Mutagenesis method of high-yield DHA schizochytrium limacinum and application of high-yield DHA schizochytrium limacinum

Abstract

The invention discloses a mutagenesis method of high-yield DHA (docosahexaenoic acid) schizochytrium limacinum and application of the high-yield DHA schizochytrium limacinum, the high-yield DHA schizochytrium limacinum is obtained by taking schizochytrium limacinum as an original strain and utilizing ultraviolet mutagenesis, and the high-yield DHA schizochytrium limacinum is schizochytrium limacinum UV1-3. The schizochytrium limacinum mutant strain UV1-3 obtained through ultraviolet mutagenesis has the characteristic of high yield of DHA, the biomass, grease and DHA yield of the schizochytrium limacinum UV1-3 are remarkably improved, salmonella typhimurium can be inhibited, and weaned piglet salmonella infection inflammation can be relieved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a mutagenesis method for high-DHA-yielding schizochytrium and application of the high-DHA-yielding schizochytrium. Background Art

[0002] DHA (docosahexaenoic acid) is one of the polyunsaturated fatty acids that are indispensable in the growth and development of the human body. It has the functions of reducing the risk of cardiovascular diseases, promoting nerve and brain development, and anti-inflammatory and anti-tumor. In the past, DHA was mainly derived from fish oil, but due to the high price of fish oil and the deterioration of the marine ecological environment, there are certain biosafety issues in obtaining DHA from fish oil.

[0003] Scizochytrium sp. is a heterotrophic marine microalgae with fast growth rate, high oil production, and rich in PUFAs, among which DHA can account for more than 50% of the total fatty acids. Industrial production has been achieved. In order to further improve the DHA production performance of Scizochytrium sp., some researchers have increased DHA production by optimizing the culture medium components and culture conditions, but no significant improvement has been achieved.

[0004] Mutation breeding is an important means of improving microalgae. Ultraviolet mutagenesis refers to a mutagenesis method that uses ultraviolet rays to cause changes in DNA structure. By forming thymine dimers between adjacent pyrimidines on the same strand of DNA, the normal pairing between bases is hindered, thereby causing mutation or death of microorganisms. Ultraviolet mutagenesis has the advantages of high mutation rate, simple operation, economical and easy to obtain equipment, and low pollution.

[0005] Due to stress from weaning and environmental factors, weaned piglets are very susceptible to intestinal pathogens, which affects their growth performance and health. Infection of weaned piglets with Salmonella can cause infectious diseases with high mortality rates, with clinical symptoms often characterized by septicemia (acute) and necrotic enteritis (chronic). These symptoms not only seriously affect the growth and health of piglets, but may also cause a large number of piglets to become ill and die, causing huge losses to the pig farming industry. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0007] The mutagenesis method of high-DHA-yielding Schizochytrium of the present invention comprises two rounds of ultraviolet mutagenesis screening, and the DHA yield of mutant strains is detected after culturing Schizochytrium by shake flask fermentation as a screening basis, so as to screen out high-DHA-yielding mutant strains.

[0008] As weaned piglets are susceptible to intestinal pathogens, Schizochytrium, which produces high levels of DHA, was added to their diets. It was found that it can effectively alleviate inflammation caused by Salmonella infection and improve intestinal health.

[0009] The culture medium composition is as follows:

[0010] Solid medium (g / L): glucose 5, yeast extract 1, peptone 1, sea crystal 15, agar powder 20, natural pH.

[0011] Liquid medium (g / L): glucose 5, yeast extract 1, peptone 1, sea crystal 15, pH natural.

[0012] Seed medium (g / L): glucose 30, yeast extract 10, peptone 2, sea crystal 15, pH natural. Fermentation medium (g / L): glucose 80, yeast extract 9.53, peptone 26.27, sea crystal 15, pH 6.0.

[0013] The mutagenesis method uses ultraviolet mutagenesis, and the test steps are as follows:

[0014] (1) Seed solution culture: Pick a loop of activated bacteria from the solid culture medium and inoculate it into a 250-mL conical flask containing 50 mL of autoclaved seed solution. Culture at 25°C and 180 rpm for 24 to 36 h to obtain the seed solution.

