A method for increasing the yield of alginate produced by brown nitrogen-fixing bacteria

Abstract

The invention discloses a method for increasing the yield of alginate produced by brown nitrogen-fixing bacteria, and belongs to the technical field of microbial fermentation. H2O2 is added during the culture of brown nitrogen-fixing bacteria. The proportion of mannuronic acid (M) in alginate obtained by the method of the invention is increased from 84.54% to 91.95%, the proportion of guluronic acid (G) is reduced from 15.46% to 8.05%, M / G is increased from 5.47 to 11.42, the acetyl content is 6.61%, the molecular weight is reduced from 2.1 million Da to 1.95 million Da, and the maximum yield of alginate can reach 13.5 g / L, which greatly improves the potential of brown nitrogen-fixing bacteria to synthesize alginate rich in mannuronic acid.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, in particular to a method for increasing the yield of alginate produced by brown nitrogen-fixing bacteria. Background Art

[0002] Alginate is a polysaccharide carbohydrate widely found in brown algae. It is also the main component of seaweed cell walls, accounting for about 40% of its dry weight. Alginate is a natural acidic polysaccharide composed of two sugar units, β-D-mannuronic acid fragment (M) and α-L-guluronic acid fragment (G), connected by 1,4 glycosidic bonds. Due to its non-toxicity, good stability, and good biocompatibility, alginate is widely used in food, medicine, cosmetics, and industrial production. Studies have shown that the physicochemical properties (viscosity, sol / gel transition properties, water absorption, etc.) and biological activity of alginate are closely related to the M / G ratio of alginate, the arrangement of uronic acid residues, molecular weight, and degree of acetylation. For example, alginate rich in M ​​segments and its oligosaccharides have good antioxidant, immunomodulatory, and anti-Alzheimer's biological activities. Therefore, the preparation of alginate with novel structure, clear degree of polymerization and M / G ratio is of great significance for discovering new biological activities and modes of action of alginate.

[0003] As we all know, most commercial alginate is extracted from brown algae. The M / G ratio and molecular weight of alginate extracted from different regions, different species, different growing seasons, and even different parts vary greatly, making it difficult to ensure stable alginate properties. In addition, it is very difficult to directly separate M-rich alginate from brown algae such as kelp. Microbial fermentation to produce alginate has the advantages of controllable conditions, no time and geographical restrictions, and directional modification, and has received widespread attention in recent years. Brown nitrogen-fixing bacteria are Gram-negative bacteria, facultative aerobic, able to convert nitrogen in the air into their own nitrogen source, with a wide range of sources, simple cultivation, and not easy to be contaminated, so they are most likely to become alginate production strains.

[0004] At present, there are few reports on increasing the yield of M-rich alginate produced by brown nitrogen-fixing bacteria, and in the existing technology, the yield of alginate with M / G≥9 synthesized by nitrogen-fixing bacteria is about 2-6g / L, which seriously limits the application of nitrogen-fixing bacteria in producing M-rich alginate. Therefore, developing a method to increase the yield of M-rich alginate produced by brown nitrogen-fixing bacteria has broad prospects. Summary of the invention

[0005] The purpose of the present invention is to provide a method for increasing the yield of alginate produced by brown nitrogen-fixing bacteria, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a method for improving the yield of alginate produced by brown nitrogen-fixing bacteria, and H2O2 is added during the cultivation process of brown nitrogen-fixing bacteria.

[0008] Preferably, the alginate is alginate rich in mannuronic acid.

[0009] Preferably, the culture process is specifically to inoculate brown nitrogen-fixing bacteria seed solution into a fermentation medium for fermentation culture, add H2O2 after 12 to 36 hours so that the final concentration of H2O2 in the fermentation liquid is 1-6 mmol / L, and continue to culture for 48 hours to obtain alginate.

[0010] Preferably, the inoculation amount of the vine nitrogen-fixing bacteria seed solution is 10% of the volume of the fermentation medium.

[0011] Preferably, the formula of the fermentation medium is: 20 g sucrose, 0.2 g potassium dihydrogen phosphate, 1.2 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate, 0.1 g calcium sulfate, 0.005 g sodium molybdate, 0.5 g yeast extract and 1 L distilled water, with a pH of 7.2±0.2.

[0012] Preferably, the fermentation culture conditions are: temperature 28°C, rotation speed 230r / min.

[0013] The invention also provides alginate obtained by fermentation according to the method.

