CN114214234A - Low-molecular-weight gellan gum production strain and screening method and application thereof - Google Patents
Low-molecular-weight gellan gum production strain and screening method and application thereof Download PDFInfo
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- A23L29/269—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of microbial origin, e.g. xanthan or dextran
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Abstract
The invention relates to the technical field of bioengineering, in particular to a low molecular weight gellan gum producing strain and a screening method and application thereof. The invention utilizes UV-ARTP composite mutagenesis technology to carry out mutagenesis on sphingomonas paucimobilis, and obtains a mutant strain M155 for producing low molecular weight gellan gum. Compared with the original strain, the gellan gum produced by the strain has the advantages that the yield is improved by 24 percent, and the molecular weight is reduced by 61 percent; and the mutant has stable genetic property. The produced low molecular weight gellan gum has stable performance, has the gel characteristics of low viscosity, high elasticity, low hardness and the like, and has excellent moisturizing effect compared with high molecular weight gellan gum; can be used in the fields of cosmetics and medicines; meanwhile, the low molecular weight gellan gum is low in adhesion and high in chewiness, is not sticky to teeth and chewy when being eaten, can endow food with better mouthfeel, and has wide application prospects in the fields of food and health care product manufacture.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a low molecular weight gellan gum producing strain and a screening method and application thereof.
Background
Gellan gum is made of gram-negative bacteriaSphingomonas paucimobilis ATCC31461 produces extracellular polysaccharide with molecular weight of 0.5-1.0 × 106Da. The microbial exopolysaccharide is used as an industrial product for microbial fermentation, and has wide application prospects in the fields of food, chemical engineering, medicine and the like. In the food industry, gellan gum is used as a thickener, stabilizer, suspending agent, gelling agent, etc. in foods such as jam, sausage, ice cream, salad dressing, jelly, dairy products, etc. In the field of medicine, gellan gum can be applied to eye drops, sustained-release medicines, coatings, tissue engineering scaffold materials and other products. In the chemical field, gellan gum can be used as an adhesive, toothpaste, air freshener, etc. At present, gellan gum is the latest one of the series of food gels available on the market, and the global demand is increasing. Therefore, the development and production of the gellan gum have extremely high commercial economic significance and market prospect.
The molecular weight is one of the basic parameters for characterizing the characteristics of the gellan gum, and is also an important index for quality detection in industrial production. However, at present, there are few reports on low molecular weight gellan gum, and the precise application of low molecular weight gellan gum is greatly limited. The traditional method for preparing low molecular weight gellan gum is a hydrolysis process comprising: physical method, chemical method and enzymolysis method, but the hydrolysis processes are mostly uncontrollable, the molecular weight distribution range of the obtained gellan gum is wide, the gellan gum is randomly distributed, the product efficacy is influenced by direct use, and the application effect of the gellan gum is seriously influenced. If the low molecular weight gellan gum with narrow molecular weight distribution is to be obtained, the fractional purification method is required again, so that the production cost is increased, the product yield is reduced, and finally the sale price of the gellan gum is high, and the application range of the gellan gum is severely restricted.
Disclosure of Invention
In view of the above, in order to solve one of the above technical problems, the present invention provides a low molecular weight gellan gum producing strain, and a screening method and application thereof. The mutant strain can be used for continuously producing the low-molecular-weight gellan gum, the produced low-molecular-weight gellan gum has uniform molecular weight distribution and lower production cost, and an application foundation is laid for the precise application of the low-molecular-weight gellan gum.
In order to realize the purpose, the invention adopts the following technical scheme:
the invention provides a low molecular weight gellan gum producing strain named asSphingomonas paucimobilssM155 (referred to as mutant strain M155 for short), which is preserved in the China Wuhan type culture Collection with the preservation number of CCTCC NO: M20211430 and the preservation unit address of: the eight-channel Wuhan university No. 299 in Wuchang district, Wuhan city, Hubei province has a preservation date of 11 months and 17 days in 2021.
The invention uses sphingomonas paucimobilis (Sphingomonas paucimobilis) ATCC31461 is the original strain, and is obtained by screening after mutagenesis treatment. Compared with the original strain, the molecular weight of gellan gum produced by the mutant strain M155 is reduced by 61%, the yield is improved by 24%, and the hereditary character in ten generations of passage is stable.
