A method for producing DHA in schizochytrium by using spirulina platensis fermentation

Abstract

The present application relates to the technical field of enzyme engineering and fermentation engineering, and particularly relates to a method for producing DHA in schizochytrium sp. by using chondrus crispus fermentation. After chondrus crispus is subjected to enzymatic saccharification and schizochytrium sp. fermentation, the concentration of DHA reaches 10.82 g / L. The present application performs enzymatic treatment on chondrus crispus, and uses the hydrolysate to stimulate schizochytrium sp. to continuously accumulate DHA. The strategy of the present application emphasizes the feasibility of commercialization of sargassum, and has the potential to expand the production scale in the near future.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of enzyme engineering and fermentation engineering, and particularly relates to a method for producing DHA in schizochytrium by using sargassum horneri fermentation. BACKGROUND

[0002] Brown algae is a promising resource for green biorefinery due to its high carbohydrate content and low lignocellulose content, and it does not require fresh water or arable land. Sargassum horneri belongs to the phylum Ochrophyta, order Fucales, family Sargassaceae, and genus Sargassum, and is also known as S. cylindricum. In recent years, the frequency of large-scale S. horneri blooms (also known as "golden tides") has increased globally.

[0003] In China, the abnormal accumulation of S. horneri during red tides mainly occurs in the Yellow Sea and the East China Sea, resulting in a large amount of plankton biomass. The presence of this invasive algal biomass has a serious impact on the local ecosystem, seaweed cultivation, fisheries, tourism, and local biodiversity. Due to the high cost of removing this invasive seaweed, it is necessary to find a way to convert it into valuable products to mitigate its impact and generate economic benefits.

[0004] Although there have been some successful applications of producing biogas, biofuels, and bioethanol from S. horneri, the yield and profit of these chemicals are still limited. Because of its composition rich in carbohydrates, proteins, and lipids, bioprocesses provide a promising way to convert S. horneri into high-value compounds, and it is crucial to develop innovative strategies to produce high-value chemicals. The potential of producing high-value chemicals from S. horneri has been verified in recent studies, indicating that the commercialization of this invasive macroalgae has a promising prospect.

[0005] Schizochytrium is a high-yield docosahexaenoic acid (DHA) producing marine fungus belonging to the family Thraustochytriaceae, which is a kind of marine heterotrophic microorganism. Microscopic observation shows that it is single-cell and spherical, and is often distributed in marine, saltwater lake, estuary, and mangrove forest. Schizochytrium can produce through heterotrophic propagation, and its growth and propagation are fast, the production is not affected by seasons, and it is suitable for large-scale cultivation in fermenters. Schizochytrium can accumulate a large amount of substances beneficial to humans, such as oil, pigment, and non-saponified substances, among which oil accounts for 50-70% of the biomass, and polyunsaturated fatty acids account for 40-70% of the total oil.

[0006] DHA and EPA in polyunsaturated fatty acids (PUFAs) have important functions such as anti-cancer, anti-inflammatory, promoting the development of nervous system and visual system, and preventing cardiovascular diseases. Therefore, the extraction of n-3 polyunsaturated fatty acids from schizochytrium has gradually become a new research hotspot. As a new biological resource for producing DHA, how to improve the DHA yield of schizochytrium has become a research hotspot. SUMMARY

[0007] To solve the above technical problems, the present application provides a method for fermenting production of DHA in Schizochytrium sp. by using C. brownii.

[0008] The method comprises using brown algae lyase and cellulase to enzymatically hydrolyze C. brownii to obtain C. brownii saccharified liquid, inoculating Schizochytrium sp. for fermentation culture, so as to achieve fermenting production of DHA in Schizochytrium sp.

[0009] Further, the Schizochytrium sp. strain is first grown in GPY medium, and after incubation for 24-48 h in the seed culture, the strain is transferred to a fermenter containing C. brownii saccharified liquid as the fermentation medium.

[0010] Further, the formula of the GPY medium is 2% glucose, 1% peptone, 0.5% yeast powder, and 2% sea salt.

[0011] Further, the concentration of the brown algae lyase and cellulase used is 3%.

[0012] Further, the inoculation is 3-5% of the Schizochytrium sp. seed liquid by volume.