[0014] Preferably, in the alginate, mannuronic acid accounts for 91.95%, guluronic acid accounts for 8.05%, M / G is 11.42, the acetyl content is 6.61%, and the average relative molecular mass is 1.95 million Da.

[0015] The present invention also provides the use of the alginate in preparing products for anti-oxidation, immunomodulation and / or anti-Alzheimer's disease.

[0016] Based on the above technical solution, the present invention has the following technical effects:

[0017] The invention discloses a method for improving the yield of alginate rich in mannuronic acid produced by brown nitrogen-fixing bacteria. The proportion of mannuronic acid (M) in the alginate obtained by the method of the invention is increased from 84.54% to 91.95%, the proportion of guluronic acid (G) is reduced from 15.46% to 8.05%, M / G is increased from 5.47 to 11.42, the acetyl content is 6.61%, the molecular weight is reduced from 2.1 million Da to 1.95 million Da, and the maximum yield of alginate can reach 13.5 g / L, thereby greatly improving the potential of brown nitrogen-fixing bacteria to synthesize alginate rich in mannuronic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is the growth curve of brown nitrogen-fixing bacteria;

[0020] Figure 2 The graph of the production of alginate produced by brown nitrogen-fixing bacteria over time;

[0021] Figure 3 This is the dry weight chart of brown nitrogen-fixing bacteria after adding H2O2 for 12 hours;

[0022] Figure 4 This is the yield graph of alginate produced by brown nitrogen-fixing bacteria with H2O2 added for 12h;

[0023] Figure 5 This is the dry weight chart of brown nitrogen-fixing bacteria after adding H2O2 for 24 hours;

[0024] Figure 6 This is the yield diagram of alginate produced by brown nitrogen-fixing bacteria with H2O2 added for 24 hours;

[0025] Figure 7 The dry weight of brown nitrogen-fixing bacteria after adding H2O2 for 36 hours;

[0026] Figure 8 This is the yield diagram of alginate produced by brown nitrogen-fixing bacteria with H2O2 added for 36h;

[0027] Fig. 9 This is the dissolved oxygen change curve of brown nitrogen-fixing bacteria;

[0028] Fig.10 This is the monosaccharide composition diagram of alginate produced by brown nitrogen-fixing bacteria in the control group;

[0029] Fig.11 This is the monosaccharide composition diagram of alginate produced by brown nitrogen-fixing bacteria under 2mmol / L H2O2 stress;

[0030] Fig.12 This is the monosaccharide composition diagram of alginate produced by brown nitrogen-fixing bacteria under 4mmol / L H2O2 stress;

[0031] Fig.13 This is the infrared spectrum of brown nitrogen-fixing bacteria extracellular polysaccharide and sodium alginate. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0038] The Latin name of the brown nitrogen-fixing bacteria described in the embodiment of the present invention is Azotobacter vinelandii, and the strain collection number is CGMCC No.28548.

[0039] Example 1

[0040] 1) Preparation of seed solution: Pick a single colony from the plate cultured at constant temperature, add it to a conical flask containing 50 mL of culture medium, and culture it at 28°C, 230 rpm for 24 h for later use;

[0041] 2) Preparation of fermentation medium: 20 g sucrose, 0.2 g potassium dihydrogen phosphate, 1.2 g potassium hydrogen phosphate, 0.2 g magnesium sulfate, 0.1 g calcium sulfate, 0.005 g sodium molybdate, 0.5 g yeast extract, 1 L distilled water, pH 7.2±0.2 to prepare fermentation medium, sterilize at 121°C for 20 min, and set aside.