In a specific embodiment, the invention also provides a method for breeding mutant strains, wherein the breeding method comprises the steps of carrying out compound mutagenesis by using UV and ARTP, and breeding a mutant strain which produces low-molecular-weight gellan gum and is stable in heredity through primary screening, secondary screening and verification.
The breeding method comprises the following steps:
(1) preparation of bacterial suspension: inoculating the starting strain into seed culture medium, shake culturing to logarithmic phase, taking out bacterial suspension, diluting with 0.9% NaCl solution to obtain bacterial liquid OD600In the range of 0.6-0.7; obtaining mutagenic bacteria suspension;
(2) ultraviolet mutagenesis and ARTP mutagenesis;
(3) primary screening: picking large, round and smooth-surfaced faint yellow single bacterial colony, activating, fermenting and culturing to produce crude gellan gum, and screening according to the gum yield compared with the original strain.
(4) Re-screening: selecting mutant strains with obviously lower molecular weight and more stable passage and identifying the stability of the strains.
Preferably, the ultraviolet mutagenesis time is 150 s, and the ARTP mutagenesis time is 25 s.
The breeding method comprises the following specific steps:
(1) UV-ARTP complex mutagenesis treatment: inoculating a starting strain from a solid culture medium to a 250 mL conical flask filled with 50 mL of seed liquid, performing shake culture at 30 ℃ and 220 r/min to logarithmic phase, taking out the bacterial suspension in the conical flask, diluting the bacterial suspension with 0.9% NaCl solution to obtain a mutant bacterial suspension, and making the OD of the bacterial solution of the mutant bacterial suspension600In the range of 0.6-0.7. And (3) sucking 5 mL of bacterial suspension mixed with the mutagenic bacterial suspension and 5% (v/v) glycerol according to the proportion of 1:1 into a sterilized glass plate with the diameter of 9 cm, and carrying out mutagenesis in an ultraviolet full-automatic mutagenic instrument under the action of magnetic stirring, wherein the wavelength is 254 nm, and the mutagenesis time is 150 s. Uniformly coating 20 mu L of the bacterial suspension subjected to ultraviolet mutagenesis on a metal slide, placing the metal slide in an ARTP mutagenesis instrument, setting the power to be 120W, the ventilation capacity to be 10 SLM and the processing time to be 25 s, placing the processed metal gasket in an EP tube filled with 980 mu L of sterile water, diluting according to ten times of gradient after fully shaking, coating 100 mu L of proper gradient diluent on a solid culture medium, and culturing for 3 d in an incubator at 30 ℃.
(2) Screening of mutagenized strains
a. Primary screening: performing primary screening treatment on the colony subjected to composite mutagenesis, selecting a large, round and smooth-surface faint yellow single colony, inoculating the faint yellow single colony into a seed solution, performing shake culture at 30 ℃ and 220 r/min to the late logarithmic phase, inoculating the seed solution cultured to the logarithmic phase into 50 mL of liquid fermentation medium according to the inoculum size of 10%, and performing constant-temperature culture at 30 ℃ and 220 r/min for 72 h to obtain a fermentation liquid; diluting the fermentation liquor ten times in volume, heating in a constant-temperature water bath kettle at 95 ℃ for 15 min, adding 2-3 times of 95% ethanol while hot, precipitating with ethanol at 4 ℃ overnight, centrifuging at 4000 r/min for 15 min, removing supernatant, collecting precipitate, and drying at 60 ℃ to constant weight to obtain crude gellan gum product with gum yield as the gum content in the fermentation liquor per unit volume.
b. Re-screening: activating and fermenting mutant strains with the primarily screened gellan gum yield changed remarkably, collecting gellan gum samples produced by different strains, and re-screening; calculating the intrinsic viscosity [ eta ] of each gellan gum sample by using Ubbelohde viscometer]According to the formula [ eta]K is 1.16 × 10 = kM α, k-3Taking alpha as 0.67 to calculate the viscosity-average molecular weight of the gellan gum; selecting mutant strains with obviously lower molecular weight and more stable passage.