[0013] Further, the preparation process of the C. brownii saccharified liquid comprises the following steps:

[0014] 1) Swelling the dry C. brownii powder with distilled water at a ratio of 1:6 (w / v);

[0015] 2) Then adding the prepared brown algae lyase and commercial cellulase to the swollen biomass;

[0016] 3) Reacting at 40°C and pH 5.0 for 6 h with stirring using a hot mixer to obtain the C. brownii saccharified liquid.

[0017] Compared with the prior art, the present application has the following technical effects:

[0018] (1) The present application provides a new method for fermenting production of DHA in Schizochytrium sp., and also provides a new method for better commercialization of degradation of invasive S. thunbergii;

[0019] (2) The self-made brown algae lyase and commercial cellulase provided by the present application have obvious enzymatic saccharification effect on C. brownii, and the glucose level reaches 16.92 g / L at a feed ratio (w / v) of 1:6. After strain enzymatic saccharification and fermentation, the DHA concentration can reach 10.82 g / L, and the research method provided by the present application has the potential for expanding the production scale in the near future;

[0020] (3) The results of the present invention emphasize the commercial feasibility of degrading the invasive S. hainanensis, with the potential for scaling up production in the near future. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 (A) Effect of different feed ratios (w / v) on the saccharification of S. wrightii. (B) Effect of cellulase dosage on the saccharification of S. wrightii;

[0022] Figure 2 MYA-1381 fermentation fourth day key plant hormone screening biomass, DHA index level;

[0023] Figure 3 MYA-1381 cell lipid droplet morphology chart;

[0024] Figure 4 MYA-1381 fermentation production of time course DHA yield, glucose concentration and biomass level of change. DETAILED DESCRIPTION

[0025] The present invention is further described in conjunction with the following examples.

[0026] Example 1 Culture of Schizochytrium sp.

[0027] The Aurantiochytrium limacinum ATCC MYA-1381 strain was obtained from the Algal Culture Collection and stored at -80°C. The activated Aurantiochytrium limacinum ATCC MYA-1381 strain was cultured in GPY medium as a seed culture. The GPY liquid medium formula is: 2% glucose, 1% peptone, 0.5% yeast extract, 2% sea salt. Solid medium: 2% glucose, 1% peptone, 0.5% yeast extract, 2% sea salt, 2% agar powder. Sterilized at 115°C for 30 min. In a sterile clean bench, a single colony of Aurantiochytrium limacinum in good growth condition was picked up with a loop and inoculated into a conical flask containing 50 mL of GPY liquid medium, and cultured at 28°C, 150 rpm on a shaker for 24-48 h. Then the strain in good growth condition was passed on as a seed liquid by microscopy to ensure the fresh growth condition of Aurantiochytrium limacinum.

[0028] Example 2 Preparation of alginate lyase

[0029] The high-activity alginate lyase strain Alyw201 (ATCC MYA-2613) was screened out MYA-2613 TMThe engineered yeast strain secreting recombinant alginate lyase Alyw201 of the application was inoculated into YPD medium (3% glucose, 2% peptone, 1% yeast extract) for expansion culture, and cultured at 25°C for 48 h. The cultured strain was then inoculated into a fermenter, and cultured at 25°C for 48 h. The culture solution in the fermenter was centrifuged to obtain the high-efficiency alginate lyase. After concentration, the final activity of the enzyme solution was 8000 U / mL.

[0030] Example 3 Preparation of saccharified liquid of S. maxima

[0031] The S. maxima used in the application was collected from the coastal waters of Rongcheng, Shandong.

[0032] 3.1 The collected material was subjected to drying, grinding and sieving

[0033] Composition analysis showed that the dried material was composed of 8.3% protein, 12.6% cellulose, 27.4% alginate and 6.4% fucoidan. The alginate lyase used in the application was prepared by fermentation of the engineered yeast strain secreting recombinant alginate lyase Alyw201 of the application. After concentration, the final activity of the enzyme solution was 8000 U / mL.