[0015] (2) Ultraviolet mutagenesis lethality curve: The bacterial solution cultured to the logarithmic phase is diluted appropriately and inoculated onto a plate, and spread evenly so that there are about 100 single colonies on each plate. The coated plates are irradiated with a 15W ultraviolet lamp at a distance of 20 cm for 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 seconds, respectively. After ultraviolet irradiation, they are placed in a 25°C incubator for 3 to 4 days. Be careful to avoid light to prevent photorepair. After the colonies grow, count the surviving colonies. Draw a lethality curve with the irradiation time as the horizontal axis and the lethality as the vertical axis, and perform mutagenesis breeding with the treatment time when the lethality is about 90%. The lethality is calculated according to the following formula:

[0016] Lethality rate = (number of colonies in the control group - number of colonies in the induced group) / number of colonies in the control group × 100%

[0017] (3) UV mutagenesis screening: The starting strain Schizochytrium sp. S31 was cultured for 24 h. After appropriate dilution, 100 μL of the bacterial solution was evenly spread on the plate. After the UV lamp was turned on and stabilized for 20 min, the coated plate was removed from the lid and irradiated with a 15W UV lamp at a distance of 20 cm for 50 s. The lid was quickly replaced, the plate was taken out, and moved to an incubator in the dark to culture for 3 to 4 days away from light.

[0018] (4) After the colonies grow, single colonies are picked for shake flask fermentation experiments (fermentation temperature is 25°C, rotation speed is 180 rpm, and total fermentation time is 96 hours). After the fermentation is completed, the biomass, oil content, and DHA production are detected. The mutant strains with high DHA production are selected and the second round of mutagenesis is carried out to screen out the high-yield DHA mutant strain UV1-3.

[0019] Detection of biomass during the screening process of mutagenesis of Schizochytrium high-yielding DHA:

[0020] Dry weight method: Take 2 mL of fermentation broth into a weighed centrifuge tube (recorded as W 离心管 ), centrifuged at 12000rpm for 20min, discarded the supernatant, added an appropriate amount of triple distilled water to wash the precipitate, centrifuged at 12000rpm for 20min, discarded the supernatant, and repeated washing twice to obtain wet cells (denoted as W 湿菌体 ) was dried in a forced air drying oven to constant weight. The calculation formula for biomass (Dry cell weight, DCW) is: DCW (g / L) = (W 离心管 -W 湿菌体 )×500.

[0021] Detection of oil content during the screening process of mutagenesis method for high-yield DHA Schizochytrium:

[0022] Drawing of standard curve of oil content: Take 5 10mL stoppered test tubes, add 2mg / mL oil n-hexane solution (blank control adds 0.2mL triple distilled water) and 98% concentrated sulfuric acid in the following volume, shake and mix, place in a 90℃ water bath for 20min, let stand and cool for 15min, add 3mL phosphate vanillin solution to the test tube, shake and mix, let stand and react for 30min, and detect the absorbance of the sample at 530nm with an enzyme marker, and use A 530nm Draw a standard curve with oil content as the horizontal axis and fat content as the vertical axis.

[0023]

[0024] Detection of oil content in fermentation broth: Take 1mL of fermentation broth and place it in a 1.5mL centrifuge, centrifuge at 12000rpm for 20min, discard the supernatant, add an appropriate amount of triple distilled water to wash the precipitate, centrifuge at 12000rpm for 20min, discard the supernatant, repeat the washing twice, add triple distilled water to the 1mL mark, blow and mix to obtain the bacterial solution. Take 0.2mL of bacterial solution and place it in a 10mL stoppered test tube, add 2mL of 98% concentrated sulfuric acid, shake and mix, place in a 90℃ water bath for 20min, let it stand and cool for 15min, add 3mL of phosphate vanillin solution to the test tube, shake and mix, let it stand for 30min, and use an enzyme marker to detect the absorbance of the sample at 530nm, and calculate the oil content in the sample according to the standard curve.

