Method for improving yield and / or stress resistance of Antarctic diatom polysaccharide

Abstract

The invention belongs to the technical field of plant culture, and particularly relates to a method for improving the yield and / or stress resistance of Antarctic diatom polysaccharide. According to the present invention, it is found that one or more of Alteromonas profundi 1K04570, Pseudoalteromonas atlantica 1C00251, Marinobacter denitrificans 1K05758 and Polaribacter marinaquae 1K00696 are co-cultured with the Antarctic diatom, such that the metabolic pathway of the Antarctic diatom can be activated, the Antarctic diatom can be efficiently stimulated to synthesize the polysaccharide, and the stress resistance of the Antarctic diatom can be synchronously improved; furthermore, by optimizing the inoculation proportion and co-culture conditions, the yield and stress resistance of the Antarctic diatom can be further improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant culture, and particularly relates to a method for improving the yield and / or stress resistance of Antarctic diatom polysaccharide. BACKGROUND

[0002] Antarctic diatom (Fragilariaceae) Phaeodactylum tricornutum ) lives in extreme environments, and its metabolic products (such as antifreeze protein, special polysaccharide, and polyunsaturated fatty acid) have unique structures and great application potential (such as medicine, cosmetics, and food additives). However, its industrialization faces two bottlenecks: (1) low polysaccharide yield in pure culture; and (2) poor stress resistance: Antarctic diatom is sensitive to large-scale industrial culture environment (such as temperature fluctuation, high light intensity, change of nutrient salt, and shear force), which easily leads to culture collapse and high production cost. The existing technologies are mostly focused on optimizing the culture medium or culture conditions (physical and chemical methods) or genetically engineering the algae itself, but the effect is limited and may cause safety supervision problems. SUMMARY

[0003] The purpose of the present application is to make up for the deficiencies of the prior art, provide a method for improving the yield and / or stress resistance of Antarctic diatom polysaccharide, improve the yield of Antarctic diatom polysaccharide, optimize the molecular structure of Antarctic diatom polysaccharide, improve the stress resistance of Antarctic diatom, and meet the requirements of commercial production.

[0004] The present application provides the application of microorganisms in improving the yield and / or stress resistance of Antarctic diatom polysaccharide, wherein the microorganisms include one or more of Alternomonas profunda Alteromonas profundi 1K04570, Pseudoalteromonas atlantica Pseudoalteromonas atlantica 1C00251, and Thalassospira sp. Marinobacter denitrificans 1K05758 and Thalassospira sp. Polaribacter marinaquae 1K00696.

[0005] The present application provides a compound microbial agent, which includes independent component A and independent component B, wherein the independent component A includes Alternomonas profunda Alteromonas profundi 1K04570. The independent component B includes Pseudoalteromonas atlantica Pseudoalteromonas atlantica 1C00251.

[0006] Preferably, the cell number ratio of Alternomonas profunda Alteromonas profundi 1K04570 and Pseudoalteromonas atlantica Pseudoalteromonas atlantica 1C00251 in the compound microbial agent is 3:5.

[0007] The present application provides the application of the compound microbial agent in improving the yield and / or stress resistance of Antarctic diatom polysaccharide.

[0008] Preferably, the stress resistance comprises antioxidant performance.

[0009] The application provides a method for improving the yield and / or stress resistance of Antarctic silicate polysaccharide, comprising co-culturing microorganisms and Antarctic silicate polysaccharide. The microorganism is the compound microbial agent described in the above technical solution, or is Alteromonas macleodii Alteromonas profundi 1K04570, Pseudoalteromonas atlantica Pseudoalteromonas atlantica 1C00251, Thalassospira denitrificans Marinobacter denitrificans 1K05758 and Thalassospira saliha Polaribacter marinaquae 1K00696.

[0010] Preferably, the cell inoculation ratio of the microorganism and Antarctic silicate polysaccharide is (1-2): (1-1000); The co-culturing temperature is 2-15°C, the light intensity is 50-500 μmol / m 2 / s, the time is 12-18 d, the light cycle is 8-16 h light and 16-8 h dark.

