CN108517338A - Method for producing arachidonic acid oil by fermenting mortierella alpina based on active oxygen regulation - Google Patents

Abstract

The invention relates to a method for regulating Mortierella alpine fermentation to produce arachidonic acid oil based on active oxygen. The Mortierella alpina strain is amplified and cultivated step by step through a test tube slant medium and a shake flask medium to prepare a seed liquid, and then inoculated in a fermenter for fermentation. The entire fermentation process is divided into two stages: a relatively high active oxygen period and a relatively low active oxygen period; in the relatively high active oxygen period, the active oxygen source is added 8 to 12 hours after the fermentation starts, and the intracellular active oxygen is controlled at 100 to 125 RFLU to stimulate rapid cell proliferation; during the period of relatively low reactive oxygen species, the intracellular reactive oxygen species are controlled at about 200 RFLU by intermittent hypoxia or adding antioxidants to reduce the loss of intracellular products. A total of 168 hours of fermentation, at the end of the Mortierella alpina cell dry weight, oil content, the percentage of arachidonic acid in the total oil, and the unit yield of arachidonic acid can reach 59.8 g/L, 60.1%, respectively. 65.7%, 23.6 g/L, the production intensity of arachidonic acid can reach 3.37 g/(L*d).

Description

A method based on reactive oxygen species to regulate the fermentation of Mortierella alpina to produce arachidonic acid oil method

technical field

The invention relates to a method for producing arachidonic acid oil by regulating the fermentation of Mortierella alpina based on active oxygen, and belongs to the technical field of microbial fermentation.

Background technique

In the past thirty years, people have paid more and more attention to dietary nutrition, and long-chain polyunsaturated fatty acids have gradually come into people's sight. Omega-6 long-chain polyunsaturated fatty acid arachidonic acid (ARA) not only plays an important role in the composition of membrane phospholipids, but also accumulates in large quantities in the brain and other nerve tissues, and can be used as a synthetic signal molecule eicosanoid It is the precursor of acid (including prostaglandin, thromboxane and leukotrienes, etc.), which is widely used in medicine and cosmetics. Especially in the field of infant food and nutritional products, since arachidonic acid oil cannot be synthesized in infants and young children, and cow's milk does not contain arachidonic acid, additional intake of arachidonic acid oil is beneficial to infant brain growth and development. very important.

At present, the process of producing arachidonic acid oil by fermentation of the microorganism Mortierella alpina has entered the stage of industrial production. A variety of effective nutrient-limited fermentation strategies have been extensively studied. For example, the nitrogen source limitation strategy widely used in oleaginous microorganisms can change the intracellular metabolic flow to the lipid synthesis pathway, and rapidly accumulate fatty acid components in large quantities. Another example is that the Zygomycetes strains belonging to Mortierella alpina will increase the content of arachidonic acid after the carbon source is exhausted, which has been used to improve the quality of oil. However, a large number of studies have shown that stress conditions such as nutrient limitation can cause the accumulation of intracellular reactive oxygen species, trigger oxidative stress, and disrupt intracellular redox homeostasis. At the same time, reactive oxygen species will attack and oxidize intracellular active components, resulting in the loss of biomass and lipids. And 90% of active oxygen originates from the leakage of oxygen molecules in the mitochondrial respiratory chain. Electron microscope pictures show that the shape of Mortierella alpina becomes extremely irregular during the aging period, which may be the main reason for the sharp increase of active oxygen. On the other hand, active oxygen has two sides to the action of microorganisms. Studies have shown that low concentrations of reactive oxygen species, as a potential intracellular signal transduction molecule, can stimulate the rapid proliferation of filamentous fungal cells. Studies have also proved that an appropriate concentration of reactive oxygen species can promote oil-producing microorganisms to achieve rapid and simultaneous accumulation of cell biomass and oil.

Therefore, in view of the disadvantages of slow growth in the early stage of fermentation, inability to simultaneously accumulate products and biomass in the middle and late stages, and the accumulation of a large amount of active oxygen, the present invention aims at the intracellular active oxygen concentration of Mortierella alpina, and implements regulation in stages to increase the arachidonic The fermentation time is shortened while the acid production is increased, the production intensity is enhanced, and the total energy consumption of stirring is reduced, providing a simple and effective new fermentation process of Mortierella alpina arachidonic acid oil, which can be used for reference by similar oil-producing microorganisms .

