Agarose preparation method based on microbial desulfurization

Abstract

The invention discloses an agarose preparation method based on microbial desulfurization, which comprises one of the following two modes: (1) placing agar in a culture solution, inoculating microorganisms, culturing at 25-40 DEG C for 20-48 hours, carrying out solid-liquid separation, and continuously transferring for 1-3 times; (2) placing agar-containing plants or extracted agar in the culture solution, inoculating microorganisms, culturing at 25-40 DEG C for 20-48 hours, carrying out solid-liquid separation, and continuously transferring for 1-3 times; the microorganism is selected from the group consisting of yarrowia lipolytica PO1h-URA, bacillus megatherium OUC-Gel (QD)-W # DS # 1-WMX, bacillus velezensis, yarrowia guilligera and achromobacter xylosoxidans. According to the method disclosed by the invention, the sulfuric acid groups in the agar are decomposed and utilized by utilizing microbial metabolism specificity, so that green, low-energy-consumption and high-purity agarose production is realized, and a foundation is laid for green production of high-quality agarose.

Description

Technical Field

[0001] The present invention relates to a method for preparing agarose based on microbial desulfurization, and belongs to the technical field of agarose preparation. Background Art

[0002] Agarose is a long-chain structure with agarobiose composed of 1,3-linked β-D-galactose and 1,4-linked α-3,6-anhydro-L-galactose as the basic monomer, and is the main component of agar. Agarose appears as a pure white powder, dissolves in hot water, and forms a gel after condensation. As a support for gel electrophoresis and an affinity chromatography carrier, it is widely used in the biochemical and medical fields. It is an important reagent for studying virus phages, bacteria, clinical tests, biochemical analysis, proteins, nucleic acids, antigens, and antibodies, and can be used for polysaccharide separation, purification, and drug preparation. The main indicators for evaluating the quality of agarose are the content of sulfate groups and the gel strength. Another component of agar, sulfated agar, contains a considerable amount of sulfate groups. During the formation of the aggregate network of agar, the agarose gel in which the hydroxyl group is replaced by a sulfate ester group will form a kink at this position, hindering the alignment of the helix, thereby affecting the formation of the double helix, making the agar form a relatively loose double helix structure and affecting the formation of aggregates, which reduces the overall gel performance of agar. The electrical neutrality of agarose is one of the important reasons for it to be used as a raw material for preparing gels in agarose gel electrophoresis. The presence of charged sulfate groups will significantly increase the electroendosmosis of agarose. Therefore, it is very necessary to remove the sulfate groups in agar during the preparation of agarose.

[0003] At present, most of the agarose produced at home and abroad uses finished agar as raw material, and the preparation process is relatively complex. The agar after chemical desulfurization needs to be separated and purified by methods such as ion exchange chromatography technology and ionic liquid method to remove agar gum to obtain agarose. Chemical desulfurization is carried out by treating agar with strong alkalis such as sodium hydroxide and washing with a large amount of water. The chemical substances generated during the chemical desulfurization process cause great pollution to the environment, have an adverse impact on life, and it is difficult to accurately control the product performance. There are problems such as high cost, complex process, and residual chemical reagents. In addition, chemical desulfurization can remove the sulfate groups at the C-6 position of L-galactose, but cannot remove the sulfate groups at the C-4 and C-6 positions of D-galactose. During the treatment of agar with strong alkali, the polysaccharide chain will be severely depolymerized and the agar product will turn brown due to the violent reaction, thus affecting the yield and gel strength of agar. Therefore, the quality of agar cannot reach the level of agarose through chemical desulfurization, and it is urgent to find a green, environmentally friendly, efficient and simple desulfurization method to replace chemical desulfurization. At present, there is no relevant report on preparing agarose by the microbial method.

[0004] CN 116445333 A discloses a Bacillus megaterium WMX and its application in agar desulfurization. This patent is the previous research result of the applicant of the present invention. It uses microorganisms to directly desulfurize agar powder, Gelidium amansii agar, Gracilaria agar, etc., and has good desulfurization effect. However, the product prepared by this patent is agar, and its method has the ability of agar desulfurization, but it is still far from preparing agarose products (sulfate group content < 0.15%, gel strength of 1.0% > 1200 g / cm 2 ), and subsequent research is still needed. On this basis, the present invention has developed a method for preparing agarose based on microbial desulfurization. Summary of the Invention

[0005] In view of the above-mentioned prior art, the present invention provides a method for preparing agarose based on microbial desulfurization.

[0006] The present invention is realized through the following technical solutions: A method for preparing agarose based on microbial desulfurization is one of the following two methods: (1) Place agar in a culture medium, inoculate microorganisms, and culture at 25 - 40 °C for 20 - 48 hours, then perform solid-liquid separation and continuously transfer 1 - 3 times; the method of continuous transfer is: after solid-liquid separation, place the obtained solid in the culture medium again, inoculate microorganisms, and culture at 25 - 40 °C for 20 - 48 hours; perform solid-liquid separation and extract the desulfurized agarose. (2) Place a plant plant containing agar or the extracted agar in a culture medium, inoculate microorganisms, and culture at 25 - 40 °C for 20 - 48 hours, then perform solid-liquid separation and continuously transfer 1 - 3 times; the method of continuous transfer is: after solid-liquid separation, place the obtained solid in the culture medium again, inoculate microorganisms, and culture at 25 - 40 °C for 20 - 48 hours; perform solid-liquid separation and extract the desulfurized agarose.