[0011] Preferably, when the microorganism is the compound microbial agent described in the above technical solution, the cell inoculation ratio of the microorganism and Antarctic silicate polysaccharide is 4:250; When the microorganism is Alteromonas macleodii Alteromonas profundi 1K04570, the cell inoculation ratio of the microorganism and Antarctic silicate polysaccharide is 1:800; When the microorganism is Pseudoalteromonas atlantica Pseudoalteromonas atlantica 1C00251, the cell inoculation ratio of the microorganism and Antarctic silicate polysaccharide is 1:150; When the microorganism is Thalassospira denitrificans Marinobacter denitrificans 1K05758, the cell inoculation ratio of the microorganism and Antarctic silicate polysaccharide is 1:50; When the microorganism is Thalassospira saliha Polaribacter marinaquae 1K00696, the cell inoculation ratio of the microorganism and Antarctic silicate polysaccharide is 2:1.

[0012] Preferably, when the microorganism is the compound microbial agent described in the above technical solution, the co-culturing temperature is 7°C, the light intensity is 220 μmol / m 2 / s, the time is 16 d, the light cycle is 14 h light and 10 h dark; When the microorganism is Alteromonas macleodii Alteromonas profundi 1K04570, the co-culturing temperature is 4°C, the light intensity is 60 μmol / m2 / s, time is 14 d, light period is 8 h light, 16 h dark; when the microorganism is Pseudoalteromonas atlantica Pseudoalteromonas atlantica 1C00251, the temperature of co-culture is 8℃, the light intensity is 180 μmol / m 2 / s, time is 18 d, light period is 12 h light, 12 h dark; when the microorganism is Marinobacter denitrificans Marinobacter denitrificans 1K05758, the temperature of co-culture is 14℃, the light intensity is 450 μmol / m 2 / s, time is 12 d, light period is 16 h light, 8 h dark; when the microorganism is Marinobacter oceanosedimenticolus Polaribacter marinaquae 1K00696, the temperature of co-culture is 6℃, the light intensity is 90 μmol / m 2 / s, time is 16 d, light period is 10 h light, 14 h dark.

[0013] Preferably, when the microorganism is the compound microbial agent in the above technical solution, Pseudoalteromonas atlantica Pseudoalteromonas atlantica 1C00251 and microalgae are inoculated in the algal culture medium to co-culture, and Pseudoalteromonas profundus Alteromonas profundi 1K04570 is inoculated after 4 d to continue co-culture; when the microorganism is Pseudoalteromonas profundus Alteromonas profundi 1K04570, Pseudoalteromonas atlantica Pseudoalteromonas atlantica 1C00251, Marinobacter denitrificans Marinobacter denitrificans 1K05758 or Marinobacter oceanosedimenticolus Polaribacter marinaquae 1K00696, the microorganism and the microalgae are inoculated in the algal culture medium to co-culture.

[0014] Beneficial effects: The present application is based on ecological interaction, and finds that one or more of Pseudoalteromonas profundus Alteromonas profundi 1K04570, Pseudoalteromonas atlantica Pseudoalteromonas atlantica 1C00251, Marinobacter denitrificans Marinobacter denitrificans 1K05758 and Marinobacter oceanosedimenticolus Polaribacter marinaquae 1K00696 are co-cultured with Antarctic diatoms, which can activate the metabolic pathway of Antarctic diatoms, efficiently stimulate the synthesis of polysaccharides of Antarctic diatoms, and synchronously improve the stress resistance of Antarctic diatoms. Further, the present application can further improve the yield and stress resistance of Antarctic diatoms by optimizing the inoculation ratio and co-culture conditions. DETAILED DESCRIPTION

[0015] The present application provides the use of microorganisms in improving the yield and / or stress resistance of Antarctic diatom silicate, wherein the microorganisms include Alternomonas profunda Alteromonas profundi 1K04570, Pseudoalteromonas atlantica Pseudoalteromonas atlantica 1C00251, Thalassospira denitrificans Marinobacter denitrificans 1K05758 and Thalassospira halifaxensis Polaribacter marinaquae 1K00696.