Contents of the invention

In view of the above-mentioned problems in the prior art, the purpose of the present invention is to propose a method based on active oxygen regulation and control of Mortierella alpina fermentation to produce arachidonic acid oil, which rebalances the concentration of active oxygen in the cells of Mortierella alpina Reactive Oxygen Requirements During Biomass, Lipid, and Arachidonic Acid Synthesis. A total of 168 hours of fermentation, at the end of the Mortierella alpina cell dry weight, oil content, the percentage of arachidonic acid in the total oil, and the unit yield of arachidonic acid can reach 59.8 g/L, 60.1 %, 65.7 %, 23.6 g/L, the production intensity of arachidonic acid can reach 3.37 g/(L*d). The method improves the yield of arachidonic acid while shortening the fermentation time and enhancing the production intensity. In addition, the intermittent hypoxia strategy can also reduce the energy consumption of stirring.

In order to achieve the above object, the technical scheme adopted in the present invention is:

A method for producing arachidonic acid oil based on active oxygen regulation and fermentation of Mortierella alpina. The Mortierella alpina strain is amplified step by step through a test tube slant medium and a shake flask medium to prepare a seed liquid, and then inoculate it in a fermenter During the fermentation process, aiming at controlling the active oxygen concentration in the cells of Mortierella alpina, the fermentation process was regulated in stages, and the entire fermentation process was divided into a relatively high active oxygen period and a relatively low active oxygen period. In the relatively high active oxygen period, by adding active oxygen sources (such as hydrogen peroxide), the intracellular active oxygen concentration was increased to 100-125 RFLU (an increase of about 40-50 %) to stimulate the accelerated growth of cells; During the period of low reactive oxygen species, intermittent hypoxia or adding antioxidants (such as sodium ascorbate) can reduce the concentration of intracellular reactive oxygen species to about 200 RFLU (a drop of about 30-40%) to reduce the loss of cell products.

The relatively high active oxygen period is the exponential growth period of Mortierella alpina fermentation, that is, 0 to 60 hours.

The active oxygen source is added during the relatively high active oxygen period, and the addition time is after the lag period ends, that is, 8 to 12 hours.

The active oxygen source is added during the relatively high active oxygen period, and the concentration of the active oxygen source in the culture medium after the addition is 50-100 μmol/L.

The active oxygen source can be hydrogen peroxide, nano-titanium dioxide particles, nano-iron particles, etc., taking hydrogen peroxide as an example, the added concentration is 50-100 μmol/L.

The relatively low active oxygen period is 61 to 168 hours of Mortierella alpina fermentation.

The intermittent hypoxia in the relatively low active oxygen period is realized by controlling ventilation and rotating speed, that is, 12 to 18 hours is a cycle, and the stirring rate and ventilation are reduced every cycle, so that the dissolved oxygen in the environment is reduced by 50 to 80%, resulting in 15 ~ 120min intermittent hypoxic environment.

The time points for adding antioxidants in the relatively low active oxygen period are 60 hours and 120 hours respectively.

The added antioxidants in the relatively low active oxygen period include sodium ascorbate, sesamol, carotene, etc. Taking sodium ascorbate as an example, the single addition amount is 5-10 g/L.

The Mortierella alpina strain used in the present invention is Mortierella alpina strain R807 (CCTCC M 2012118), and similar oleaginous microorganisms can also be used in practice.

The control principle of the present invention is as follows: the fermentation step is divided into two stages (relatively high active oxygen period and relatively low active oxygen period), and different control processes are adopted for different stages. In the period of relatively high active oxygen, the seed liquid has just been connected to the fermenter, the cell viability is low, and there is a growth stagnation period of about 8 to 12 hours, after which an appropriate amount of active oxygen source (such as hydrogen peroxide) is added to stimulate the rapid proliferation of cells , which is conducive to the accumulation of Mortierella alpina biomass and shortens the fermentation cycle. When the late stage of growth index is reached, the biomass reaches a certain level, and the synthesis of intracellular arachidonic acid oil accelerates, and the product damage caused by the accumulation of active oxygen needs to be dealt with. One can reduce the oxidative damage caused by reactive oxygen species by adding an appropriate amount of antioxidants (such as sodium ascorbate); the other is to reduce the reactive oxygen species produced by the respiratory chain from the source and reduce intracellular activity by intermittently reducing the stirring rate and ventilation. oxygen concentration. Both can be used alone or in combination.