(3) Determination of molecular weight and yield stability of excellent mutant strain
Selecting excellent mutant strains from a solid plate, inoculating the excellent mutant strains to a solid culture medium, culturing at the temperature of 30 ℃ for 2 d, recording as 1 generation, and subculturing the excellent mutant strains to 10 generations on the solid culture medium plate under the same condition; and (3) measuring the yield and the molecular weight of the 1-10 generation excellent mutant strain according to the primary screening and secondary screening methods of the mutant strain in the step (2).
The invention improves the mutation efficiency through UV-ARTP composite mutagenesis, and has strong mutagenesis purpose and high practicability. The optimized mutagenesis conditions of the invention are as follows: the ultraviolet mutagenesis time is 150 s, and the ARTP mutagenesis time is 25 s. Through UV and ARTP 2 mutagenesis, the mutagenesis lethality rate is more than or equal to 90 percent. Under the condition, the mutagenic strain producing the low molecular weight gellan gum is more easily obtained.
The invention also provides application of the mutant strain M155 in producing low molecular weight gellan gum.
The invention also provides application of the mutant strain M155 in preparation of food, cosmetics, health products or medicines.
Compared with the prior art, the invention has the beneficial effects that:
the invention utilizes ultraviolet mutagenesis and normal pressure room temperature plasma composite mutagenesis technology (UV-ARTP) to carry out mutagenesis on sphingomonas paucimobilis to obtain a mutant strain M155 for producing low molecular weight gellan gum. Compared with the original strain, the gellan gum produced by the strain has the advantages that the yield is improved by 24%, the molecular weight is reduced by 61%, and the mutant has stable genetic performance. The produced low molecular weight gellan gum has stable performance, has the gel characteristics of low viscosity, high elasticity, low hardness and the like, and has excellent moisturizing effect compared with high molecular weight gellan gum; can be used in the fields of cosmetics and medicines; meanwhile, the low molecular weight gellan gum is low in adhesion and high in chewiness, is not sticky to teeth and chewy when being eaten, can endow food with better mouthfeel, and has wide application prospects in the fields of food and health care product manufacture. Therefore, the invention lays an important theoretical and application foundation for the industrial and accurate production of the low molecular weight gellan gum and the accurate application thereof.
Drawings
FIG. 1 is a graph of lethality; in the figure, A is an ultraviolet mutagenesis lethality curve, and B is an ARTP lethality curve;
FIG. 2 is a graph comparing gellan gum production after primary screening of mutant strains;
FIG. 3 is a graph showing the viscosity-average molecular weight of gellan gum produced by rescreened mutant strains;
FIG. 4 is a genetic stability plot of viscosity average molecular weight and yield over ten passages of mutant strain M155;
FIG. 5 is a graph of elution patterns and molar masses of L-GG versus I-GG;
FIG. 6 is a graph comparing the yields of L-GG and I-GG;
FIG. 7 is a graph of the rheology of L-GG versus I-GG, where A is a static apparent viscosity graph and B is a dynamic rheology graph;
FIG. 8 is a qualitative graph of L-GG and I-GG.
Detailed Description
The invention is further illustrated by the following examples: those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention. The experimental methods in the examples, which are not indicated for specific conditions, are carried out according to conventional conditions; the reagents and biomaterials used, unless otherwise specified, are commercially available.
Starting strains: sphingomonas paucimobilis ATCC31461, purchased from American Type Culture Collection (ATCC) under the accession number ATCC @ 31461.
Seed culture medium: 1 g/L of yeast powder, 3 g/L of beef extract, 5 g/L, NaCl 5 g/L of tryptone and 5 g/L of sucrose.