[0016] The microorganisms described in the present application are all preserved in the Marine Culture Collection of China (MCCC). The present application provides a compound microbial agent, which comprises independent component A and independent component B, wherein the independent component A comprises Alternomonas profunda Alteromonas profundi 1K04570; and the independent component B comprises Pseudoalteromonas atlantica Pseudoalteromonas atlantica 1C00251.

[0017] As an embodiment, the compound microbial agent described in the present application comprises Alternomonas profunda Alteromonas profundi 1K04570 and Pseudoalteromonas atlantica Pseudoalteromonas atlantica 1C00251 at a cell number ratio of 3:5. The present application compounding Alternomonas profunda Alteromonas profundi 1K04570 and Pseudoalteromonas atlantica Pseudoalteromonas atlantica 1C00251 at a certain ratio, Alternomonas profunda Alteromonas profundi 1K04570 and Pseudoalteromonas atlantica Pseudoalteromonas atlantica Alteromonas profundi 1C00251 synergistically, further improving the yield and stress resistance of Antarctic diatom silicate.

[0018] The present application provides the use of the compound microbial agent described in the above technical solution in improving the yield and / or stress resistance of Antarctic diatom silicate.

[0019] As an embodiment, the Antarctic diatom described in the present application comprises Antarctic diatom. As an embodiment, the stress resistance described in the present application comprises antioxidant performance. As an embodiment, the antioxidant performance described in the present application comprises DPPH clearance ability The present application provides a method for improving the yield and / or stress resistance of Antarctic diatom silicate, which comprises co-culturing microorganisms and Antarctic diatom silicate. The microorganisms are the compound microbial agent described in the above technical solution, or Alternomonas profunda Pseudoalteromonas atlantica 1K04570, Pseudoalteromonas atlantica Marinobacter denitrificans Polaribacter marinaquae 1C00251, Thalassospira denitrificansAlteromonas profundi 1K05758 and Psychrobacter maritimus Pseudoalteromonas atlantica 1K00696.

[0020] As an embodiment, the ratio of the inoculation amount of the microorganism and the Antarctic diatom is (1-2) : (1-1000); as another embodiment, the ratio of the inoculation amount of the microorganism and the Antarctic diatom is 2:1 or 1:(50-800); as another embodiment, the ratio of the inoculation amount of the microorganism and the Antarctic diatom is 1:(62.5-150).

[0021] As an embodiment, the temperature of the co-culture is 2-15℃; as another embodiment, the temperature of the co-culture is 4-14℃; as another embodiment, the temperature of the co-culture is 6-18℃. As an embodiment, the light intensity of the co-culture is 50-500 μmol / m 2 2; as another embodiment, the light intensity of the co-culture is 60-450 μmol / m 2 2; as another embodiment, the light intensity of the co-culture is 90-180 μmol / m 2 2. As an embodiment, the time of the co-culture is 12-18 d; as another embodiment, the time of the co-culture is 14-16 d. As an embodiment, the light cycle of the co-culture is 8-16 h light, 16-8 h dark; as another embodiment, the light cycle of the co-culture is 10-12 h light, 14-12 h dark.

[0022] As an embodiment, when the microorganism is the compound microbial agent described in the above technical solution, the ratio of the inoculation amount of the microorganism and the Antarctic diatom is 4:250. As an embodiment, when the microorganism is Alteromonas piechaudii Marinobacter denitrificans 1K04570, the ratio of the inoculation amount of the microorganism and the Antarctic diatom is 1:800. As an embodiment, when the microorganism is Pseudoalteromonas atlantica Polaribacter marinaquae Alteromonas profundi 1C00251, the ratio of the inoculation amount of the microorganism and the Antarctic diatom is 1:150. As an embodiment, when the microorganism is Thalassospira denitrificans Pseudoalteromonas atlantica 1K05758, the ratio of the inoculation amount of the microorganism and the Antarctic diatom is 1:50. As an embodiment, when the microorganism is Psychrobacter maritimus Marinobacter denitrificans 1K00696, the ratio of the inoculation amount of the microorganism and the Antarctic diatom is 2:1.

