Method for increasing content of beta-carotene in mucor circinelloides by utilizing illumination

Abstract

The invention discloses a method for improving the content of beta-carotene in mucor circinelloides (CBS 277.49) by utilizing illumination.

Description

Technical Field

[0001] The invention belongs to the technical field of food science, and particularly relates to a method for increasing the content of beta-carotene in Mucor circinelloides by utilizing light. Background Art

[0002] β-carotene is a type of natural pigment that is widely present in nature. It is a natural powerful antioxidant and free radical scavenger. It has the effects of protecting eyesight, preventing cirrhosis and liver cancer, and improving one's own immunity.

[0003] At present, the sources of β-carotene mainly include plants and microorganisms. Compared with plant sources that are greatly affected by seasons and have high production costs, efficient and low-cost microbial sources have great potential in future industrial applications. Mucor circinelloides is a model strain for β-carotene synthesis, with simple fermentation, clear genetic background and perfect genetic operation system.

[0004] Light is an important environmental factor that participates in the regulation of the growth, development and physiological processes of most species and is an essential source of information for the light response mechanism of fungi. In many organisms, the biosynthesis of β-carotene is regulated by light. When synthesizing β-carotene in Mucor circinelloides, continuous and constant light is usually used. However, the light adaptation mechanism in the organism may make it difficult to increase the yield of β-carotene, which is not suitable for long-term industrial production. Changing the constant light pattern may help stimulate Mucor circinelloides to synthesize β-carotene and increase the yield. Summary of the invention

[0005] The object of the present invention is to provide a method for increasing the content of beta-carotene in Mucor circinelloides by utilizing light.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A method for increasing the content of beta-carotene in Mucor circinelloides by using a light pattern that gradually increases the light intensity, comprising the following steps: (1) activation culture: inoculating Mucor circinelloides onto a YGB solid culture medium for activation culture; (2) seed culture: inoculating Mucor circinelloides onto a YNB liquid culture medium for seed culture; (3) fermentation culture: inoculating the seed liquid into a K&R culture medium at a 10% inoculation amount for fermentation culture, and performing light treatment during the process; and (4) pigment extraction: extracting beta-carotene at the end of the culture.

[0008] As a preferred embodiment, the components of the YGB solid culture medium are: 10 g / L peptone, 3 g / L yeast extract, 20 g / L glucose and 20 g / L agar powder.

[0009] As a preferred embodiment, the YNB liquid culture medium comprises 1.5 g / L ammonium sulfate, 1.5 g / L glutamic acid, 0.5 g / L yeast nitrogen base, and 10 g / L glucose. Nicotinic acid / thiamine is added to 1 μg / ml at room temperature after high pressure sterilization at 115°C for 20 min.

[0010] As a preferred embodiment, the seed liquid is a liquid of Mucor circinelloides in the logarithmic growth phase.

[0011] As a preferred embodiment, the components of the K&R medium are 50g / L glucose, 2.0g / L L-ammonium tartrate, 7.0g / L KH2PO4, 2.0g / L Na2HPO4, 1.5g / L MgSO4·7H2O, 1.5g / L yeast powder, 0.1g / LCaC12·2H2O, 0.008g / L FeCl3·6H2O, 0.001g / L ZnSO4·7H2O, 0.0001g / L CuSO4·5H2O, 0.0001g / L Co(NO3)2·6H2O, 0.0001g / L MnSO4·5H2O. KOH is used to adjust the pH to 6.20, and the glucose is sterilized separately before mixing with the other components.

[0012] As a preferred embodiment, the fermentation culture conditions are 200 rpm and 26°C.

[0013] As a preferred embodiment, the light treatment is performed by gradually increasing the light intensity after 60 hours of fermentation culture.

[0014] As a preferred embodiment, the mode of gradually increasing light intensity is to use blue light irradiation, with an initial light intensity of 250 lux, and then increase by 250 lux every 6 hours until the 24th hour.

[0015] As a preferred embodiment, the fermentation culture time is 84 hours.

[0016] As a preferred embodiment, the method for extracting pigments is: collecting the bacteria and using filter paper to absorb as much water as possible, adding liquid nitrogen to fully grind, vacuum freeze-drying, mixing with an organic solvent, leaching, centrifuging to obtain the supernatant, nitrogen blowing, and re-dissolving with an organic solvent.

[0017] As a preferred embodiment, the extraction organic solvent is methanol and petroleum ether; the re-dissolution organic solvent is chloroform / isopropanol (2 / 1, v / v).