[0007] The microorganisms are selected from one of the following 5 species: Yarrowia lipolytica PO1h-URA; Priestia megaterium OUC-Gel(QD)-W#DS#1-WMX; Bacillus velezensis; Meyerozyma guilliermondii; Achromobacter xylosoxidans. The specific conditions of each strain are as follows: Yarrowia lipolytica PO1h-URA, a kind of hemiascomycetous yeast, is widely distributed in nature, has a strong ability to decompose hydrophobic substrates, is a non-pathogenic bacterium, and is recognized as Generally Recognized as Safe (GRAS) by the US Food and Drug Administration (FDA). The biological characteristics of this strain are as follows: colonies are round, beige, wrinkled, with a hairy edge, dry in texture, 4.0 - 6.0 mm. Cells are single, spherical, oval, or sausage-shaped, 2.5 - 4.0 × 4.0 - 11.2 μm. There are true and false hyphae; the asexual reproduction method is multilateral budding.

[0008] Bacillus megaterium OUC-Gel(QD)-W#DS#1-WMX, which is the Bacillus megaterium WMX disclosed in CN 116445333 A, is the research result of the applicant of the present invention in the early stage. The biological characteristics of this strain are as follows: rod-shaped, with a round end, single or arranged in short chains, 1.2 - 1.5 μm × 2.0 - 4.0 μm, strictly aerobic, motile, Gram-positive, spores 1.0 - 1.2 μm × 1.5 - 2.0 μm, oval, central or subterminal.

[0009] Bacillus velezensis, the cells are straight rods, often arranged in pairs or chains, with round or square ends. Most cells show Gram-positive staining in young cultures and move with peritrichous flagella. Each cell produces one spore and can adapt to many adverse environments. Aerobic or facultatively anaerobic. Chemotrophic heterotrophs, with fermentative or respiratory metabolic types.

[0010] Meyerozyma guilliermondii, the cells are oval, elliptical to cylindrical, with a bacterial ring and island-like pellicles produced, colonies are buttery, milky white, smooth or wrinkled on the surface, reflective, with an etched or fringed edge, and have false hyphae.

[0011] Achromobacter xylosoxidans, Gram-negative, colony size 2 - 3 mm, yellow, moist, raised, opaque, with a neat edge.

[0012] Further, in the above (1), the product form of agar is agar powder, agar flakes or agar strips.

[0013] Further, in the above (2), the plants containing agar are selected from any one or more of Gracilaria, Gelidium amansii, and Porphyra; the plants are fresh plants or dried plants.

[0014] Further, in the above (2), the agar is extracted from plants containing agar, and the extraction method may be as follows: take the plant plants containing agar, mix them with water at a solid-liquid ratio of 1:35, extract gum at 100-115°C for 30 minutes, filter while it is hot with a 200-mesh gauze, cool to obtain agar gel particles, or cool and dry to obtain dry agar.

[0015] Further, the culture medium is a culture medium existing in the prior art. Specifically, the component composition of the culture medium may be composed of a carbon source, inorganic salts, sulfur-free trace elements, and water. More specifically, the component composition of the culture medium is as follows: 2% glucose; inorganic salts (KH 2 PO 4 , 4.0 g / L; NH 4 Cl, 12.16 g / L; K 2 HPO 4 , 3.19 g / L; MgCl 2 ·6H 2 O, 0.83 g / L; CaCl 2 ·2H 2 O, 4.5×10 -2 g / L); sulfur-free trace elements [biotin, 5 μg / L; VB1, 5 μg / L; disodium ethylenediaminetetraacetate dihydrate (EDTA), 0.05 g / L; FeCl 3 , 0.0083 g / L; ZnCl 2 , 0.0004 g / L; CuCl 2 ·2H 2 O, 0.00013 g / L; CoCl 2 ·6H 2 O, 0.0001 g / L; H 3 BO 3 , 0.0001 g / L; MnCl 2 ·4H 2 O, 0.000016 g / L; Na 2 MoO 4 , 0.0003 g / L]; the balance is water.

[0016] Further, in the above (1), the addition amount of agar in the culture medium is 0.1% - 5%, preferably 4%.

[0017] Further, in the above (2), the addition amount of the plant plants containing agar in the culture medium is 0.2% - 5%, preferably 2% and 4%.

[0018] Further, the microorganism is inoculated in the form of a seed solution, and the inoculation amount is 1% - 10% (volume ratio).

[0019] Furthermore, the specific preparation method is as follows: Add 2 g of agar (agar powder, agar strips, agar flakes, agar gel particles or dry agar) to 50 mL of culture medium, add 5 mL of microbial bacterial liquid, and culture at 30 °C or 37 °C for 24 - 48 hours; perform solid-liquid separation to obtain desulfurized agarose.

[0020] Furthermore, the specific preparation method is as follows: Add 2 g of dry plant to 50 mL of culture medium, add 5 mL of microbial bacterial liquid, and culture at 30 °C or 37 °C for 48 hours; separate the solid with a filter cloth, mix the solid with water at a solid-liquid ratio of 1:35, and extract the gum at 115 °C for 30 minutes; filter while it is hot with a 200-mesh gauze, cool and dry to obtain agarose.

[0021] Further, to eliminate the influence of possible miscellaneous bacteria on the microorganisms, the raw materials (agar or plant plants containing agar) can be first soaked in a sodium hypochlorite solution and then added to the culture medium. The specific method of the soaking treatment can be: Take 2 g of raw materials, place them in a 250 mL conical flask, add 100 mL of 0.025% sodium hypochlorite solution, sterilize by shaking in a constant temperature shaker for 40 min (30 °C, 220 rpm), take out, wash three times with sterilized deionized water in a laminar flow bench, add 50 mL of deionized water, and shake in a constant temperature shaker (30 °C, 220 rpm) for 24 h to remove residual chlorine.

[0022] The agarose prepared by using the above-mentioned method for preparing agarose based on microbial desulfurization.