[0025] Detection of DHA content during the screening of high-DHA-producing Schizochytrium mutagenesis methods:

[0026] (1) Take 1 mL of fermentation broth and 1.5 mL of centrifuge, centrifuge at 12000 rpm for 20 min, discard the supernatant, add an appropriate amount of triple distilled water to wash the precipitate, centrifuge at 12000 rpm for 20 min, discard the supernatant, and repeat the washing twice; (2) Transfer the bacterial precipitate to a 15 mL stoppered test tube, add 2 mL of 0.5 mol / L potassium hydroxide-methanol solution, mix by pipetting, and place in a 60°C water bath for 30 min, shaking and mixing 3 times during the process to fully break the cell wall; (3) After cooling to room temperature, add 14% boron trifluoride methanol solution in a volume of 2 times that of sodium hydroxide-methanol, shake and mix, and place in a 60°C water bath for 30 min, shaking during the process (4) After cooling to room temperature, add 2 mL of GC-grade n-hexane and shake vigorously to mix; (5) Add 4 mL of saturated sodium chloride solution, shake and mix, and let stand to wait for stratification; (6) Pipette the upper layer into a new 2 mL centrifuge tube; (7) Add n-hexane and extract repeatedly until the upper organic phase is colorless; (8) Add a small amount of anhydrous sodium sulfate to the DHA n-hexane solution to remove water, and filter the solution to remove impurities; (9) Dilute the sample to an appropriate multiple, add methyl ester standard to make the final concentration of 1 mg / mL, place it in a gas phase vial, use gas chromatography to detect the DHA content, and calculate the DHA content in the fermentation broth.

[0027] The gas chromatograph model used in the present invention is Shimadzu gas chromatograph GC-2010-plus, and the gas chromatography conditions are as follows: (1) chromatographic column: capillary column Ag.lent 112-88A7 HP-88: 30m×250μm×0.20μm; (2) detector: hydrogen flame ionization FID detector; (3) column box temperature program setting: initial temperature is 100°C, retained for 1min, heated to 200°C at a rate of 25°C / min, then heated to 280°C at a rate of 8°C / min and maintained for 5min, injection port temperature is 280°C, detector temperature is 300°C; (4) combustion gas path is hydrogen and air, nitrogen is used as carrier gas, and injection volume is 1μL. Nonadecanoic acid methyl ester is used as internal standard, and DHA in the sample is qualitatively analyzed by comparing with the retention time and peak area of ​​DHA methyl ester standard, and the DHA content can be calculated.

[0028] The present invention publicly deposits the high-yield DHA mutant strain UV1-3 obtained by mutagenesis in the Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No: 6.009, a classification name of Schizochytrium sp., and a deposit date of September 11, 2024.

[0029] The invention also adds the high-DHA-yielding Schizochytrium mutant UV1-3 to the weaned pig feed after fermentation to inhibit the inflammation of the weaned pig infected with Salmonella.

[0030] Beneficial effects of the present invention: The Schizochytrium mutant strain UV1-3 obtained by ultraviolet mutagenesis has the characteristic of high DHA production, and the biomass, oil and DHA production of Schizochytrium UV1-3 are significantly improved, and can inhibit Salmonella typhimurium (S. typhimurium) and alleviate the inflammation of Salmonella infection in weaned piglets. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following briefly introduces the drawings required for describing the embodiment, wherein:

[0032] Figure 1 This is the mortality curve of Schizochytrium under UV irradiation in Example 1 of the present invention;

[0033] Figure 2 is the relative lipid content of the mutant strain obtained by UV mutagenesis in Example 2 of the present invention;

[0034] Figure 3 is the relative DHA yield of the mutant strain obtained by UV mutagenesis in Example 2 of the present invention;

[0035] Figure 4 is the relative lipid content of the mutant strain obtained by the second round of UV mutagenesis in Example 3 of the present invention;

