium sulfate, 2 g / L potassium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 10 g / L sodium glutamate, 2 mg / L zinc sulfate heptahydrate, 0.04 mg / L cobalt chloride hexahydrate, 4 mg / L copper sulfate pentahydrate, 2 mg / L nickel sulfate hexahydrate, 10 mg / L iron sulfate heptahydrate, 4 mg / L manganese chloride tetrahydrate, 0.04 mg / L sodium molybdate dihydrate, and the solvent is water.

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