[0023] The method for preparing agarose based on microbial desulfurization of the present invention uses the specific metabolism of microorganisms to decompose and utilize the sulfate groups in agar, realizing the production of agarose with green, low energy consumption and high purity. The microorganisms used are highly efficient desulfurizing strains verified by screening. During the fermentation process, the strains secrete desulfurase into the culture medium to remove the sulfate groups in agar, releasing free sulfate radicals as a sulfur source for the growth of the strains. The strains secrete more enzymes to desulfurize during the growth process, forming a positive cycle. When the degree of desulfurization is high enough, agarose meeting the requirements of electrophoresis can be obtained. The present invention provides a new way for the high-value utilization of red algae plants, which not only helps to promote the development and progress of the domestic agar industry, but also contributes to the development of large-scale high-value seaweed aquaculture, laying a foundation for the green production of high-quality agarose.

[0024] All the terms and phrases used in the present invention have the general meanings well-known to those skilled in the art. Description of the Drawings

[0025] Figure 1 : Growth curve of the bacterial community.

[0026] Figure 2 : Dilution coating diagram of the microbial community.

[0027] Figure 3 : Schematic diagram of the sulfur content in agar after single colony fermentation.

[0028] Figure 4 : Schematic diagram of the agar gel strength after single colony fermentation.

[0029] Figure 5 : Schematic diagram of the detection results of sulfur content and gel strength after Yarrowia lipolytica ferments agar strips and agar powder.

[0030] Figure 6 : OD of Yarrowia lipolytica after fermentation for 1 - 4 times 600 value.

[0031] Figure 7 : Schematic diagram of the detection results of sulfur content after Yarrowia lipolytica ferments for 1 - 4 times.

[0032] Figure 8 : Schematic diagram of the detection results of gel strength after Yarrowia lipolytica ferments for 1 - 4 times.

[0033] Figure 9 : OD of Bacillus megaterium WMX after fermentation for 1 - 4 times 600 value.

[0034] Figure 10 : Schematic diagram of the detection results of sulfur content and gel strength after Bacillus megaterium WMX ferments for 4 times.

[0035] Figure 11 : Schematic diagram of the results of agarose gel electrophoresis. Among them, the samples in lanes 1, 2, 3, and 4 are standard proteins with lengths of 2000 bp, 5000 bp, 10000 bp, and 15000 bp respectively; the agaroses of a, b, and c are agarose purchased from the market, the agarose prepared by the present invention, and agar from Gelidium amansii respectively. Detailed implementation manners

[0036] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0037] The instruments, reagents, and materials involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, and materials existing in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods and detection methods existing in the prior art.

[0038] In the present invention, unless otherwise specified, the percentages refer to weight / volume ratios, with the unit of g / mL.

[0039] The agar powder and agar strips used in the present invention are both obtained by conventional market purchase. Agar powder and agar strips are common agar products extracted from Gelidium amansii or Gracilaria lemaneiformis. Commercially available agar powder and agar strips are usually obtained from Gracilaria lemaneiformis as the raw material and through alkali treatment.

[0040] The components of the culture medium (inorganic sulfur-free liquid medium) used in the present invention are as follows: 2% glucose; inorganic salts (KH 2 PO 4 , 4.0 g / L; NH 4 Cl, 12.16 g / L; K 2 HPO 4 , 3.19 g / L; MgCl 2 ·6H 2 O, 0.83 g / L; CaCl 2 ·2H 2 O, 4.5×10 -2 g / L); sulfur-free trace elements [biotin, 5 μg / L; VB1, 5 μg / L; disodium ethylenediaminetetraacetate dihydrate (EDTA), 0.05 g / L; FeCl 3 , 0.0083 g / L; ZnCl 2 , 0.0004 g / L; CuCl 2 ·2H 2 O, 0.00013 g / L; CoCl 2 ·6H 2 O, 0.0001 g / L; H 3 BO 3 ,0.0001 g / L; MnCl 2 ·4H 2 O, 0.000016 g / L; Na 2 MoO 4 , 0.0003 g / L]; the balance is water.

[0041] The components of the inorganic carbon-free liquid medium used in the present invention are as follows: inorganic salts (KH 2 PO 4 , 4.0 g / L; NH 4 Cl, 12.16 g / L; K 2 HPO 4 , 3.19 g / L; MgCl 2 ·6H 2 O, 0.83 g / L; CaCl 2 ·2H 2O, 4.5×10 -2 g / L); sulfur-free trace elements [biotin, 5 μg / L; VB1, 5 μg / L; disodium ethylenediaminetetraacetate dihydrate (EDTA), 0.05 g / L; FeCl 3 , 0.0083 g / L; ZnCl 2 , 0.0004 g / L; CuCl 2 ·2H 2 O, 0.00013 g / L; CoCl 2 ·6H 2 O, 0.0001 g / L; H 3 BO 3 , 0.0001 g / L; MnCl 2 ·4H 2 O, 0.000016 g / L; Na 2 MoO 4 , 0.0003 g / L]; the balance is water.

[0042] Example 1 Screening of target flora, isolation and verification of single colonies, and desulfurization effect for the microbial desulfurization method applied to agar and red algae plants Samples were taken from seawater and tidal flats in Qingdao, Shandong Province, lake water and soil on the west coast campus of Ocean University of China (including the original microorganisms on the raw material Gelidium amansii), and the original preserved flora in the laboratory. The samples were cultured in an inorganic sulfur-free liquid medium (enrichment culture in a 30 °C or 37 °C water bath). The medium used was a Gelidium amansii liquid screening medium with unsterilized Gelidium amansii as the sole sulfur source, and its composition was: 0.5% unsterilized Gelidium amansii, and the balance was an inorganic sulfur-free liquid medium.