[0036] Figure 5 is the relative DHA production of the mutant strain obtained by the second round of UV mutagenesis in Example 3 of the present invention;

[0037] Figure 6 The biomass, total oil content, and DHA yield of the mutant strains UV1-1 and UV1-3 of Example 4 of the present invention after five passages;

[0038] Figure 7 The biomass, oil, and DHA yields obtained by fermentation of mutant strains UV1-3 and WT in Example 5 of the present invention;

[0039] Figure 8 This is the metabolic map of differentially expressed genes related to fatty acid metabolism between WT and UV1-3 in Example 5 of the present invention; Fig. 9 The effect of UV1-3 on the colon morphology of weaned piglets treated with Salmonella;

[0040] Fig.10 The effect of UV1-3 on the expression of tight junction genes in the colon of weaned piglets treated with Salmonella;

[0041] Fig.11Effects of UV1-3 on gene expression of colon inflammatory factors in weaned piglets treated with Salmonella after Salmonella infection. DETAILED DESCRIPTION

[0042] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0043] Embodiment 1: determination of ultraviolet mutagenesis method

[0044] The starting strain Schizochytrium sp.S31 was cultured for 24 hours. After appropriate dilution, 100 μL of bacterial solution was evenly spread on the plate (so that there were about 100 bacteria on the plate). After the UV lamp was turned on and stabilized for 20 minutes in advance, the coated plate was removed from the lid and irradiated with a 15W UV lamp at a distance of 20 cm for 50 seconds. The lid was quickly covered, the plate was taken out, and moved into the incubator in the dark to culture for 3 to 4 days away from light. After the colony grew, a larger single colony was picked for a shake flask fermentation experiment. After the fermentation was completed, the biomass, oil content, and DHA production were tested.

[0045] Test results: Figure 1 This is the lethality curve of Schizochytrium under UV irradiation. The mutation rate increases with the extension of UV irradiation time. Within 0-40s, the lethality increases rapidly with the extension of treatment time; after 40s, the lethality increases slowly with the extension of treatment time; when the treatment time increases to 60s, the lethality is close to 100%. When the lethality is between 85% and 95%, a higher positive mutation rate can be obtained. When the UV treatment is 50s, the lethality is 87.63%, close to 90%, so the UV irradiation time of 50s is selected as the mutagenesis time.

[0046] Example 2: First round of UV mutagenesis to screen for DHA high-yielding mutants

[0047] The original strain Schizochytrium sp.S31 was subjected to ultraviolet mutagenesis to obtain 24 mutants, which were named UV1 to UV24. The mutants and the original strain were fermented to obtain the biomass, oil content and DHA yield.

[0048] Detection: Among the 24 mutants obtained, 13 were positive mutations and 11 were negative mutations. Figure 2 The relative oil content of the mutants obtained by ultraviolet mutagenesis. The fermentation oil content of the 9 mutants UV1, UV2, UV3, UV6, UV7, UV8, UV9, UV11, and UV12 is more than 30% higher than that of the wild strain, and the fermentation oil content of the 4 mutants UV1, UV7, UV9, and UV11 is more than 50% higher than that of the wild strain. Figure 3The relative DHA yields of the mutants obtained by ultraviolet mutagenesis are: UV1, UV3, UV9, and UV11. The fermentation DHA yields of these four mutants are more than 30% higher than that of the wild strain, and the fermentation DHA yield of UV1 is more than 50% higher than that of the wild strain.

[0049] Example 3: Second round of UV mutagenesis to screen for DHA high-yielding mutants

[0050] The mutant strains UV1, UV3, UV9, and UV11 were subjected to a second UV mutagenesis, and the larger colonies that grew were selected for shake flask fermentation experiments.

[0051] Detection: Figure 4 is the relative oil content of the mutants obtained by the second round of UV mutagenesis. After the second round of UV mutagenesis, only the total oil content of UV1-1 showed an upward trend compared with its starting strain UV1, while the total oil content of other mutants was lower than their respective starting strains. Figure 5 The relative DHA production of the mutants obtained by the second round of UV mutagenesis. After the second round of UV mutagenesis, only UV1-1, UV1-2, and UV1-3 had significantly higher DHA production than their starting strain UV1, while the DHA production of other mutants was lower than their respective starting strains.