[0023] As an implementation form, when the microorganism is the compound microbial agent in the technical solution, the temperature of the co-culture is 7℃, the light intensity is 220 μmol / m 2 / s, the time is 16 d, the light period is 14 h light, and 10 h darkness. As an implementation form, when the microorganism is Alternomonas litoralis Polaribacter marinaquae 1K04570, the temperature of the co-culture is 4℃, the light intensity is 60 μmol / m 2 / s, the time is 14 d, the light period is 8 h light, and 16 h darkness. As an implementation form, when the microorganism is Pseudoalteromonas atlantica Pseudoalteromonas atlantica 1C00251, the temperature of the co-culture is 8℃, the light intensity is 180 μmol / m 2 / s, the time is 18 d, the light period is 12 h light, and 12 h darkness. As an implementation form, when the microorganism is Thalassospira denitrificans Alteromonas profundi Alteromonas profundi 1K05758, the temperature of the co-culture is 14℃, the light intensity is 450 μmol / m 2 / s, the time is 12 d, the light period is 16 h light, and 8 h darkness. As an implementation form, when the microorganism is Thalassospira arctica Pseudoalteromonas atlantica 1K00696, the temperature of the co-culture is 6℃, the light intensity is 90 μmol / m 2 / s, the time is 16 d, the light period is 10 h light, and 14 h darkness.

[0024] As an implementation form, when the microorganism is the compound microbial agent in the technical solution, Pseudoalteromonas atlantica Marinobacter denitrificans 1C00251 and the microalgae are inoculated in the algal culture medium at the same time to perform co-culture, and Alternomonas profunda Polaribacter marinaquae 1K04570 is inoculated after 4 d to continue the co-culture. As an implementation form, when the microorganism is Alternomonas profunda Alteromonas profundi 1K04570, Pseudoalteromonas atlantica Pseudoalteromonas atlantica 1C00251, Thalassospira denitrificans Marinobacter denitrificans Polaribacter marinaquae 1K05758, or Thalassospira arctica Pseudoalteromonas atlantica 1K00696, the microorganism and the microalgae are inoculated in the algal culture medium at the same time to perform co-culture.

[0025] In order to further illustrate the present application, the method for improving the yield of Antarctic siliceous polysaccharide and / or stress resistance provided by the present application is described in detail below in combination with examples, but they cannot be understood as limitations to the protection scope of the present application.

[0026] Example 1 Deep-source alternitoplasmosis Alteromonas profundi 1K04570 and Antarctic diatom (CCMP2561, deposited at the National Collection of Marine Algae and Microorganisms in the United States) were co-cultured in algal culture medium (F2 medium) at a cell ratio of 1:150 for 14 days at a temperature of 8°C and a light intensity of 180 μmol / m². 2 / s, the photoperiod (L:D) is 12h:12h.

[0027] Example 2 Atlantic pseudoalternium Alteromonas profundi Cells 1C00251 and Antarctic diatoms were co-cultured in algal culture medium (F2 medium) at a cell ratio of 1:800 for 18 days at a temperature of 4°C and a light intensity of 60 μmol / m². 2 / s, with a photoperiod (L:D) of 8h:16h.

[0028] Example 3 Denitrified seaweed Escherichia coli 1K05758 and Antarctic diatoms were co-cultured in algal culture medium (F2 medium) at a cell ratio of 1:50 for 12 days at a temperature of 14℃ and a light intensity of 450 μmol / m². 2 / s, the photoperiod (L:D) is 16h:8h.