Beneficial effects of the present invention:

(1) To solve the problem of slow growth in the early stage of fermentation, appropriately increase the concentration of active oxygen in cells to stimulate rapid cell proliferation, which is conducive to the accumulation of Mortierella alpina biomass and shorten the fermentation cycle.

(2) In view of the different accumulation rates of biomass, oil and arachidonic acid in the middle and late stages of fermentation, the inability to reach the accumulation peak at the end of fermentation, and the accumulation of a large amount of active oxygen to lose products, etc., by adding antioxidants or implementing intermittent hypoxia regulation , which rebalanced the active oxygen demand in the process of biomass, lipid and arachidonic acid synthesis, so that the yield and production intensity of arachidonic acid reached 23.6 g/L and 3.37 g/(L*d), respectively. Compared with "A method for producing arachidonic acid oil by regulating Mortierella alpina fermentation based on dissolved oxygen" (CN104278107A), the fermentation period is shortened by 22%, the yield of arachidonic acid is increased by 14%, the production intensity is increased by 47.2%, and the production Efficiency is greatly improved.

(3) The process involved in the present invention is easy to operate, and at the same time, it reduces the total energy consumption of stirring by about 15% compared with "A method for producing arachidonic acid oil by regulating the fermentation of Mortierella alpina based on dissolved oxygen" (CN104278107A), which is more conducive to Industrial production. At present, the trial operation has been completed at the level of 25m³ fermentation tank, and good benefits have been obtained.

Description of drawings

Figure 1: The process flow of the method for regulating the fermentation of Mortierella alpina to produce arachidonic acid oil based on active oxygen.

Detailed ways

The present invention will be described in detail below in combination with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

The culture medium adopted in the following examples is as follows:

Test tube slant medium (PDA slant medium): 200g of fresh peeled potatoes, boiled and timed for 30min, filtered through 4 layers of gauze to remove solids, added 20g of glucose, 20g of agar, distilled water to 1L, natural pH, extinguished at 115°C Bacteria 30min.

Seed medium: glucose 30g/L, yeast extract 6g/L, KH ₂ PO ₄ 3g/L, pH natural, sterilized at 115°C for 30min.

Fermentation medium: glucose 80g/L, yeast extract 11g/L, KH ₂ PO ₄ 3.8g/L, NaNO ₃ 3.4g/L, MgSO ₄ 7H ₂ O 0.5g/L, natural pH, sterilized at 115°C for 30min .

The strain used in the examples is: Mortierella alpina strain R807 (CCTCC M 2012118), and other similar oleaginous microbial strains can also be used.

Example 1. Based on active oxygen regulation and control of 7.5 L tank Mortierella alpina fermentation to produce arachidonic acid oil

1. Activation of strains and preparation of seed solution: Mortierella alpina strain R807 (CCTCC M 2012118) was selected as the starting strain, and the preserved strains were inserted into PDA slant medium, and placed in an incubator at 25°C Cultivate for 10 days, transfer to a 500mL grooved bottle containing 100mL seed medium and culture in a constant temperature shaker, the inoculum size is 10% (v/v, 10mL), the culture conditions are 125rpm, the temperature is 25°C, and the culture time is 1 -2 days.