Fermentation medium: 30 g/L of sucrose, 0.2 g/L of yeast powder and 2 g/L, KH g of tryptone2PO4 1 g/L、K2HPO41.5 g/L、MgSO4 0.6 g/L。
Example 1:
(1) UV-ARTP composite mutagenesis treatment
a. Preparation of mutagenic bacteria suspension: selecting a loopful of original strain from a solid culture medium, inoculating the loopful of original strain into a 250 mL conical flask filled with 50 mL seed culture medium, performing shake culture at 30 ℃ and 220 r/min until the logarithmic phase, taking out the bacterial suspension in the conical flask, and diluting the bacterial suspension by using 0.9% NaCl solution to ensure that the OD (origin-to-degree) of bacterial liquid of the bacterial suspension is600In the range of 0.6-0.7; obtaining mutant bacterial suspension.
b. Ultraviolet mutagenesis: mixing the mutagenic bacteria suspension and 5% (v/v) glycerol according to the proportion of 1:1, placing the mixture in a vortex oscillator under the aseptic condition for oscillation and shaking up, taking 5 mL of the bacteria suspension, placing the bacteria suspension in a sterilized plate with the diameter of 9 cm, and carrying out mutagenesis in an ultraviolet full-automatic mutagenic instrument under the action of magnetic stirring, wherein the wavelength is set to be 254 nm, and the mutagenesis time is respectively set to be 30, 60, 90, 120, 150, 180 and 210 s. Diluting the ultraviolet mutation treated bacterial liquid according to a ten-fold gradient, and taking a proper gradient (the dilution gradient is 10)-4The number of colonies on the solid medium grown with the bacterial suspension on the gradient was within 300) 100. mu.L of the diluted solution was applied to the solid medium and cultured in an incubator at 30 ℃ for 3 days. The best mutagenesis treatment condition is determined according to a lethality curve by taking sphingomonas paucimobilis without mutagenesis treatment as a control. Lethality (%) = (number of colonies of control group-number of colonies of treatment group)/number of colonies of control group × 100%.
ARTP mutagenesis: mixing the mutant bacterial suspension with 5% (v/v) glycerol at a ratio of 1:1, and placing in a vortex oscillator under aseptic conditionShaking, uniformly smearing 20 μ L of the bacterial suspension on a metal slide, placing in an ARTP mutagen apparatus, setting power at 120W, ventilation at 10 SLM, and processing for 10, 15, 20, 25, 30 and 35 s. Placing the treated metal gasket into an EP tube filled with 980 mu L of sterile water, fully vibrating, diluting according to a tenfold gradient, and taking the dilution gradient as 10-4The diluted solution (2) was applied to a solid medium in an amount of 100. mu.L, and cultured in an incubator at 30 ℃ for 3 days. And (3) determining the optimal mutagenesis treatment condition according to a lethality curve by taking the original strain sphingosine paucimobilis thalli as a control.
FIG. 1 is a graph of lethality; in the figure, A is an ultraviolet mutagenesis lethality curve, and B is an ARTP lethality curve; as can be seen from FIG. 1, when the UV mutagenesis time is 150 s, the lethality is 84%, and when the mutagenesis time is 180 s, the lethality is 91%; when the ARTP mutagenesis time is 25 s, the lethality rate is 90 percent; in order to ensure the survival of more strains, the ultraviolet mutagenesis time is selected to be 150 s in the subsequent mutagenesis process, and the ARTP mutagenesis time is selected to be 25 s as the proper mutagenesis dosage.
d. Compound mutagenesis: mixing the mutagenic bacteria suspension and 5% (v/v) glycerol according to the proportion of 1:1, placing the mixture in a vortex oscillator under the aseptic condition for oscillation and shaking up, taking 5 mL of the shaken bacteria suspension, placing the bacteria suspension into a sterilized glass plate with the diameter of 9 cm, and carrying out mutagenesis in an ultraviolet full-automatic mutagenic apparatus under the action of magnetic stirring, wherein the wavelength is 254 nm, and the mutagenesis time is 150 s. Uniformly coating 20 mu L of bacterial suspension subjected to ultraviolet mutagenesis on a metal slide, placing the metal slide in an ARTP mutagenesis instrument, setting the power to be 120W, the ventilation capacity to be 10 SLM and the processing time to be 25 s, placing the processed metal gasket in an EP tube filled with 980 mu L of sterile water, fully vibrating, diluting according to ten times of gradient, and taking the dilution gradient to be 10 mu L-4The diluted solution (2) was applied to a solid medium in an amount of 100. mu.L, and cultured in an incubator at 30 ℃ for 3 days.