[0042] 3) Fermentation culture: Inoculate the brown nitrogen-fixing bacteria seed solution into the fermentation medium at an inoculation rate of 10% of the volume of the fermentation medium, with a fermentation temperature of 28°C, a rotation speed of 230 r / min, and a pH of 7.2±0.2. Take 2 mL of the fermentation solution at intervals of 10,000 r / min, centrifuge at 4°C for 5 min, remove the supernatant, add 1 mL of distilled water to wash the bacteria, centrifuge again, repeat this step 3 times, pour out the supernatant, dry the bacteria at 105°C and weigh them, and obtain the growth curve of the brown nitrogen-fixing bacteria, as shown in the figure. Figure 1 As shown in the figure, brown nitrogen-fixing bacteria reached the stable stage in 48 hours. Take another 10mL fermentation broth, centrifuge at 8000r / min for 10min, take the supernatant, add 3 times the volume of 95% ethanol to precipitate, collect the precipitate, dry and weigh. Figure 2 As shown, it can be determined that the fermentation time of brown nitrogen-fixing bacteria is 48h. The brown nitrogen-fixing bacteria seed solution is inoculated into the fermentation medium at an inoculation rate of 10% of the volume of the fermentation medium. The fermentation temperature is 28°C, the rotation speed is 230r / min, and the pH is 7.2±0.2. After culturing for 36h, 1mol / L H2O2 is added to make the final concentration of H2O2 in the fermentation broth 1mmol / L, 2mmol / L, 3mmol / L, 4mmol / L, 5mmol / L and 6mmol / L, respectively. The culture is continued for 48h, and the fermentation broth is centrifuged at 8000r / min for 10min. The supernatant is concentrated 5 times, and 3 times the volume of 95% ethanol is added for alcohol precipitation. The precipitate is collected and dried to obtain alginate. The yield of alginate produced by brown nitrogen-fixing bacteria is shown in the table. Figure 8 When H2O2 was added for 36 hours, there was no significant effect on the dry weight of the bacteria. When the H2O2 concentration was 4 mmol / L, the extracellular polysaccharide production could reach up to 13.5 g / L. The dissolved oxygen electrode was used to detect the changes in dissolved oxygen during the fermentation of brown nitrogen-fixing bacteria. The results are shown in Fig. 9 .

[0043] Example 2

[0044] 1) Preparation of seed solution: Pick a single colony of brown nitrogen-fixing bacteria from a plate cultured at a constant temperature, add it to a conical flask containing 50 mL of culture medium, and culture it at 28°C and 230 rpm for 24 h for later use;

[0045] 2) Preparation of fermentation medium: 20 g sucrose, 0.2 g potassium dihydrogen phosphate, 1.2 g potassium hydrogen phosphate, 0.2 g magnesium sulfate, 0.1 g calcium sulfate, 0.005 g sodium molybdate, 0.5 g yeast extract, 1 L distilled water, pH 7.2±0.2 to prepare fermentation medium, sterilize at 121°C for 20 min, and set aside.

[0046] 3) Fermentation culture: Inoculate the brown nitrogen-fixing bacteria seed solution into the fermentation medium at a 10% inoculation rate, with a fermentation temperature of 28°C, a rotation speed of 230r / min, and a pH of 7.2±0.2. After culturing for 12 hours, add 1mol / L H2O2 to make the final concentration of H2O2 in the fermentation liquid 1mmol / L, 2mmol / L, 3mmol / L, 4mmol / L, 5mmol / L and 6mmol / L, respectively. Continue to culture for 48 hours, centrifuge the fermentation liquid at 8000r / min for 10 minutes, concentrate the supernatant 5 times, add 3 times the volume of 95% ethanol for alcohol precipitation, and collect the precipitate, which is alginate. Weigh and obtain the alginate yield as shown in the following figure: Figure 4 As shown, when H2O2 was added for 12 h, the alginate production in all experimental groups was lower than that in the control group.

[0047] Example 3

[0048] 1) Preparation of seed solution: Pick a single colony from the plate cultured at constant temperature, add it to a conical flask containing 50 mL of culture medium, and culture it at 28°C, 230 rpm for 24 h for later use;

[0049] 2) Preparation of fermentation medium: 20 g sucrose, 0.2 g potassium dihydrogen phosphate, 1.2 g potassium hydrogen phosphate, 0.2 g magnesium sulfate, 0.1 g calcium sulfate, 0.005 g sodium molybdate, 0.5 g yeast extract, 1 L distilled water, pH 7.2±0.2 to prepare fermentation medium, sterilize at 121°C for 20 min, and set aside.

[0050] 3) Fermentation culture: Inoculate the brown nitrogen-fixing bacteria seed solution into the fermentation medium at a 10% inoculation rate, with a fermentation temperature of 28°C, a rotation speed of 230r / min, and a pH of 7.2±0.2. After culturing for 24 hours, add 1mol / L H2O2 to make the final concentration of H2O2 in the fermentation liquid 1mmol / L, 2mmol / L, 3mmol / L, 4mmol / L, 5mmol / L and 6mmol / L, respectively. Continue to culture for 48 hours, centrifuge the fermentation liquid at 8000r / min for 10 minutes, concentrate the supernatant 5 times, add 3 times the volume of 95% ethanol for precipitation, and collect the precipitate, which is alginate. Weigh and the yield of alginate produced by brown nitrogen-fixing bacteria is shown in Figure 6 When the final concentration of H2O2 added for 24 hours was 2mmol / L, the alginate production was 4.31g / L, which was slightly higher than that of the control group, but not significantly higher.