[0018] The method of the present invention is easy to operate, simple and feasible, and has good effects. Gradually increasing the light intensity can effectively stimulate the synthesis of β-carotene in Mucor circinelloides, mainly because continuous constant light causes the synthesis of β-carotene in Mucor circinelloides to produce light adaptation, resulting in the β-carotene synthesis-related genes returning to the expression level before light stimulation after a short-term high expression. The method of gradually increasing the light intensity further increases the light intensity before Mucor circinelloides adapts to the current light intensity, so that the β-carotene synthesis-related genes are always in an activated state and maintain a high level of expression, which is conducive to the continuous and efficient synthesis of β-carotene. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The biomass of Mucor circinelloides CBS 277.49 changes over time under different light regimes

[0020] Figure 2 The β-carotene content of Mucor circinelloides CBS 277.49 changes over time under different light modes DETAILED DESCRIPTION

[0021] The method of the present invention is further described below in conjunction with the accompanying drawings and specific examples. The following examples and drawings are only for illustrative purposes, but the embodiments of the present invention are not limited thereto.

[0022] The experimental methods in the following examples without specific experimental conditions are usually carried out according to conventional experimental conditions or the experimental conditions recommended by the manufacturer. The materials and reagents used are commercially available unless otherwise specified.

[0023] The strain used in the present invention is Mucor circinelloides CBS 277.49, which was purchased from Guangdong Institute of Microbiology.

[0024] The method for determining biomass of the present invention is: collecting bacterial liquid, filtering, freeze-drying using a vacuum freeze dryer (-40° C., 12 h) and weighing.

[0025] The method for determining the content of beta-carotene adopted by the present invention is HPLC determination method. The detection conditions are: Hypersil GOLD C18 reverse phase column (2.1mm×100mm, 3μm, ThermoFisher, USA). The mobile phase is acetonitrile / isopropanol (85 / 15, v / v), the flow rate is 0.15mL / min, the column temperature is 30°C, the sample injection volume is 3μL, and the standard injection volume is 0.5μL. The detector is an ultraviolet detector and a photodiode array detector, the detection wavelength is 450nm, and the beta-carotene standard is used for quantitative determination. The beta-carotene and lycopene standards are dissolved in chloroform (concentration is 1mg / mL) and filled with nitrogen for protection. When injecting, isopropanol is used to dilute according to the ratio of 2:1 (v / v), and it is prepared and used immediately.

[0026] The data in the following examples are all the results of at least 3 repeated experiments.

[0027] Example 1

[0028] (1) Activation culture

[0029] First, prepare YPG solid medium, whose ingredients are: 10g / L peptone, 3g / L yeast extract, 20g / L glucose and 20g / L agar powder. Inoculate Mucor circinelloides into YPG solid medium for activation at 26±2℃ until a single colony grows. Pick a single colony and re-inoculate it into YPG solid medium for spore production. Rinse the mycelium with sterile water and obtain spore liquid after filtering through a filter membrane. Count the spores using a hemocytometer and adjust the concentration of the spore suspension to 10 with sterile water. 7 sp / mL.

[0030] (2) Seed culture

[0031] Prepare YNB liquid medium, whose ingredients are: 1.5g / L ammonium sulfate, 1.5g / L glutamic acid, 0.5g / L yeast nitrogen base, 10g / L glucose, autoclave at 115°C for 20min, and add nicotinic acid / thiamine to 1μg / ml at room temperature. Inoculate the spore liquid into the YNB liquid medium at 10% inoculum, and culture at 26±2°C, 200rpm for 24h.

[0032] (3) Fermentation culture

[0033] Prepare K&R medium, whose ingredients are: 1.5g / L ammonium sulfate, 1.5g / L glutamic acid, 0.5g / L yeast nitrogen source, 20g / L glucose, sterilize at 115℃ for 20min, and then add nicotinic acid / thiamine to 1μg / ml at room temperature. Inoculate the seed liquid into K&R medium at 10% inoculum for fermentation, at 26±2℃, 200rpm, and culture in the dark for 84h;

[0034] (4) Extraction and determination of β-carotene

[0035] The cells were collected and the water was absorbed as much as possible with filter paper, and liquid nitrogen was added to fully grind them. After vacuum freeze drying, they were extracted with methanol and petroleum ether, centrifuged to obtain the supernatant, dried with nitrogen, and then reconstituted with chloroform / isopropanol (2 / 1, v / v). The β-carotene content was determined by HPLC. The detection conditions were: Hypersil GOLD C18 reverse phase column (2.1mm×100mm, 3μm, ThermoFisher, USA). The mobile phase was acetonitrile / isopropanol (85 / 15, v / v), the flow rate was 0.15mL / min, the column temperature was 30℃, the sample injection volume was 3μL, and the standard injection volume was 0.5μL. The detector was a UV detector and a photodiode array detector, the detection wavelength was 450nm, and the β-carotene standard was used for quantification. β-carotene and lycopene standards were dissolved in chloroform (concentration of 1 mg / mL) and protected by nitrogen. They were diluted with isopropanol at a ratio of 2:1 (v / v) during injection and were prepared and used immediately.