[0029] Example 4 marine polar bacteria Pseudoalteromonas atlantica 1K00696 and Antarctic diatoms were co-cultured in algal culture medium (F2 medium) at a cell ratio of 2:1 for 16 days at a temperature of 6℃ and a light intensity of 90 μmol / m². 2 / s, with a photoperiod (L:D) of 10h:14h.

[0030] Example 5 Atlantic pseudoalternium Alteromonas profundi 1C00251 and Antarctic diatoms were co-cultured in algal culture medium (F2 medium) at a cell ratio of 5:800. On the 4th day of co-culture, *Alternaria solani* was inoculated. Alteromonas profundi 1K04570 was co-cultured for a total of 16 days at a temperature of 7℃ and a light intensity of 220 μmol / m². 2 / s, photoperiod (L:D) is 14h:10h, *Alternaria alterniflora* Pseudoalteromonas atlantica Alteromonas profundi1K04570 and Antarctic diatom cell number ratio of 3:800.

[0031] Comparative Example 1 E. coli Pseudoalteromonas atlantica CGMCC 1.1569 and Antarctic diatom were inoculated into algal medium (F2 medium) at a cell number ratio of 1:100 for co-culture, the co-culture time was 14d, the temperature was 8℃, the light intensity was 180 μmol / m 2 / s, and the light period (L:D) was 12h:12h.

[0032] Comparative Example 2 Co-culture was carried out in the manner of Example 2, the only difference being that Pseudoalteromonas atlantica Marinobacter denitrificans 1C00251 and Antarctic diatom cell inoculation amount ratio was 50:1.

[0033] Comparative Example 3 Co-culture was carried out in the manner of Example 1, the only difference being that Alteromonas deephove Polaribacter marinaquae Polaribacter marinaquae 1K04570 and Antarctic diatom cell inoculation amount ratio was 1:5000.

[0034] Comparative Example 4 Co-culture was carried out in the manner of Example 4, the only difference being that the co-culture temperature was 18℃.

[0035] Comparative Example 5 Co-culture was carried out in the manner of Example 3, the only difference being that the co-culture light intensity was 25 μmol / m 2 / s.

[0036] Test Example 1 1. Sample collection: After the co-culture of Examples 1-5 and Comparative Examples 1-5 was completed, the supernatant and precipitate (algal cells) were collected by centrifugation at 4℃, 8000xg for 15 min.

[0037] 2. Polysaccharide content detection Under the action of concentrated sulfuric acid, polysaccharides are hydrolyzed into monosaccharides and rapidly dehydrated to form furfural derivatives, which are then condensed with phenol to form colored compounds, and the color depth is proportional to the sugar content. The absorbance value of the colored compound was measured at a certain wavelength, and the total sugar content in the sample was calculated by comparing the standard curve or known concentration of sugar solution. According to this principle, the polysaccharide content was detected, and the specific steps were as follows: (1) Standard curve drawing: weigh the glucose control product, dissolve it with deionized water, dilute it to 6 different concentrations of control solution at a dilution ratio of 1:1. Take 0.5 mL of the control solution, add 0.3 mL of phenol solution, mix well, then quickly add 1.8 mL of concentrated sulfuric acid, mix well, and place at room temperature for 20 min. An orange yellow color appears. Scan the standard solution with a spectrophotometer and measure the A value at the maximum absorption wavelength (490 nm). Use the corresponding reagent as a blank to draw the standard curve.

[0038] (2) Exopolysaccharide (EPS) detection: Take 0.5 mL of the supernatant collected in step 1, add 0.3 mL of phenol solution, mix well, then quickly add 1.8 mL of concentrated sulfuric acid, mix well, and place at room temperature for 20 min. Scan the solution with a spectrophotometer and measure the A value at the maximum absorption wavelength. Enter the standard curve in step (1) to calculate the exopolysaccharide content. The results are shown in Table 1.