(2) Screening of mutagenized strains
a. Primary screening: and (3) carrying out primary screening treatment on the colony subjected to composite mutagenesis, selecting a large, round and smooth-surface faint yellow single colony, inoculating the faint yellow single colony into a seed solution, carrying out shake cultivation at 30 ℃ and 220 r/min until the late logarithmic phase, inoculating the seed solution cultured to the logarithmic phase into 50 mL of liquid fermentation medium according to the inoculum size of 10%, and carrying out constant-temperature cultivation for 72 h at 30 ℃ and 220 r/min to obtain a fermentation liquid. Diluting the fermentation liquor ten times in volume, heating in a constant-temperature water bath kettle at 95 ℃ for 15 min, adding 2-3 times of 95% ethanol while hot, precipitating with ethanol at 4 ℃ overnight, centrifuging at 4000 r/min for 15 min, removing supernatant, collecting precipitate, and drying at 60 ℃ to constant weight to obtain crude gellan gum product with gum yield as the gum content in the fermentation liquor per unit volume. After UV-ARTP mutagenesis, 210 mutant strains are obtained in total, wherein 54 mutant strains with high or low glue yield are stored for subsequent verification. For comparison, the crude product of gellan gum of the original strain was prepared by the same method, and the yield was calculated to be 4.56 g/L.
FIG. 2 is a graph comparing gellan gum production after primary screening of mutant strains; as can be seen from FIG. 2, compared with the gellan gum yield of the original strain of 4.56 g/L, the gellan gum yield produced by fermentation of 54 mutant strains is significantly changed, and the mutant strain can be used for subsequent re-screening.
b. Re-screening: and (3) activating and fermenting the 54 mutant strains with the gellan gum yield changed remarkably after primary screening, and collecting gellan gum samples produced by different strains. Weighing a certain amount of crude gellan gum, and adding 50 mmol/L DMSO/NaNO3As a solvent, a gellan gum solution with a gellan gum concentration of 1 mg/mL was prepared. Magnetically stirring in a constant-temperature magnetic stirring water bath kettle at 80 deg.C for 2 hr to dissolve gellan gum. With DMSO/NaNO3Adding 9.0 mL of solvent into the solution (50 mmol/L) as solvent, and measuring the outflow time t in a constant temperature bath at the constant temperature of 25 +/-0.1 DEG C0Repeating the steps for three times, wherein the flow-out time of each time is different from 0.2 second or more, and taking the average value. Then adding 1.0 mL of 1 mg/mL gellan gum solution, diluting to 0.1 mg/mL gellan gum solution, and measuring the flow-out time t by the same method1. Respectively calculating the intrinsic viscosity [ eta ] of each gellan gum solution by a single-point method]According to the formula [ eta]K is 1.16 × 10 = kM α, k-3And α is 0.67. The viscosity average molecular weight of the gellan gum was calculated. Selecting mutant strains with obviously lower molecular weight and more stable passage.
FIG. 3 is a graph showing the viscosity-average molecular weight of gellan gum produced by rescreened mutant strains; as can be seen from FIG. 3, the viscosity average molecular weight of the mutant strain M155 was 12444.66. + -. 350.51 Da, which was lower than that of most other mutant strains. The strain has high gellan gum yield, meets the industrial production requirement of low molecular weight gellan gum, and selects the high-yield low molecular weight mutant strain M155 as an excellent strain.
(3) Determination of molecular weight and yield stability of Excellent mutant strains
After picking out the mutant strain M155 from the solid plate, inoculating the mutant strain into a solid medium, culturing at 30 ℃ for 2 d, recording as 1 generation, and carrying out subculture on the solid medium plate to 10 generations under the same condition. And (3) respectively carrying out yield and molecular weight determination on the mutant strains M155 of 1-10 generations according to the mutant strain screening method in the step (2). FIG. 4 is a genetic stability plot of viscosity average molecular weight and yield over ten passages of mutant strain M155; as can be seen from FIG. 4, the mutant strain has relatively stable yield and molecular weight generation within ten generations of M155. The obtained mutant strain M155 has the viscosity average molecular weight of 12444.66 +/-350.51 Da, the average yield of 5.68 +/-0.09 g/L and the improvement of 24 percent.