[0036] Three groups of parallel tests were performed under the same conditions, and the results are shown in Table 1: Table 1 Fermentation results of Example 1 Parallel batches β-carotene content after 72h fermentation (μg / g) β-carotene content after 84h fermentation (μg / g) 1 21.20 46.30 2 38.17 75.46 3 38.47 61.19

[0037] Example 2

[0038] (1) Activation culture (same as Example 1)

[0039] (2) Seed culture (same as Example 1)

[0040] (3) Fermentation culture

[0041] The same as Example 1, except that the culture was first placed in the dark for fermentation for 60 hours, and then continuously irradiated with constant blue light, with a light intensity of 1000 lux and a lighting time of 24 hours.

[0042] (3) Extraction and determination of β-carotene (same as in Example 1)

[0043] Three groups of parallel tests were performed under the same conditions, and the results are shown in Table 2: Table 2 Fermentation results of Example 2 Parallel batches β-carotene content after 72h fermentation (μg / g) β-carotene content after 84h fermentation (μg / g) 1 758.28 370.18 2 720.47 420.91 3 680.85 358.45

[0044] Example 3

[0045] (1) Activation culture (same as Example 1)

[0046] (2) Seed culture (same as Example 1)

[0047] (3) Fermentation culture

[0048] The same as Example 1, except that the culture was first placed in the dark for fermentation for 60 hours, and then irradiated with blue light. The lighting mode was to gradually increase the light intensity. The initial light intensity was 250 lux, and then increased by 250 lux every 6 hours until the 24th hour.

[0049] (3) Extraction and determination of β-carotene (same as in Example 1)

[0050] Three groups of parallel tests were performed under the same conditions, and the results are shown in Table 3: Table 3 Fermentation results of Example 3 Parallel batches β-carotene content after 72h fermentation (μg / g) β-carotene content after 84h fermentation (μg / g) 1 440.52 635.90 2 407.76 704.31 3 461.61 686.33

Claims

1. A lighting pattern for increasing the content of β-carotene in Mucor circinelloides, characterized in that: The following steps are involved: (1) Activation culture: inoculate Mucor circinelloides onto YGB solid culture medium for activation culture; (2) Seed culture: inoculate onto YNB liquid culture medium for culture; (3) Fermentation culture: inoculate the seed liquid into K&R culture medium at a 10% inoculation rate for fermentation culture, and perform light treatment during the process; (4) Pigment extraction: extract β-carotene at the end of the culture.

2. The method according to claim 1, characterized in that The components of the YGB solid culture medium are: 10 g / L peptone, 3 g / L yeast extract, 20 g / L glucose and 20 g / L agar powder.

3. The method according to claim 1, characterized in that The YNB liquid culture medium comprises 1.5 g / L ammonium sulfate, 1.5 g / L glutamic acid, 0.5 g / L yeast nitrogen base, and 10 g / L glucose. Nicotinic acid / thiamine is added to 1 μg / ml at room temperature after high-pressure sterilization at 115° C. for 20 min.

4. The method according to claim 1, characterized in that The seed liquid is a liquid of Mucor circinelloides in the logarithmic growth phase.

5. The method according to claim 1, characterized in that The K&R medium comprises 50 g / L glucose, 2.0 g / L L-ammonium tartrate, 7.0 g / L KH2PO4, 2.0 g / L Na2HPO4, 1.5 g / L MgSO4·7H2O, 1.5 g / L yeast powder, 0.1 g / L CaC12·2H2O, 0.008 g / L FeCl3·6H2O, 0.001 g / L ZnSO4·7H2O, 0.0001 g / L CuSO4·5H2O, 0.0001 g / L Co(NO3)2·6H2O, and 0.0001 g / L MnSO4·5H2O. The pH is adjusted to 6.20 by KOH, and the glucose is sterilized separately before being mixed with the other components.

6. The method according to claim 1, characterized in that The fermentation culture conditions are 200 rpm and 26°C.

7. The method according to claim 1, characterized in that The light treatment is performed by gradually increasing the light intensity after 60 hours of fermentation culture.

8. The method according to claim 7, characterized in that The mode of gradually increasing the light intensity is to use blue light irradiation, with the initial light intensity of 250 lux, and then increase by 250 lux every 6 hours until the 24th hour.

9. The method according to claim 1, characterized in that: The fermentation culture time is 84h.

10. The method according to claim 1, characterized in that The pigment extraction method comprises the following steps: collecting the bacterial cells and using filter paper to absorb water as much as possible, adding liquid nitrogen to fully grind, vacuum freeze-drying, mixing with an organic solvent, leaching, centrifuging to obtain a supernatant, blowing with nitrogen, and re-dissolving with an organic solvent.

11. The method according to claim 10, characterized in that The extraction organic solvent is methanol and petroleum ether; the re-dissolution organic solvent is chloroform / isopropanol (2 / 1, v / v).

Images