[0052] Example 4: Genetic stability analysis

[0053] The mutant strains UV1-1 and UV1-3 were subcultured for five times, and shake flask fermentation experiments were carried out on them to detect their biomass, oil content and DHA production.

[0054] Detection: Figure 6 The biomass, total oil content, and DHA production of mutant strains UV1-1 and UV1-3 after five generations. Figure 6 -a) Grease quantity ( Figure 6 -b) and DHA production ( Figure 6 -c) was steadily higher than that of the starting strain, and the DHA production did not show a significant decrease with the increase in the number of generations, showing good genetic stability; the DHA production of the mutant strain UV1-1 decreased since the second generation and was stable from the 2nd to the 5th generation, but the production was not as good as that of UV1-3.

[0055] Example 5: Comparison of fermentation performance between UV1-3 and Schizochytrium sp. S31

[0056] The wild strain and UV1-3 were subjected to shake flask fermentation experiments to compare the fermentation characteristics of the two strains. After 96 hours of fermentation, samples were collected and the biomass, total oil content, and DHA production of the wild strain and UV1-3 were detected, and the oil content (% of DCW) and DHA content (% of DCW) were calculated.

[0057] Detection: Figure 7 The biomass, oil and DHA yields obtained by fermentation of mutant strain UV1-3 and wild strain. The biomass (P<0.05), total oil content and its proportion (P<0.001), and DHA yield and its proportion (P<0.001) of UV1-3 were significantly higher than those of the wild strain, which were 31g / L, 28.08%, 14.50%, 8.71g / L, and 4.49g / L, respectively, which were 8.14%, 19.12%, 20.84%, 28.82%, and 30.56% higher than those of the wild strain.

[0058] The fatty acid composition of the wild strain and UV1-3 was detected and analyzed respectively.

[0059] Test results: Compared with the wild strain, the contents of pentadecanoic acid (C15:0), palmitic acid (C16:0), and heptadecanoic acid (C17:0) in UV1-3 were significantly decreased (P<0.001), and the content of DHA was significantly increased (P<0.01), exceeding that of the wild strain by 13.00%, which was consistent with the previous fermentation results.

[0060] The fatty acid composition of the wild strain and mutant UV1-3 is shown in Table 1:

[0061] Table 1

[0062]

[0063]

[0064] Example 6: Transcriptome analysis

[0065] The WT and UV1-3 strains were cultured for 60 hours according to the fermentation method, and 2 mL of fermentation broth was taken respectively, centrifuged at 4 ° C and 12000 rpm for 10 minutes, the supernatant was discarded, and washed three times with PBS solution to obtain bacterial precipitates, which were immediately placed in liquid nitrogen for quick freezing for more than 30 minutes and moved to a -80 ° C refrigerator for standby use. The total RNA of Schizochytrium was extracted using the UNIQ-10 column Trizol kit. The extracted total RNA was incubated with DNaseI at 37 ° C for 30 minutes to remove the genomic DNA.

[0066] RNA quality analysis, library construction, and sequencing were completed by Shanghai Meiji Biotechnology Co., Ltd.