[0043] After multiple transfers (multiple transfers were used to eliminate the influence of sulfur-containing compounds in the original samples), a flora that grew stably at 30 °C and 37 °C was screened. This flora was cultured for 5 days at 30 °C or 37 °C, and its growth curve is as Figure 1 shown (the ordinate is the OD of the culture solution 600 ). The flora was further screened and spread on a screening plate, as Figure 2 shown. Strains that could utilize the sulfate group on agar to grow were selected, and after re-selection based on the size and morphology of the colonies, they were streaked and purified on the plate 2 - 3 times. A total of 16 single colonies were screened and named: Z01 - Z08, Z71 - Z78.

[0044] Pick the above single colonies and explore the removal effect on sulfate groups on agar. Use the laboratory-preserved strains Bacillus megaterium WMX and PO1h-URA as fermentation controls: Inoculate the single bacteria into an inorganic sulfur-free liquid medium and culture for 24 hours, then inoculate into an agar strip liquid screening medium (prepared by adding 2 g of agar strips to 50 mL of inorganic sulfur-free liquid medium) and ferment at 30 °C or 37 °C for 5 days (strains Z01-Z08 are fermented at 30 °C, and strains Z71-Z78 are fermented at 37 °C). After fermentation, wash and separate the agar strips (initial agar) and the bacteria with ultrapure water, dry them, add 1 mol / L hydrochloric acid, and perform acid hydrolysis at 115 °C under high temperature and high pressure in an autoclave for 4 hours. After acid hydrolysis, filter with a 0.22 μm filter membrane, measure the content of free sulfate in the solution by ion chromatography, and calculate the agar desulfurization rate. The results are as Figure 3 shown.

[0045] Explore the effect on the gel strength of agar: Weigh 1.0% of the agar sample and add an appropriate amount of deionized water, heat and dissolve it in a microwave oven. Take 3 mL of the dissolved solution and place it in a cylindrical container with a diameter of 2 cm and a height of 1.5 cm. Cool it to solidify at room temperature and then age it overnight in a 4 °C refrigerator. Take it out the next day and use a texture analyzer to measure the gel strength of the agar. The detection parameters are: detection speed: 60 mm / min; puncture depth: 5 mm; probe radius: 3 mm. The results are as Figure 4 shown.

[0046] Figure 3 The results show that strains Z01, Z75, Z76, Z77, and Z78 grow well at 30 °C and 37 °C, and it can be further speculated that they can make good use of the sulfate groups in agar. After fermentation, measure the sulfur content and gel strength of the agar treated by microorganisms. After treatment with strains Z01, Z75, and Z78, compared with WMX (sulfur content 0.69%, gel strength 729.38 g / cm 2 ) and PO1h-URA (sulfur content 0.59%, gel strength 580.03 g / cm 2 ), their sulfur content decreases. The sulfur content of strain Z01 decreases from 1.18% to 0.31%, the sulfur content of strain Z75 decreases from 1.18% to 0.35%, and the sulfur content of strain Z78 decreases from 1.18% to 0.49%. The desulfurization rates are 73.73%, 70.34%, and 58.47% respectively; the gel strength increases significantly. The gel strength of strain Z01 increases from 362.02 g / cm 2 to 976.42 g / cm 2 , the gel strength of strain Z75 increases from 362.02 g / cm 2 to 952.45 g / cm 2 , and the gel strength of strain Z78 increases from 362.02 g / cm 2 to 817.44 g / cm2 (The determination of gel strength mentioned in this article is for a 1.0% content), and the gel strength increased by 169.71%, 163.09%, and 125.79% respectively. Strains such as Z02, Z05, and Z07 had no desulfurization effect on the sulfate groups on agar, and no further research was conducted subsequently.

[0047] After the above process, to prevent the strains from utilizing the degradation products during fermentation, it is necessary to verify whether the strains grow using agar as a carbon source. The above strains were respectively inoculated into an inorganic carbon-free liquid medium, and treated agar strips were added. The initial weight was recorded, and fermentation was carried out at 30 °C and 37 °C for 5 days. After completion, they were washed, separated, dried, weighed, and the sulfur content and gel strength were measured. The results showed that the selected single strains did not grow using agar as a carbon source and did not degrade the agar raw material.

[0048] Single colonies Z01, Z75, and Z78 with obvious desulfurization effects were picked, and 16S rDNA identification and NCBI comparison were carried out. The identification results were: single colonies Z01, Z75, and Z78 were Bacillus velezensis, Meyerozyma guilliermondii, and Achromobacter xylosoxidans respectively. These 3 wild-type single strains and the laboratory-preserved Yarrowia lipolytica PO1h-URA and Bacillus megaterium WMX strains were selected for subsequent experiments.

[0049] Example 2 Preparation of agarose by fermentation desulfurization of agar strips and agar powder with Yarrowia lipolytica Weigh 2 g of agar strip or 2 g of agar powder sample into a 250 mL conical flask, add 100 mL of 0.025% sodium hypochlorite solution, shake and sterilize in a constant temperature shaker for 40 min (30 °C, 220 rpm), take out and wash the agar three times with sterilized deionized water in a laminar flow hood, add 50 mL of deionized water, and shake in a constant temperature shaker (30 °C, 220 rpm) for 24 h to remove the residual chlorine.

[0050] Add 2 g each of the above agar strips and agar powder treated with sodium hypochlorite solution to 50 mL of inorganic sulfur-free liquid medium respectively to prepare an agar powder sulfur source inorganic medium and an agar strip sulfur source inorganic medium.

[0051] Take 5 μL of the bacterial liquid (Yarrowia lipolytica PO1h-URA) in the bacteria preservation tube and streak it on a YPD solid plate, place it in a constant temperature incubator at 30 °C for 24 h, pick the colonies on the plate, and shake-flask culture overnight (30 °C, 220 rpm) with an inorganic sulfur-free liquid medium to obtain a seed solution.