2. Inoculate the seed liquid obtained in step 1 into a 7.5L tank (New Brunswick Scientific, USA), the liquid volume is 5L, the inoculum volume is 10% (v/v, 500mL), and the culture temperature is controlled at 25°C. During the 0-60 hour period of fermentation, it is a relatively high active oxygen period. At the beginning of fermentation, when the seed solution was just put into the fermenter, there was a growth stagnation period of 12 hours, after which 50 μmol/L hydrogen peroxide was added to control the intracellular active oxygen at about 110 RFLU. The period of 60-168 hours of fermentation is the stage of low active oxygen, which is the stage of accumulation of arachidonic acid oil. Taking 12 hours as a cycle, reduce the stirring and ventilation for 45 minutes in each cycle, so that the dissolved oxygen will drop by 50-70%, resulting in intermittent hypoxia. And add sodium ascorbate 5 g/L and 8 g/L at 60 hours and 120 hours, respectively. The intracellular active oxygen was controlled at 180-200 RFLU, the ventilation was controlled at about 2VVM, and the rotation speed was controlled at 150 rpm.

When the fermentation was cultivated to 168 hours, the fermentation was finished, and samples were taken for detection. The dry weight of Mortierella alpina cells at the end point, the oil content, the content of arachidonic acid, and the unit yield of arachidonic acid can reach 59.8 g/L, 60.1%, 65.7%, and 23.6 g/L respectively, and the production of arachidonic acid The strength can reach 3.37 g/(L*d). The total stirring energy consumption decreased by about 15% during fermentation.

Example 2. Arachidonic acid oil produced by fermentation of Mortierella alpina in a 25 m³ tank based on active oxygen regulation

1. Activation of strains and preparation of seed solution: Mortierella alpina strain R807 (CCTCC M 2012118) was selected as the starting strain, and the preserved strains were inserted into PDA slant medium, and placed in an incubator at 25°C Cultured for 10 days, transferred to a 500 mL grooved bottle containing 100 mL seed medium and cultured in a constant temperature shaker, the inoculum volume was 10% (v/v, 10 mL), the culture conditions were 125 rpm, the temperature was 25°C, The cultivation time is 2 days, and the primary seed liquid is prepared. Then, according to the inoculum volume of 10% (v/v), the primary seed solution was inserted into a 150 L seed tank, the cultivation conditions were 175 rpm, the temperature was 25°C, and the cultivation time was 1 day to prepare the secondary seed solution. Next, all the prepared secondary seed liquids were put into a 5000 L seed tank, the cultivation conditions were 125 rpm, the temperature was 25°C, and the cultivation time was 18 hours to prepare the tertiary seed liquids.

2. Put all the three-stage seed liquid obtained in step 1 into a 25 m³ fermenter, and cultivate at a temperature of 25°C. During the 0-60 hour period of fermentation, it is a relatively high active oxygen period. At the beginning of fermentation, when the seed liquid was just put into the fermenter, there was a growth stagnation period of 10 hours, after which 80 μmol/L hydrogen peroxide was added to control the intracellular active oxygen at about 125 RFLU. The period of 60-168 hours of fermentation is the stage of low active oxygen, which is the stage of accumulation of arachidonic acid oil. Among them, during the period of 60-120 hours, the agitation and ventilation were reduced for 30 min in each cycle of 12 hours; during the period of 120-168 hours, the agitation and aeration were reduced for 60 min in a cycle of 18 hours. And add sodium ascorbate 5 g/L at 60 hours and 120 hours respectively. The intracellular active oxygen was controlled at about 210 RFLU, the ventilation was controlled at about 2VVM, and the rotation speed was controlled at 175 rpm.

When the fermentation was cultivated to 168 hours, the fermentation was finished, and samples were taken for detection. The dry weight of Mortierella alpina cells at the end point, the oil content, the content of arachidonic acid, and the unit yield of arachidonic acid can reach 56.3 g/L, 56.4%, 59.1%, and 18.8 g/L respectively, and the production of arachidonic acid Strength 2.69 g/(L*d). The total stirring energy consumption decreased by about 17% during fermentation.

In the above examples, the intracellular active oxygen concentration was measured by the DCFH-DA method:

Take 10 ml of cell culture solution, centrifuge at 10,000 g at 4°C for 10 minutes, and wash twice with sterile water. At room temperature in a dark room, 5 μL of DMSO solution with a concentration of 10 μmol/L DCFH-DA was added. Incubate in a dark environment for 30 minutes, centrifuge to take the supernatant, and wash twice with pH 7.4 PBS solution. Shake and resuspend, use a microplate reader to detect, the excitation wavelength is 488nm, and the emission wavelength is 525nm. The resulting fluorescence intensity was divided by the biomass in the culture solution to obtain the relative fluorescence intensity, which was regarded as the active oxygen concentration. pH 7.4 PBS solution was used as negative control.