[0067] Detection: Pathway significant enrichment analysis was performed on the differentially expressed genes between UV1-3 and S31. Through GO enrichment analysis of genes in fatty acid metabolism-related pathways, a total of 12 genes were enriched, among which the transcription of genes related to lipid metabolism, cellular oxidoreductase activity, and lipid decomposition metabolism showed significant changes; KEGG enrichment analysis results showed that genes involved in fatty acid degradation, pyruvate metabolism, unsaturated fatty acid biosynthesis, fatty acid biosynthesis, fatty acid elongation, etc. had significant differences. The above differentially expressed genes were made into differentially expressed gene metabolism maps. Figure 8 The following is a metabolic map of differentially expressed genes related to fatty acid metabolism between WT and UV1-3. Compared with the starting strain, the transcription levels of long-chain acyl-CoA synthetase (ACSL), long-chain acyl-CoA synthetase (SLC27A2), and enoyl-ACP reductase (FabI) ​​in UV1-3 were significantly downregulated, and the transcription level of acetyl-CoA C-acetyltransferase (ACAT) was significantly upregulated, the ability to synthesize FAS products decreased, the utilization of acetyl CoA and NADPH was reduced, and the production of acetyl CoA increased, providing more substrates for the synthesis of DHA by the PKS pathway; the transcription levels of alkB and Thioesterase superfamily member 4 (THEM4) were significantly increased, and the synthesis of long-chain unsaturated fatty acids increased, which was consistent with the increase in DHA production; cytochrome oxidase (Cytochrome The transcription levels of Cox subunits Cox2, Cox3, and CoxA were significantly downregulated, and the bacteria tended to synthesize DHA using the PKS pathway that does not require oxygen, and weakened the synthesis of products in the direction of FAS that requires oxygen. The transcription levels of histidine ammonia-lyase (HAL) and proC were significantly downregulated, the generation of α-ketoglutarate by amino acid cleavage was reduced, and the TCA cycle was weakened, which was conducive to the metabolism towards fatty acid synthesis.

[0068] Example 7: Effect of Schizochytrium on the intestinal health of weaned piglets infected with Salmonella (Salmonella typhimurium SL1344 strain)

[0069] Materials and methods:

[0070] Animal husbandry and treatment: 21 28-day-old weaned piglets of three-way crossbreed (Durham × Long × Da) were purchased from a pig farm in Jiangsu Province and raised on the farm. The room temperature was maintained at 25±1℃ during the experiment. Water and food were not limited. The formal experiment was carried out after 7 days of adaptation. The entire experimental period was 28 days.

[0071] Twenty-one piglets were randomly divided into three groups: control group (CON), Salmonella treatment group (ST), and Salmonella + Schizochytrium group (UV1-3). The feeding conditions were as follows: control group (CON): fed with a basic diet; Salmonella treatment group (ST): fed with a basic diet; Salmonella + Schizochytrium group (UV1-3): the basic diet was supplemented with 1% (w / w) mutant strain UV1-3 fermentation freeze-dried powder (containing about 3x10 10 CFU / g), and after 21 days of feeding, 10 mL 10 9 CFU / mL of Salmonella typhimurium SL 1344, and the control group was fed with the same amount of culture medium. Sampling was carried out on the 28th day of the experiment after 7 days of treatment. The piglets were fasted for 12 hours before sampling. The composition of the diet in this experiment is shown in Table 2.

[0072] Table 2

[0073]

[0074] Each kilogram of aPermix contains: Vitamin A: 12000IU, Vitamin D3: 3000IU, Vitamin E: 3000IU, Vitamin K3: 6mg, Vitamin B2: 10mg, Vitamin B6: 5mg, Vitamin B12: 0.5mg, Niacin: 70mg, Pantothenic Acid: 550mg, Folic Acid: 40mg, Biotin: 5mg, Choline: 30mg, Manganese: 40mg, Iron: 200mg, Zinc: 300mg, Iodine: 2mg, Copper: 350mg, Selenium: 10mg.

[0075] On the 28th day of the experiment, blood was collected in several 1.5 mL centrifuge tubes soaked with sodium heparin, centrifuged at 3000 rpm for 15 min, and the upper plasma was aspirated into a new centrifuge tube and stored in a -20°C refrigerator for testing.

[0076] After blood collection, the piglets were killed, and the colon of each piglet was cut off at the same position. The colon contents were gently washed with sterile saline, and the intestinal mucosa was gently scraped off with a sterile glass slide, placed in a 2mL centrifuge tube, quickly transferred to liquid nitrogen, and then transferred to a -80℃ refrigerator for storage until examination. Another section of the colon at the same position was cut off and fixed in 4% paraformaldehyde as a tissue sample for examination.