[0052] Take 2 portions of the seed solution (5 mL each) and inoculate them into 50 mL of agar powder sulfur source inorganic medium and 50 mL of agar strip sulfur source inorganic medium respectively, and culture them in a constant temperature shaker (30 °C, 220 rpm), and regularly measure the growth OD600 Value. After the strain grew to the stationary phase (fermentation for 12 h), the agar powder and agar strips were taken out, washed three times with deionized water, dried in an electrothermal blast drying oven, and after drying, the sulfur content and gel strength of the agar were measured for analysis and comparison.

[0053] The results are as Figure 5 shown. When using agar powder for fermentation desulfurization, its sulfur content decreased from 1.18% to 0.72%, a decrease of 39.36%, and the gel strength increased from 362.02 g / cm 2 to 471.81 g / cm 2 , an increase of 30.33%. When using agar strips for fermentation desulfurization, the sulfur content decreased from 1.18% to 0.58%, a decrease of 50.59%, and the gel strength increased from 362.02 g / cm 2 to 565.09 g / cm 2 , an increase of 56.09%.

[0054] Example 3: Continuous transfer desulfurization of agar by Yarrowia lipolytica to prepare agarose The quality of the agar after the first desulfurization by the strain was not high enough. In order to further improve the desulfurization rate and then improve the quality of the agar, making the desulfurized agar close to the properties of agarose and enabling effective application in the fields of biotechnology, etc., the present invention adopts a continuous transfer method to continue desulfurization. After the first desulfurization of the strain, the nutrients in the culture medium are consumed and the growth of the strain enters the decline phase. At this time, the treated agar is transferred into a new culture medium solution and a fresh seed solution with growth vitality, so as to realize the secondary desulfurization of the agar by the strain and then improve the quality of the agar.

[0055] Use Yarrowia lipolytica to desulfurize agar by continuous transfer: Inoculate 5 mL of the seed solution (prepared in Example 2) into 50 mL of an agar strip sulfur source inorganic medium, and perform shake flask culture in a constant temperature shaking incubator (30 °C, 220 rpm). During the fermentation process, regularly measure the growth OD 600 value. After the strain grew to the stationary phase (fermentation for 12 h), perform solid-liquid separation (pour out the liquid), add 50 mL of a new agar strip sulfur source inorganic medium and 5 mL of the seed solution, and repeat this transfer process until the strain no longer grows significantly (ferment for 4 times in total, that is, transfer continuously 3 times, and each fermentation is 12 h). The OD 600 values of Yarrowia lipolytica during fermentation 1-4 times are as Figure 6 shown, the sulfur content detection results of Yarrowia lipolytica during fermentation 1-4 times are as Figure 7 shown, and the gel strength detection results of Yarrowia lipolytica during fermentation 1-4 times are as Figure 8 shown.

[0056] From Figure 6It can be seen that when fermented once, the highest OD 600 value is 0.51, while when fermented 4 times, the OD 600 value is 0.31. As the number of fermentation times increases, the growth OD 600 value of Yarrowia lipolytica decreases successively, indicating that the sulfur in agar is continuously consumed by the strain. After 4 times of fermentation, Figure 7 、 Figure 8 show that the sulfur content of agar decreases from 1.18% to 0.04%, a decrease of 96.61%, and the gel strength increases from 362.02 g / cm 2 to 959.63 g / cm 2 , an increase of 165.08%. It can be seen that each time of subculture can effectively improve the properties of agar. Compared with the alkali method for desulfurization, the method of the present invention has the characteristics of environmental friendliness, can remove sulfate groups that cannot be removed by the alkali method for desulfurization, makes the desulfurization more thorough, and agar close to the quality of agarose can be obtained without separation and purification of agar gum and agarose.

[0057] Example 4 Preparation of agarose by continuous subculture desulfurization of agar by Bacillus megaterium WMX Bacillus megaterium WMX is a strain screened by the inventor's team in the early stage using agar as the sole sulfur source, and it has been verified that it can effectively improve the quality of agar. In order to further verify the effectiveness of the continuous subculture desulfurization method, agar strips were desulfurized by the continuous subculture method using Bacillus megaterium WMX. The higher the cell density of the strain, the more sulfur source it utilizes, and the more sulfate groups in agar can be removed.

[0058] The method of continuous subculture is the same as that in Example 3: 5 mL of the seed liquid of Bacillus megaterium WMX was inoculated into 50 mL of the agar strip sulfur source inorganic medium, and flask shaking culture was carried out in a constant temperature shaking incubator (30 °C, 220 rpm). During the fermentation process, the growth OD 600 value was measured regularly. After the strain grew to the stationary phase (fermentation for 12 h), the liquid was poured out, and 50 mL of the new agar strip sulfur source inorganic medium and 5 mL of the seed liquid were added. This subculture process was repeated until the strain no longer grew significantly (fermented 4 times in total, that is, subcultured 3 times, each fermentation for 12 h). The OD 600 values of Bacillus megaterium WMX during fermentation 1 - 4 times are as Figure 9 shown, and the sulfur content detection results and gel strength detection results of Bacillus megaterium WMX after 4 times of fermentation are as Figure 10 shown.

[0059] It can be seen from Figure 9 that when fermented once, the highest OD 600 value is 0.73, while when fermented 4 times, the OD 600The value is only 0.19. As the number of fermentation times increases, the growth OD of Bacillus megaterium WMX 600 value decreases successively, indicating that the sulfur in the agar is continuously consumed by the strain. It can be seen from Figure 10 that after 4 times of fermentation, the sulfur content of the agar decreases from 1.18% to 0.13%, a decrease of 88.98%. The gel strength increases from 362.02 g / cm 2 to 1067.78 g / cm 2 , an increase of 194.95%. And each time of subculture can effectively improve the properties of the agar. By comparison, the desulfurization rate of Bacillus megaterium WMX is lower than that of Yarrowia lipolytica, but the gel strength is higher than that of Yarrowia lipolytica, which indicates that using different microorganisms to desulfurize agar has different effects on the properties of agar.