The strain is preserved in the Wuhan type culture preservation center in China, the preservation number is CCTCC NO: M20211430, and the preservation unit address is as follows: the eight-channel Wuhan university No. 299 in Wuchang district, Wuhan city, Hubei province has a preservation date of 11 months and 17 days in 2021.
Example 2: preparation of low molecular weight gellan gum
(1) The activation and seed liquid culture method comprises the following steps: selecting a ring of bacteria from a solid culture medium at 4 ℃, streaking the bacteria on the solid culture medium, culturing the bacteria for 2 d at 30 ℃, selecting a ring of single colony, inoculating the single colony into a seed culture medium, and performing shake culture at 30 ℃ and 200 r/min until the late logarithmic phase.
(2) The liquid fermentation culture method comprises the following steps: transferring the seed liquid into a fermentation culture medium according to the inoculation amount of 10% (V/V), and performing shake cultivation at 30 ℃ and 220 r/min for 3 d.
(3) Culturing the mutant strain M155 by activated seed liquid, carrying out liquid shake flask fermentation to obtain fermentation liquid, diluting the fermentation liquid by ten times of volume, heating for 15 min in a constant-temperature water bath kettle at 95 ℃, adding 2-3 times of volume of 95% ethanol while the fermentation liquid is hot, carrying out alcohol precipitation at 4 ℃ overnight, centrifuging for 15 min at 4000 r/min, removing supernatant, collecting precipitate, and drying at 60 ℃ to constant weight to obtain a crude gellan gum product. Weighing a certain amount of crude gellan gum product, preparing the crude gellan gum product into a solution of 2 mg/mL, removing proteins by using a trichloroacetic acid method, dialyzing by using a dialysis bag with the molecular weight cutoff of 6000-plus 8000 Da, changing water once every 4 h, dialyzing for 48 h, collecting gellan gum polysaccharide solution in the dialysis bag, and drying in vacuum to obtain low-molecular-weight gellan gum (L-GG).
For comparison, the gellan gum (I-GG) is produced by fermentation by using the original strain ATCC31461 as a raw material by adopting a preparation method which is the same as that of the L-GG preparation process.
Example 3: measurement of Gellan gum molecular weight and yield
(1) Determination of viscosity average molecular weight (Da): weighing a certain amount of crude gellan gum, and adding 50 mmol/L DMSO/NaNO3And (3) preparing a gellan gum solution with a final gellan gum concentration of 1 mg/mL as a solvent, and magnetically stirring for 2 hours in a constant-temperature magnetic stirring water bath kettle at 80 ℃ to fully dissolve the gellan gum for later use. 9.0 mL DMSO/NaNO was added to the Ubbelohde viscometer3The solution (50 mmol/L) was subjected to a constant temperature of 25. + -. 0.1 ℃ in a thermostatic bath to determine the flow-out time t0Repeating the steps for three times, wherein the flow-out time of each time is different from 0.2 second or more, and taking the average value. Adding 1.0 mL of gellan gum solution, diluting to 0.1 mg/mL gellan gum solution, and measuring the outflow time t by the same method1. Measuring the intrinsic viscosity [ eta ] of gellan gum solution by single-point method]According to the formula [ eta]The viscosity-average molecular weight of the gellan gum was calculated by = kM α, and the results are shown in table 1. k is 1.16 × 10-3And α is 0.67.
(2) Weight average molecular weightMwAnd number average molecular weightMnThe determination of (1): method for separately measuring two types of gellan gum by using size exclusion chromatography combined with multi-angle laser scattering method (SEC-MALLS)Mw、Mn. The chromatographic conditions are as follows: mobile phase 0.1 mol/L NaCl + 0.02 mol/L sodium azide; the flow rate is 0.5 mL/min; column temperature: 35 ℃ is carried out.
Table 1 is a comparison table of molecular weight determinations of I-GG and L-GG. As shown in Table 1, the viscosity-average molecular weight of gellan gum (L-GG) produced by fermentation with the mutant strain M155,MwAndMnout ofThe molecular weight of the gellan gum (I-GG) produced by the strain is respectively reduced to a large extent, specifically 37.8%, 44.6% and 61.0%. It can be seen that the gellan gum produced by the mutant strain M155 has a lower molecular weight, and a low molecular weight gellan gum is obtained in the present application.