[0077] Experimental results: After the Salmonella treatment group, the concentrations of proinflammatory cytokines IL-6 (P<0.05) and IL-1β (P<0.01) in the piglets' plasma increased significantly. Adding 1% Schizochytrium to the diet significantly reduced the increased levels of plasma IL-6 and IL-1β caused by Salmonella infection (P<0.01).

[0078] CON ST UV1-3 P-value IL-1 (U / L) 26.93±0.29 <![CDATA[35.04±0.99 ＊＊ ]]> <![CDATA[27.66±2.61 ## ]]> 0.01 IL-6 (U / L) 596.18±35.75 <![CDATA[758.91±58.63 ＊ ]]> <![CDATA[553.22±38.61 ## ]]> 0.02

[0079] Data are expressed as mean ± standard error; *: compared with CON group, #: compared with ST group; *P<0.05, **P<0.01, n=7

[0080] The H&E stained sections of the colon of weaned piglets were obtained and photographed under a microscope. Fig. 9 As shown in the figure, the number of goblet cells in the colon of the Salmonella-treated group decreased, the infiltration of inflammatory cells increased, some villi were broken, and the intestinal mucosa was damaged. Adding 1% Schizochytrium to the diet increased the number of goblet cells in the colon, reduced the infiltration of inflammatory cells, and improved the morphology and integrity of the intestinal mucosa.

[0081] The colon tight junction protein-related genes were tested, and the results were as follows Fig.10 The transcription levels of tight junction protein genes Occludin (P<0.05), Claudin2 (P<0.001), and Claudin3 (P<0.01) in the colon of the Salmonella-treated group were significantly reduced, and the addition of 1% Schizochytrium to the diet significantly increased the transcription levels of Occludin (P<0.05), Claudin1 (P<0.01), and Claudin2 (P<0.05) in the colon of piglets, improving the colon barrier structure and function.

[0082] The results of the detection of genes related to colon inflammatory factors are as follows Fig.11 As shown. The transcription levels of NLRP3, MCP-7, COX2 (P<0.05), IL-1β, IL-6, IL-7α (P<0.01), iNOS, TNNF-α (P<0.001) in the colon of the Salmonella treatment group were significantly increased; the addition of 1% Schizochytrium to the diet significantly reduced the transcription levels of IL-7β, iNOS, COX2 (P<0.05), and NF-κB (P<0.01) in the colon of piglets, which can significantly alleviate the increased expression of inflammatory factors caused by Salmonella infection.

[0083] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for mutagenesis of high-DHA-producing Scizochytrium sp., characterized in that: Taking Schizochytrium as the starting strain, ultraviolet mutagenesis was used to obtain a high-DHA Schizochytrium, and the high-DHA Schizochytrium was Schizochytrium UV1-3.

2. The method according to claim 1, characterized in that: The starting strain is Schizochytrium sp.S31, and its deposit number is ATCC 20888.

3. The method according to claim 1 or 2, characterized in that: The ultraviolet power of the ultraviolet mutagenesis is 15-20w, and the irradiation time is 50-80s.

4. The method according to claim 3, characterized in that: The ultraviolet mutagenesis includes subjecting the high-yield DHA mutant strain screened after the first ultraviolet mutagenesis to a second ultraviolet mutagenesis.

5. An application of high-DHA Schizochytrium in the production of DHA, characterized in that: The high-DHA-producing Schizochytrium is Schizochytrium UV1-3.

6. The use according to claim 5, characterized in that: The production of DHA comprises fermentation and cultivation of Schizochytrium UV1-3, the fermentation temperature is 25-30° C., and the fermentation time is 96-120 hours.

7. Use of the high-DHA-producing Schizochytrium according to claim 5 in inhibiting Salmonella.

8. Use of the high-DHA-producing Schizochytrium according to claim 5 in the preparation of a bacterial agent for treating colitis caused by Salmonella.

9. Use of the high-DHA-producing Schizochytrium according to claim 5 in the preparation of a feed additive for treating colitis in poultry and livestock caused by Salmonella.

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