[0060] Example 5 Continuous subculture desulfurization of agar by Bacillus velezensis to prepare agarose Bacillus velezensis is the optimal strain screened in the present invention. In Example 1, the screening result shows that the sulfur content is 0.31% and the gel strength is 976.42 g / cm 2 . In order to further verify the effectiveness of the continuous subculture desulfurization method, the agar strips were desulfurized by this strain using the continuous subculture method.

[0061] The method of continuous subculture is the same as that in Example 3: Inoculate 5 mL of the seed liquid of Bacillus velezensis into 50 mL of the agar strip sulfur source inorganic medium, and perform shake flask culture in a constant temperature shaking incubator (37 °C, 220 rpm). During the fermentation process, regularly measure the growth OD 600 value. After the strain grows to the stationary phase (fermentation for 12 h), pour out the liquid, add 50 mL of the new agar strip sulfur source inorganic medium and 5 mL of the seed liquid, and repeat this subculture process until the strain no longer grows significantly (ferment for 4 times in total, that is, subculture 3 times, and each fermentation is 12 h).

[0062] After 4 times of fermentation by Bacillus velezensis, the sulfur content of the agar decreases from 1.18% to 0.09%, a decrease of 92.37%. The gel strength increases from 362.02 g / cm 2 to 1202.76 g / cm 2 , an increase of 232.22%. The desulfurization rate of Bacillus velezensis is slightly lower than that of Yarrowia lipolytica but stronger than that of Bacillus megaterium WMX, and the gel strength is significantly higher than that of Yarrowia lipolytica and Bacillus megaterium WMX.

[0063] Example 6 Continuous subculture desulfurization of Gelidium amansii plants by Bacillus velezensis to prepare agarose Weigh 2 g of the washed and air-dried Gelidium amansii plants into a 250 mL conical flask, add 100 mL of 0.025% sodium hypochlorite solution, sterilize by shaking in a constant-temperature shaker for 40 min (30 °C, 220 rpm), take it out and wash it three times with sterilized deionized water in a laminar flow hood, add 50 mL of deionized water, and shake in a constant-temperature shaker (30 °C, 220 rpm) for 24 h to remove residual chlorine.

[0064] Add 2 g of the Gelidium amansii plants soaked in the sodium hypochlorite solution to 50 mL of inorganic sulfur-free liquid medium to prepare an inorganic medium for Gelidium amansii plants with sulfur source.

[0065] Inoculate 5 mL of the seed liquid of Bacillus velezensis into 50 mL of the inorganic medium for Gelidium amansii plants with sulfur source, and carry out shake flask culture in a constant-temperature shaking shaker (37 °C, 220 rpm). During the fermentation process, regularly measure the growth OD 600 value. After the strain grows to the stationary phase (fermentation for 12 h), pour out the liquid, add 50 mL of fresh inorganic medium for Gelidium amansii plants with sulfur source and 5 mL of seed liquid, and repeat this transfer process until the strain no longer grows significantly (ferment for 3 times in total, that is, transfer continuously for 2 times, and ferment for 12 h each time). Set a control group without inoculating Bacillus velezensis.

[0066] After fermentation, take out the Gelidium amansii plants, wash them three times with deionized water, mix them with water according to the solid-liquid ratio of 1:35, extract gum at 115 °C for 30 minutes, filter while it is hot with a 200-mesh gauze, cool and dry to obtain agarose, measure the sulfur content and gel strength of the agarose, and conduct analysis and comparison.

[0067] Results: After continuous transfer and fermentation for 2 times, the sulfur content in the Gelidium amansii plants decreased from 2.95% to 0.12%, a decrease of 95.93%, and the gel strength increased from 440.42 g / cm 2 to 1402.59 g / cm 2 , an increase of 218.47%, reaching the agarose level.

[0068] Example 7 Continuous transfer desulfurization of Gelidium amansii for agar extraction by Bacillus velezensis to prepare agarose Weigh the washed dry Gelidium amansii plants, mix them with water according to the solid-liquid ratio of 1:35, extract gum at 115 °C for 30 minutes, filter while it is hot with a 200-mesh gauze, cool and dry to obtain dry Gelidium amansii agar.

[0069] Weigh 2 g of dried Gracilaria agar into a 250 mL conical flask, add 100 mL of 0.025% sodium hypochlorite solution, sterilize it by shaking in a constant temperature shaker for 40 min (30 °C, 220 rpm), take it out and wash the agar three times with sterilized deionized water in a laminar flow hood, add 50 mL of deionized water, and shake it in a constant temperature shaker (30 °C, 220 rpm) for 24 h to remove residual chlorine.

[0070] Add 2 g of the Gracilaria agar soaked in the sodium hypochlorite solution to 50 mL of an inorganic sulfur-free liquid medium to prepare a Gracilaria agar sulfur source inorganic medium.

[0071] Inoculate 5 mL of the seed liquid of Bacillus velezensis into 50 mL of the Gracilaria agar sulfur source inorganic medium, and perform shake flask culture in a constant temperature shaking shaker (37 °C, 220 rpm). During the fermentation process, regularly measure the growth OD 600 value. After the strain grows to the stationary phase (fermentation for 12 h), pour out the liquid, add 50 mL of the new Gracilaria plant sulfur source inorganic medium and 5 mL of the seed liquid, and repeat this transfer process until the strain no longer grows significantly (ferment for 3 times in total, that is, transfer continuously for 2 times, and ferment for 12 h each time). Set a control group without inoculating Bacillus velezensis.