[0051] result:

[0052] 1. Comparative Examples 1-3 show that when H2O2 concentration of 4 mmol / L was added at 36 h, the yield of alginate produced by brown nitrogen-fixing bacteria was the highest. This is because compared with the addition of H2O2 at 12 h and 24 h, 36 h is in the middle and late stages of the logarithmic growth of brown nitrogen-fixing bacteria. At this time, the biomass of the bacteria has reached a certain level, and the toxicity of H2O2 is weakened ( Figure 3 , Figure 5 and Figure 7 ), at the same time, a large amount of O2 is produced after H2O2 is decomposed by the bacteria, which increases the dissolved oxygen content of the fermentation liquid, thereby increasing the yield of alginate produced by brown nitrogen-fixing bacteria.

[0053] 2. The monosaccharide composition and M / G ratio of alginate produced by nitrogen-fixing bacteria prepared in Example 1 were determined by pre-column derivatization with PMP (1-phenyl-3-methyl-5-pyrazolone) HPLC. Figure 10-12 ), and infrared spectroscopy was performed on alginate and sodium alginate produced by brown nitrogen-fixing bacteria ( Fig.13 ), the monosaccharide composition and M / G ratio of alginate produced by brown nitrogen-fixing bacteria are shown in Table 1.

[0054] Table 1: Monosaccharide composition and M / G ratio of alginate produced by brown nitrogen-fixing bacteria

[0055]

[0056] As shown in Table 1, the proportion of mannuronic acid in alginate produced by brown nitrogen-fixing bacteria in the control group was 84.54%, and the M / G ratio was 5.47 ( Fig.10 ), the proportion of mannuronic acid in the alginate produced by brown nitrogen-fixing bacteria under 2mmol / L H2O2 stress was 89.4%, and the M / G ratio was 8.43 ( Fig.11 ), the proportion of mannuronic acid in the alginate produced by brown nitrogen-fixing bacteria under 4mmol / L H2O2 stress was 91.95%, and the M / G ratio was 11.42 ( Fig.12 ), indicating that H2O2 did increase the proportion of M in brown nitrogen-fixing bacteria. The infrared spectrum results showed that compared with sodium alginate, the characteristic absorption peak of acetyl in the extracellular polysaccharide of brown nitrogen-fixing bacteria was 1738.18 cm -1 ( Fig.13 ), indicating that the alginate produced by brown nitrogen-fixing bacteria contains acetyl groups. The acetyl content of alginate produced by brown nitrogen-fixing bacteria was 6.61% by acid-base titration. It is well known that increasing dissolved oxygen will inhibit the activity of polymerase Alg8 in alginate produced by brown nitrogen-fixing bacteria, while promoting the activity of alginate lyase AlgL, which will reduce the final molecular weight of alginate and reduce viscosity. After adding H2O2, the average relative molecular mass of alginate produced by brown nitrogen-fixing bacteria dropped from 2.1 million Da to 1.95 million Da.

[0057] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for increasing the yield of alginate produced by brown nitrogen-fixing bacteria, characterized in that: Add H2O2 during the cultivation of brown nitrogen-fixing bacteria; The alginate is alginate rich in mannuronic acid; The culture process specifically includes inoculating brown nitrogen-fixing bacteria seed solution into a fermentation medium for fermentation culture at a rotation speed of 230 r / min, adding H2O2 after 36 hours so that the final concentration of H2O2 in the fermentation liquid is 4 mmol / L, and continuing to culture for 48 hours to obtain alginate.

2. The method according to claim 1, characterized in that The inoculation amount of the brown nitrogen-fixing bacteria seed solution is 10% by volume.

3. The method according to claim 1, characterized in that The formula of the fermentation medium is: 20 g sucrose, 0.2 g potassium dihydrogen phosphate, 1.2 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate, 0.1 g calcium sulfate, 0.005 g sodium molybdate, 0.5 g yeast extract and 1 L distilled water, and the pH is 7.2±0.

2.

4. The method according to claim 1, characterized in that: The fermentation culture conditions are: temperature 28° C., rotation speed 230 r / min.

5. Alginate obtained by fermentation according to any one of claims 1 to 4.

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