[0034] 3.2 Enzymatic saccharification

[0035] Preparation of enzymatic liquid: The dried S. maxima powder was swelled in distilled water at different ratios of S. maxima powder:distilled water of 1:4 to 1:10 (w / v);

[0036] Then the alginate lyase prepared above and commercial cellulase (Cellic CTec2) were added to the swelled biomass.

[0037] The reaction was carried out at 40°C and pH 5.0 for 6 h with stirring using an Eppendorf thermomixer to prepare the saccharified liquid of S. maxima.

[0038] In order to promote the hydrolysis of S. maxima powder, the amount of alginate lyase added was set at 3% by volume.

[0039] In order to evaluate the effect of supplementing cellulase on the release of glucose and mannitol, Cellic CTec2 was added at different doses of 0.5% to 6%, and the mixture was hydrolyzed under the same conditions.

[0040] By adding excess alginate lyase (3%) to the enzymatic liquid to reduce the viscosity and promote the rapid release of glucose and mannitol, simultaneous saccharification and cell wall polysaccharide destruction was achieved, as shown in Figure 1As shown in Figure A, the levels of glucose and mannitol changed continuously with the change in the solid-liquid ratio. Considering both the glucose and mannitol levels and the yield, insufficient solution volume resulted in inadequate contact between the raw materials and the liquid, poor enzymatic hydrolysis, and incomplete reaction. Excessive liquid volume led to the presence of excess alginate lysin in the solution and also affected the concentration of reducing sugars, which was detrimental to subsequent fermentation. The results showed that a feed-to-liquid ratio of 1:6 (w / v) yielded the best results, with glucose and mannitol reaching 15.38 g / L and 1.45 g / L, respectively.

[0041] Subsequently, the effect of adding cellulase was further investigated based on a 1:6 feed ratio (w / v) and 3% alginate lyase to determine the maximum hydrolysis rate. Glucose yield increased with increasing cellulase dosage, reaching a maximum of 18.36 g / L with the addition of 3% cellulase, while mannitol yield was 1.62 g / L. Figure 1 B). In addition, higher enzyme dosages did not lead to higher glucose production, indicating that cellulose was completely hydrolyzed.

[0042] Example 4: Fermentation culture of Schizochytrium

[0043] The fermentation medium for Schizochytrium MYA-1381 was prepared using copper algae saccharification broth. The seed culture from Example 1 was transferred to the fermentation medium at a 5% inoculum size, and fermentation was carried out in a 10L fermenter. Fermentation was conducted at 28°C, with the pH automatically adjusted to 6.0 by adding 0.1M NaOH. The aeration rate and stirring speed were set to 1.0 vvm and 600 rpm, respectively. Samples were taken every 24 hours for a total of 5 days. Daily changes in biomass (cell dry weight), glucose concentration, DHA level, and the levels of total sugar and uronic acid in the fermentation broth were measured.

[0044] When copper algae saccharification solution was used as the fermentation medium for Schizochytrium, the fungus could grow normally and rapidly. For example... Figure 3 As shown, when Schizochytrium uses the hydrolysate of Columbium as the fermentation medium, lipids continuously accumulate inside the cells, and the lipid droplets are normal in shape and fill the entire cell.

[0045] like Figure 4 As shown, glucose is continuously consumed during the fermentation of Schizochytrium, and is almost completely depleted after 5 days of fermentation. The study found that biomass reached its highest level at 4 days of fermentation, and analysis of its lipid content revealed a DHA concentration of up to 10.82 g / L. The effect of the saccharified broth on biomass and DHA production is a result of the synergistic effect of key plant hormones and carbon sources such as glucose in the saccharified broth.

[0046] Example 5: Screening of key plant hormones in copper algae saccharification solution

[0047] In recent years, plant hormones have been shown to be an effective method for promoting the accumulation of various microalgae carotenoids and polyunsaturated fatty acids. The present application considers that some of the plant hormones present in the copper algae sugar liquor have a certain effect on the accumulation of DHA in Schizochytrium, so the present application focuses on the following 5 plant hormones: abscisic acid (ABA), gibberellin (GA3), indole acetic acid (IAA), indole formaldehyde (ICA), and salicylic acid (SA). First, the experimental group with 5 different plant hormones (1.5 mg / L) and the copper algae sugar liquor fermentation group and the control group were stimulated to culture, and the indicators were determined on the 4th day of fermentation.