Based on the above examples, it can be seen that a method for producing arachidonic acid oil based on active oxygen regulation and control of Mortierella alpina fermentation of the present invention is simple and operable, and reduces the production rate of arachidonic acid oil while increasing the output of arachidonic acid oil. The fermentation cycle greatly improves the production intensity and reduces the total energy consumption of stirring during fermentation.

Claims (8)

1. a kind of method for producing arachidonic acid oil of being fermented based on active oxygen regulation and control Mortierella alpina, by Mortierella alpine trichoderma strain Amplify culture step by step by test tube slant culture medium and Shake flask medium, prepares seed liquor, be then inoculated in fermentation tank and carry out Fermented and cultured, it is characterised in that：During the fermentation, to control a concentration of target of Mortierella alpina intracellular reactive oxygen species generation, to fermentation Process is regulated and controled stage by stage, and entire fermentation process is divided into opposite highly reactive form of oxygen phase and opposite 2 stages of low activity oxygen phase； Intracellular activity oxygen concentration is promoted to 100 ~ 125 RFLU by the opposite highly reactive form of oxygen phase by addition active oxygen source by 65 RFLU, Stimulate cell tachyauxesis；In the opposite low activity oxygen phase by antioxidant addition and intermittence low-oxygen, intracellular activity is reduced Oxygen concentration reduces cellular products loss by 350 RFLU to 200 RFLU or so.

2. the method for producing arachidonic acid oil according to claim 1 of being fermented based on active oxygen regulation and control Mortierella alpina, It is characterized in that：The exponential phase of growth that the opposite highly reactive form of oxygen phase ferments for Mortierella alpina, i.e., 0 ~ 60 hour, the phase To 61 ~ 168 hours that the low activity oxygen phase is Mortierella alpina fermentation.

3. the method for producing arachidonic acid oil according to claim 1 of being fermented based on active oxygen regulation and control Mortierella alpina, It is characterized in that：The active oxygen source be a series of substances that can promote cell intracellular activity oxygen concentration, such as hydrogen peroxide, Nano-titania particle, nano-iron particle etc., after the addition time is lag phase, i.e., 8 ~ 12 hours.

4. the method for producing arachidonic acid oil according to claim 1 of being fermented based on active oxygen regulation and control Mortierella alpina, It is characterized in that：It is described to be with respect to the interim active oxygen source additive amount of highly reactive form of oxygen：By taking hydrogen peroxide as an example, addition a concentration of 50 ~ 100 μmol/L。

5. the method for producing arachidonic acid oil according to claim 1 of being fermented based on active oxygen regulation and control Mortierella alpina, It is characterized in that：The intermittence low-oxygen of the opposite low activity oxygen phase is by controlling ventilation and rotating speed or addition antioxidant At least one of mode realize.

6. the method for producing arachidonic acid oil according to claim 5 of being fermented based on active oxygen regulation and control Mortierella alpina, It is characterized in that：The intermittence low-oxygen of the opposite low activity oxygen phase is addition antioxidant, and addition time point is respectively 60 small When and 120 hours.

7. the method for producing arachidonic acid oil according to claim 5 of being fermented based on active oxygen regulation and control Mortierella alpina, It is characterized in that：The intermittence low-oxygen of the opposite low activity oxygen phase is realized by controlling ventilation with rotating speed, with 12 ~ 18 Hour is a cycle, and each cycle reduces stir speed (S.S.) and ventilation, so that environment dissolved oxygen is reduced by 50 ~ 80%, causes 15 ~ 120 Min intermittence low-oxygen environments.

8. the method for producing arachidonic acid oil according to claim 5 of being fermented based on active oxygen regulation and control Mortierella alpina, It is characterized in that：The antioxidant of the opposite low activity oxygen phase addition is sodium ascorbate, sesamol, carrotene etc., with anti- For bad hematic acid sodium, single additive amount is 5 ~ 10 g/L.