[0072] After fermentation, separate the solid and liquid to obtain desulfurized agarose. After drying, measure the sulfur content and gel strength of the agarose and conduct analysis and comparison.

[0073] Results: After continuous transfer fermentation for 2 times, the sulfur content in Gracilaria agar decreased from 2.92% to 0.06%, a decrease of 97.95%. The gel strength increased from 480.21 g / cm 2 to 1402.59 g / cm 2 , an increase of 192.08%, reaching the agarose level.

[0074] Example 8 Continuous transfer desulfurization of Gracilaria - extracted agar gel particles by Bacillus velezensis to prepare agarose Weigh the washed dried Gracilaria plants, mix them with water at a solid - liquid ratio of 1:35, extract the gum at 115 °C for 30 minutes, filter while it is hot with a 200 - mesh gauze to obtain agar liquid, and obtain Gracilaria agar gel particles after cooling.

[0075] According to the dry and wet weight standard curve, weigh the agar gel particles corresponding to 2 g of dry weight, crush them and add them to 100 mL of 0.025% sodium hypochlorite solution, shake and sterilize in a constant temperature shaker for 40 min (30 °C, 220 rpm), take out and wash the agar three times with sterilized deionized water in a laminar flow cabinet, add 50 mL of deionized water, and shake in a constant temperature shaker (30 °C, 220 rpm) for 24 h to remove residual chlorine.

[0076] Add 2 g of the agar gel particles of Gelidium amansii treated with sodium hypochlorite solution to 50 mL of inorganic sulfur-free liquid medium to prepare an inorganic medium with Gelidium amansii agar gel particles as the sulfur source.

[0077] Inoculate 5 mL of the seed liquid of Bacillus velezensis into 50 mL of the inorganic medium with Gelidium amansii agar gel particles as the sulfur source, and carry out shake flask culture in a constant temperature shaking shaker (37 °C, 220 rpm). During the fermentation process, regularly measure the growth OD 600 value. After the strain grows to the stationary phase (fermentation for 12 h), pour out the liquid, add 50 mL of the new inorganic medium with Gelidium amansii agar gel particles as the sulfur source and 5 mL of the seed liquid, and repeat this transfer process until the strain no longer grows significantly (ferment for 3 times in total, that is, transfer continuously for 2 times, and each fermentation is 12 h). Set a control group without inoculating Bacillus velezensis.

[0078] After fermentation, separate the solid and liquid to obtain desulfurized agarose, dry it and measure the sulfur content and gel strength of the agarose for analysis and comparison.

[0079] Results: After continuous transfer fermentation for 2 times, the sulfur content of the Gelidium amansii agar gel particles decreased from 2.89% to 0.14%, a decrease of 95.16%, and the gel strength increased from 420.66 g / cm 2 to 1252.59 g / cm 2 , an increase of 197.77%, reaching the agarose level.

[0080] Example 9 Preparation of agarose by continuous transfer desulfurization of Gelidium amansii plants with Meyerozyma guilliermondii According to the method of Example 6, inoculate the seed liquid of Meyerozyma guilliermondii into the inorganic medium with Gelidium amansii plants as the sulfur source, and the others are the same as in Example 6.

[0081] Results: After continuous transfer fermentation for 2 times, the sulfur content in the Gelidium amansii plants decreased from 2.95% to 0.13%, a decrease of 95.59%, and the gel strength increased from 440.42 g / cm 2 to 1202.59 g / cm 2 , an increase of 173.06%, reaching the agarose level.

[0082] Example 10 Preparation of agarose by continuous transfer desulfurization of agar extracted from Gelidium amansii by Meyerozyma guilliermondii According to the method of Example 7, the seed liquid of Meyerozyma guilliermondii was inoculated into the Gelidium amansii agar sulfur source inorganic medium, and the others were the same as in Example 7.

[0083] Result: After continuous transfer fermentation for 2 times, the sulfur content in Gelidium amansii agar decreased from 2.92% to 0.10%, a decrease of 96.58%, and the gel strength increased from 480.21 g / cm 2 to 1352.33 g / cm 2 , an increase of 181.61%, reaching the agarose level.

[0084] Example 11 Preparation of agarose by continuous transfer desulfurization of Gelidium amansii agar gel particles by Meyerozyma guilliermondii According to the method of Example 8, the seed liquid of Meyerozyma guilliermondii was inoculated into the agar gel particle sulfur source inorganic medium, and the others were the same as in Example 8.

[0085] Result: After continuous transfer fermentation for 2 times, the sulfur content in Gelidium amansii agar gel particles decreased from 2.89% to 0.08%, a decrease of 97.23%, and the gel strength increased from 420.66 g / cm 2 to 1222.50 g / cm 2 , an increase of 190.61%, reaching the agarose level.

[0086] Example 12 Comparison of the effects of different strains on the continuous transfer desulfurization of Gelidium amansii agar to prepare agarose Five strains (i.e., 3 wild-type single bacteria screened in Example 1, and the Yarrowia lipolytica PO1h-URA and Bacillus megaterium WMX strains preserved in the laboratory) were used for continuous transfer of Gelidium amansii agar. The method was the same as in Example 7. After two consecutive transfers, the measured results of the sulfur content and gel strength of Gelidium amansii agar are shown in Table 1.

[0087] Table 1 Measured results of sulfur content and gel strength

[0088] The results showed that Gelidium amansii agar treated by continuous transfer desulfurization fermentation with 5 strains could all meet the agarose index, and the treatment effect of Bacillus velezensis was the best.