[0039] (3) Intracellular polysaccharide (IPS) detection: Take the precipitate collected in step 1, add 5 mL of deionized water, and sonicate (ice bath, power 300 W, 3 s on, 5 s off, for 5 min). Extract in a boiling water bath for 2 h. After cooling, centrifuge at 10000 x g at 4°C for 15 min to collect the supernatant. Add 3 volumes of absolute ethanol to the supernatant and let it stand at 4°C overnight. The next day, centrifuge at 10000 x g at 4°C for 15 min to collect the precipitate. Dissolve the precipitate in deionized water and dilute to 5 mL. Take 0.5 mL of the solution, follow the method in step (2), measure the absorbance, and enter the standard curve to calculate the intracellular polysaccharide content. The results are shown in Table 1.

[0040] Table 1 Polysaccharide yield (mg / L) of Antarctic diatom under different co-culture methods

[0041] Note: No microorganism was inoculated, only Antarctic diatom was cultured in F2 medium at 8°C, light intensity 180 μmol / m 2 2, light period 12 h:12 h for 14 days. The EPS yield was 80 mg / L, the IPS yield was 60 mg / L, and the total polysaccharide yield was 140 mg / L.

[0042] According to Table 1, the polysaccharide yield of Antarctic diatom co-cultured with microorganisms in Examples 1-5 was significantly higher than that of Comparative Example 1. Among them, the polysaccharide yield of Example 5 was higher in a synergistic way with Alteromonas macedii Marinobacter denitrificans 1K04570 and Pseudoalteromonas atlantica Polaribacter marinaquae 1C00251. Compared with Escherichia coli, Alteromonas macedii Marinobacter denitrificans1K04570, Pseudoalteromonas atlantica Pseudoalteromonas atlantica 1C00251, Marinobacter denitrificans ​ ​ 1K05758 and Psychrobacter maritimus ​ 1K00696 can induce the synthesis of Antarctic diatom polysaccharide. In addition, the inoculation ratio of microorganisms is also crucial for polysaccharide production. In the control group 2, the ratio of microorganisms was too high, resulting in competitive inhibition. In the control group 3, the ratio of microorganisms was too low, which could not induce the synthesis of polysaccharide. In the control group 4, the culture temperature was not appropriate. In the control group 5, the light intensity was not appropriate. All of these resulted in low polysaccharide production. The effective symbiotic relationship between microorganisms and Antarctic diatom is the key to high polysaccharide production.

[0043] 3. Polysaccharide molecular weight detection The average molecular weight and molecular weight distribution PDI of polysaccharide were determined by GPC gel chromatograph. The specific steps are as follows: (1) Take the supernatant collected in step 1, and make sure there is no aggregation.

[0044] (2) Ultrafiltration concentration: Concentrate the sample through a 10 kDa ultrafiltration membrane to remove small molecular impurities.

[0045] (3) Use PL aquagel-OH, combined with a multi-angle light scattering detector (MALS), to detect the absolute molecular weight and distribution information of polysaccharide. Polystyrene was used as the standard to establish the correspondence between molecular weight and retention time. According to the standard curve, the weight average molecular weight (Mw) and number average molecular weight (Mn) were calculated, and the PDI (width index of molecular weight distribution) was calculated according to the formula PDI = Mw / Mn. The closer the PDI value is to 1, the narrower the molecular weight distribution and the better the uniformity. The results of weight average molecular weight and PDI are shown in Table 2.

[0046] Table 2 Polysaccharide molecular weight detection results of Antarctic diatom under different co-cultivation methods

[0047] According to Table 2, the polysaccharide molecular weight produced by Antarctic diatom under different co-cultivation methods is significantly different. The polysaccharide produced by Psychrobacter maritimus ​ 1K00696 is 450 kDa, while the polysaccharide produced by Marinobacter denitrificans ​ ​ 1K05758 is 2200 kDa. This reflects the difference in the regulation of Antarctic diatom metabolic pathways by different strains, resulting in the synthesis of polysaccharides with different degrees of polymerization. ​1K00696 (Example 4) produced the narrowest molecular weight distribution (PDI = 1.3), indicating that its structure was relatively uniform. The PDI of the group of Example 5 (synergistic co-culture) was higher (2.4), indicating that the product was a mixture of polysaccharides of two different molecular weight ranges, which was a complex polysaccharide. Comparative Example 2 could not measure the accurate molecular weight because the endogenous enzymes degraded the polysaccharide due to the apoptosis or lysis of the Antarctic diatom cells caused by bacterial competition.