[0048] As shown in Figure 2 , the 5 plant hormones can effectively stimulate the growth of Aurantiochytrium limacinum ATCC MYA-1381 strain, but only increasing IAA, ICA and SA can significantly promote the accumulation of polyunsaturated fatty acid DHA. In the culture medium added with IAA, ICA and SA, the DHA yield is 9.53 g / L, 9.34 g / L and 9.46 g / L respectively. The results show that IAA, ICA and SA are the key plant hormones in the copper algae sugar liquor for promoting the accumulation of polyunsaturated fatty acid DHA.

[0049] Example 6 Determination method of various indicators

[0050] Biomass determination: 40 ml of fermentation broth in the fermentation medium was collected in a 50 ml centrifuge tube, centrifuged at 8000 x g for 5 min, the supernatant was discarded, and the cells were washed with distilled water for 2-3 times. The cell mass was placed in an 80°C oven together with the centrifuge tube until the weight was constant, and the increase in the weight of the centrifuge tube was the biomass of the strain.

[0051] Oil extraction: 40 ml of fermentation broth in the fermentation medium was collected in a 50 ml centrifuge tube, centrifuged at 8000 x g for 5 min, the supernatant was discarded, and 8 ml of 50% (v / v) HCl was added to the centrifuge tube, and digested at 80°C for 4 h. Then 16 ml of extraction solution (methanol: chloroform = 1:1) was added, and mixed well by inverting, so as to be fully extracted. Centrifuged at 8000 x g for 5 min, the lower layer was transferred to a new centrifuge tube, and an equal volume of 0.1 M NaCl solution was added, and the upper layer was discarded, and the lower layer was transferred to a flask, and evaporated to dryness at 80°C using a rotary evaporator, and the weight of the produced oil was determined.

[0052] Fatty acid composition analysis: The oil was methyl esterified and then subjected to GC-MS analysis. Instrument model: Agilent 7890A / 5975C Chromatographic column: Agilent HP-INNOWax Polyethylene Glyco (30 m x 250 μm x 0.25 μm). The initial temperature was 100°C, and then the temperature was increased to 240°C at a rate of 15°C / min for 10 min. Each peak was introduced into a mass spectrometer for analysis, and then the mass spectrum was compared in a database to obtain the structure closest to the substance in each peak.

[0053] The glucose index was determined using the DSH colorimetric method.

[0054] The raw materials and equipment used in the present application are conventional raw materials and equipment in the art unless otherwise specified; the methods used in the present application are conventional methods in the art unless otherwise specified.

[0055] The above description is only a preferred embodiment of the present application, and does not limit the present application in any way. Any simple modification, change, and equivalent transformation of the above embodiment based on the technical essence of the present application are still within the protection scope of the technical solution of the present application.

Claims

1. A method for producing DHA in Schizochytrium sp. by fermentation using C. glomerata, characterized by: The method comprises the following steps: using brown alginate lyase and cellulase to enzymatically hydrolyze Chondrus ocellatus to obtain Chondrus ocellatus saccharification liquid, inoculating Schizochytrium for fermentation culture, and achieving fermentation production of DHA in Schizochytrium, wherein the specific process is that Schizochytrium strain is first grown in GPY culture medium, the strain is transferred to a fermenter containing Chondrus ocellatus saccharification liquid as a fermentation medium after being incubated in seed culture for 24-48 h, The GPY culture medium formula is 2% glucose, 1% peptone, 0.5% yeast powder and 2% sea salt, The concentration of the brown alginate lyase and the cellulase is 3%, The inoculation is 3-5% of Schizochytrium seed liquid by volume, The preparation process of the Chondrus ocellatus saccharification liquid comprises the following steps: 1) Swelling dry Chondrus ocellatus powder at a ratio of Chondrus ocellatus powder:distilled water (w / v) of 1:6 (w / v); 2) Then, brown alginate lyase and commercial cellulase are added to the swollen biomass; 3) The reaction is carried out at 40℃ and pH 5.0 for 6 h, and a hot mixer is used for stirring, so as to obtain Chondrus ocellatus saccharification liquid.

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