[0089] Example 13 Gel electrophoresis results of agarose Weigh 1 g of agarose (the agarose prepared in Example 7), add it to 100 mL of 1×TAE solution (50×TAE diluted 50 times), add 5 μL of Redsafe staining solution, heat it in a microwave oven until completely dissolved, take it out and shake well. Slowly pour the dissolved solution into the gel-making plate with the sample comb inserted, wait for about 20 minutes to solidify, and then gently pull out the sample comb. Place the prepared gel in the nucleic acid electrophoresis tank, add 10 μL of sample (standard protein) to each well with a pipette, cover the lid of the nucleic acid electrophoresis tank, set the nucleic acid electrophoresis program to 150 V, 400 mA, 15 minutes. After the electrophoresis is completed, take out the gel and place it in the gel imaging system for observation and recording. Use agar from Gelidium amansii (prepared in Example 7) as a control and commercially available agarose as a control.

[0090] The schematic diagram of the agarose gel electrophoresis results is as Figure 11 shown. From the clarity and mobility of the nucleic acid electrophoresis, the commercially available agarose ( Figure 11 a in it) has the best effect, with the highest clarity and the longest migration distance at the same time. The agarose after desulfurization by microbial fermentation of the present invention ( Figure 11 b in it) has a clarity and migration distance similar to those of agarose, higher clarity than the original agar ( Figure 11 c in it), and faster migration speed. This is because after the sulfate groups on the agar are removed, the ion content decreases and its electroendosmosis decreases.

[0091] The above results show that the continuous transfer desulfurization by the microbial method has a good effect on the removal of sulfate groups in agar, and the desulfurized agar is close to the level of agarose. Compared with the chemical method for preparing agarose, it does not require separation and purification, saves the process flow and cost, and is of great significance for promoting the preparation of agarose by the microbial method, and has great research value and development prospects.

[0092] The above embodiments are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope disclosed herein. Modifications that are obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A method for preparing agarose based on microbial desulfurization, characterized in that: One of the following two methods: (1) placing agar in a culture solution, inoculating microorganisms, culturing at 25 to 40° C. for 20 to 48 hours, separating the solid from the liquid, and continuously transferring 1 to 3 times; the continuous transfer method is: after the solid-liquid separation, placing the obtained solid in a culture solution, inoculating microorganisms, and culturing at 25 to 40° C. for 20 to 48 hours; separating the solid from the liquid, and extracting to obtain desulfurized agarose; (2) placing the agar-containing plant or the extracted agar in a culture solution, inoculating the microorganism, culturing at 25 to 40° C. for 20 to 48 hours, separating the solid from the liquid, and continuously transferring 1 to 3 times; the continuous transfer method is: after the solid-liquid separation, placing the obtained solid in a culture solution, inoculating the microorganism, and culturing at 25 to 40° C. for 20 to 48 hours; separating the solid from the liquid, and extracting the desulfurized agarose; The microorganism is selected from one of the following five types: Yarrowia lipolytica PO1h-URA; Priestia megaterium OUC-Gel(QD)-W#DS#1-WMX; Bacillus velezensis; Meyerozyma guilliermondii; Achromobacter xylosoxidans.

2. The method for preparing agarose based on microbial desulfurization according to claim 1, characterized in that: In the above (1), the product form of agar is agar powder, agar flakes or agar strips.

3. The method for preparing agarose based on microbial desulfurization according to claim 1, characterized in that: In (2), the agar-containing plant is selected from any one or more of Gracilaria, Agar, and Porphyra; and the plant is a fresh plant or a dried plant.

4. The method for preparing agarose based on microbial desulfurization according to claim 1, characterized in that: In the above (2), the extracted agar is extracted from agar-containing plants.

5. The method for preparing agarose based on microbial desulfurization according to claim 4, characterized in that: The extraction method is as follows: take agar-containing plant plants, mix them with water in a solid-liquid ratio of 1:35, extract the gel at 100-115°C for 30 minutes, filter with 200-mesh gauze while hot, cool to obtain agar gel particles, or cool and dry to obtain dry agar.

6. The method for preparing agarose based on microbial desulfurization according to claim 1, characterized in that: The components of the culture medium are as follows: 2% glucose; KH2PO4, 4.0 g / L; NH4Cl, 12.16 g / L; K2HPO4, 3.19 g / L; MgCl2·6H2O, 0.83 g / L; CaCl2·2H2O, 4.5×10 -2 g / L; biotin, 5μg / L; VB1, 5μg / L; disodium ethylenediaminetetraacetic acid dihydrate, 0.05 g / L; FeCl3, 0.0083 g / L; ZnCl2, 0.0004 g / L; CuCl2·2H2O, 0.00013 g / L; CoCl2·6H2O, 0.0001 g / L; H3BO3, 0.0001 g / L; MnCl2·4H2O, 0.000016 g / L; Na2MoO4, 0.0003g / L; the balance is water.

7. The method for preparing agarose based on microbial desulfurization according to claim 1, characterized in that: In (1), the amount of agar added to the culture medium is 0.1% to 5%; Or: In (2), the amount of agar-containing plant plants added to the culture solution is 0.2% to 5%.

8. The method for preparing agarose based on microbial desulfurization according to claim 1, characterized in that: The specific preparation method is: add 2 g of agar powder, agar strips, agar slices, agar gel particles or dry agar to 50 mL of culture medium, add 5 mL of microbial culture solution, and culture at 30°C or 37°C for 24 to 48 hours; separate the solid and liquid to obtain desulfurized agarose.

9. The method for preparing agarose based on microbial desulfurization according to claim 1, characterized in that: The specific preparation method is as follows: add 2 g of dry plants to 50 mL of culture medium, add 5 mL of microbial culture medium, and culture at 30°C or 37°C for 48 hours; separate the solid with filter cloth, mix the solid with water at a solid-liquid ratio of 1:35, and extract the gel at 115°C for 30 minutes; filter with 200-mesh gauze while hot, and cool and dry to obtain agarose.

10. Agarose prepared by the method for preparing agarose based on microbial desulfurization according to any one of claims 1 to 9.

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