[0048] 4. DPPH antioxidant scavenging rate detection DPPH is a stable free radical, which is purple in ethanol solution and has a maximum absorption peak at 517 nm. When an antioxidant is present, the DPPH free radical is scavenged, the solution color becomes lighter, and the absorbance decreases. The change in absorbance can be used to evaluate the antioxidant capacity of the antioxidant. DPPH antioxidant scavenging rate was detected based on this principle.

[0049] (1) Test sample: dilute the supernatant of step 1 with deionized water to 0.5% (w / v).

[0050] (2) Test method: add 1 mL of test sample to a colorimetric tube, then add 2 mL of 0.1 mmol / L DPPH working solution, and dilute to the mark line with anhydrous ethanol. After shaking and mixing, react for 30 min at room temperature in the dark. Use anhydrous ethanol as a blank group, and use distilled water instead of polysaccharide solution for the control group. Use ultraviolet-visible spectroscopy to measure the absorbance of the sample solution at 517 nm.

[0051] (3) Calculate the scavenging rate: calculate the DPPH free radical scavenging rate (%) = (A0-A sample) / A0x100 according to the formula; where A0 is the absorbance of DPPH at 517 nm without adding the sample; A sample is the absorbance of DPPH at 517 nm after adding the sample. The results are shown in Table 3.

[0052] Table 3 DPPH antioxidant scavenging rate (%) of polysaccharides obtained under different co-culture methods

[0053] The group with high polysaccharide yield and special molecular structure has stronger antioxidant activity. As can be seen from Table 3, the scavenging rate of Example 3 (Denitrifying marine rod-shaped bacteria ​ 1K05758) is the highest (78%), which may be closely related to the polysaccharide structure containing carotenoid derivatives produced under high temperature and high light stress. Example 2 (Pseudoalteromonas atlantica ​1C00251) and Example 5 (two bacteria synergy) also showed strong antioxidant activity (65%, 70%), which was related to its acidic sugar and complex structure. The clearance rate of Comparative Examples 1-5 group was generally low (≤22%), because the polysaccharide was only a basic metabolic product of Antarctic diatom, lacking special functional groups with antioxidant activity.

[0054] According to the above, it can be seen that the technical scheme provided by the present application can improve the yield and stress resistance of Antarctic diatom polysaccharide.

[0055] Although the above examples make a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which all belong to the protection scope of the present application.

Claims

1. The application of microorganisms in improving Antarctic diatom polysaccharide yield and / or stress resistance, characterized in that, The microorganisms include *Alternaria delta*. Alteromonas profundi 1K04570, *Pseudomonas aeruginosa* Pseudoalteromonas atlantica 1C00251, Denitrified Marine Bacteria Marinobacter denitrificans 1K05758 and marine polar bacteria Polaribacter marinaquae One or more of 1K00696.

2. A compound microbial agent, comprising independent component A and independent component B, characterized in that, The independent component A includes *Alternaria solani*. Alteromonas profundi 1K04570; The independent component B includes *Pseudomonas aeruginosa*. Pseudoalteromonas atlantica 1C00251.

3. The compound microbial agent according to claim 2, characterized in that, The compound bacterial agent contains *Alternaria alterniflora*. Alteromonas profundi 1K04570 and Atlantic pseudoalteromonas Pseudoalteromonas atlantica The cell ratio of 1C00251 was 3:

5.

4. The application of the compound bacterial agent according to claim 2 or 3 in improving the yield and / or stress resistance of Antarctic diatom polysaccharides.

5. The application according to claim 1 or 4, characterized in that, The stress resistance includes antioxidant properties.

6. A method for increasing the yield and / or stress resistance of Antarctic diatom polysaccharides, characterized in that, Co-culture of microorganisms and Antarctic diatoms; The microorganism is the compound bacterial agent as described in claim 2 or 3, or it is a deep-source alterniform bacteria. Alteromonas profundi 1K04570, *Pseudomonas aeruginosa* Pseudoalteromonas atlantica 1C00251, Denitrified Marine Bacteria Marinobacter denitrificans 1K05758 and marine polar bacteria Polaribacter marinaquae One or more of 1K00696.

7. The method according to claim 6, characterized in that, The cell inoculation ratio of the microorganisms and Antarctic diatoms is (1~2) cells: (1~1000) cells; The co-culturing temperature was 2–15℃, and the light intensity was 50–500 μmol / m². 2 / s, the duration is 12~18 days, the light cycle is 8~16 hours of light and 16~8 hours of darkness.

8. The method according to claim 6, characterized in that, When the microorganism is the compound bacterial agent as described in claim 2 or 3, the cell inoculation ratio of the microorganism to Antarctic diatoms is 4:

250. When the microorganism is *Alternaria solani* Alteromonas profundi At 1K04570, the cell inoculation ratio of microorganisms to Antarctic diatoms was 1:

800. When the microorganism is *Pseudomonas aeruginosa* Pseudoalteromonas atlantica At 1C00251, the cell inoculation ratio of microorganisms to Antarctic diatoms was 1:

150. When the microorganism is denitrifying seaweed Marinobacter denitrificans At 1K05758, the cell inoculation ratio of microorganisms to Antarctic diatoms was 1:

50. When the microorganism is marine polar bacillus Polaribacter marinaquae At 1K00696, the cell inoculation ratio of microorganisms to Antarctic diatoms was 2:

1.

9. The method according to claim 6, characterized in that, When the microorganism is the compound bacterial agent as described in claim 2 or 3, the co-culturing temperature is 7°C and the light intensity is 220 μmol / m². 2 / s, the time is 16 days, the light cycle is 14 hours of light and 10 hours of darkness; When the microorganism is *Alternaria solani* Alteromonas profundi At 1K04570, the co-culturing temperature was 4℃ and the light intensity was 60 μmol / m². 2 / s, the time is 14 days, the light cycle is 8 hours of light and 16 hours of darkness; When the microorganism is *Pseudomonas aeruginosa* Pseudoalteromonas atlantica At 1C00251, the co-culturing temperature was 8℃ and the light intensity was 180 μmol / m². 2 / s, the time is 18 days, the light cycle is 12 hours of light and 12 hours of darkness; When the microorganism is denitrifying seaweed Marinobacter denitrificans At 1K05758, the co-culturing temperature was 14℃ and the light intensity was 450 μmol / m². 2 / s, the time is 12 days, the light cycle is 16 hours of light and 8 hours of darkness; When the microorganism is marine polar bacillus Polaribacter marinaquae At 1K00696, the co-culturing temperature was 6℃ and the light intensity was 90 μmol / m². 2 / s, the time is 16 days, the light cycle is 10 hours of light and 14 hours of darkness.

10. The method according to any one of claims 6 to 9, characterized in that, When the microorganism is the compound bacterial agent as described in claim 2 or 3, *Pseudomonas aeruginosa* is simultaneously inoculated into the algal culture medium. Pseudoalteromonas atlantica 1C00251 was co-cultured with microalgae, and 4 days after inoculation, it was inoculated with *Alternaria alterniflora*. Alteromonas profundi 1K04570 will continue to be co-cultured; When the microorganism is *Alternaria solani* Alteromonas profundi 1K04570, *Pseudomonas aeruginosa* Pseudoalteromonas atlantica 1C00251, Denitrified Marine Bacteria Marinobacter denitrificans 1K05758 or marine polar bacillus Polaribacter marinaquae At 1K00696, microorganisms and microalgae were simultaneously inoculated into the algal culture medium for co-culture.

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