TABLE 1 molecular weights of I-GG and L-GG
| Sample (I) | I-GG | L-GG |
| Viscosity average molecular weight (Da) | 20014±401.97 | 12444.66±350.51 |
| Mw (g/mol) | 2.079×105 | 1.151×105 |
| Mn (g/mol) | 2.074×105 | 8.092×104 |
| Mw/Mn | 1.003 | 1.423 |
The molecular weights of the two gellan gums were analyzed by Size Exclusion Chromatography (SEC). The L-GG and the I-GG are respectively put into a 0.1M sodium chloride aqueous solution, and SEC measurement is carried out in an environment at 25 ℃. FIG. 5 is a graph of the elution pattern and molar mass of L-GG versus I-GG. As shown in fig. 5, a single peak close to normal distribution without a shoulder was observed in the SEC elution mode, indicating that they have relatively high uniformity within the defined molar mass range and that the molecular weight distribution is also relatively uniform.
(3) And (3) measuring the yield of the gellan gum: weighing a proper amount of gellan gum fermentation liquor respectively, diluting by ten times of volume, heating in a constant-temperature water bath kettle at 95 ℃ for 15 min, adding 2-3 times of volume of 95% ethanol to precipitate gellan gum, precipitating overnight at 4 ℃, centrifuging at 4000 r/min for 15 min, discarding supernatant, collecting precipitate, drying at 60 ℃ to constant weight, and obtaining a crude product of gellan gum, wherein the gum yield is the gum content in the fermentation liquor per unit volume. FIG. 6 is a graph comparing the yields of L-GG and I-GG; as shown in fig. 6, the crude gum yield of the mutant strain M155 was increased by 24% over that of the starting strain. The yield of the mutant strain is greatly improved, and a foundation is laid for the subsequent industrial production of the low molecular weight gellan gum.
Example 3: 16S DNA sequence analysis of mutant M155 strain producing low molecular weight gellan gum
The sequences of rDNA 16s primers 27F and 1492R are shown in SEQ ID NO: 1. SEQ ID NO: 2, namely: 5 '-agaagtttgattctcggctcag-3' and 5 '-ggttactctgttacgagctt-3'; carrying out PCR amplification on a mutant strain M155 for producing low-molecular-weight gellan gum, wherein the reaction conditions are as follows: pre-denaturation at 94 ℃ for 10 min, denaturation at 94 ℃ for 30 s, annealing at 58 ℃ for 30 s, and extension at 72 ℃ for 40 s, and the cycle is 34 times. And (3) after the PCR product is verified to be qualified by agarose gel electrophoresis, sending the PCR product to Shanghai Jili biotechnology limited company for sequencing. Sequencing results were analyzed by blast software alignment in NCBI. The nucleotide sequence of the mutant strain M155 is shown as SEQ ID NO: 3, mutant strain M155 and Sphingomonas paucimobilis: (Sphingomonas elodeaATCC 31461) was 100%, indicating that the mutant strain was still sphingomonas paucimobilis.
Example 4: rheological Properties of Low molecular weight Gellan produced by mutant Strain M155
Respectively taking original gellan gum (I-GG) obtained by shaking flask fermentation of an original strain and low molecular weight gellan gum (L-GG) obtained by shaking flask fermentation of a mutant strain M155 as samples. Accurately weighing 0.3 g of gellan gum sample, adding 100 mL of deionized water, swelling for 24 h at room temperature, placing in a constant-temperature magnetic stirring water bath kettle at 80 ℃, magnetically stirring for 2 h until the gellan gum sample is completely dissolved, and heating 5% CaCl while hot after the sample is dissolved21 mL of the solution was added and 80 ℃ deionized water was added to replace the water lost by evaporation. And respectively placing the prepared samples on a rheometer test board, wherein the rheometer test adopts a parallel plate mode, and the diameter of a flat plate is 40 mm, and performing static rheological property and dynamic viscoelasticity test.
FIG. 7 is a graph of the rheology of L-GG versus I-GG; wherein A is a static apparent viscosity map and B is a dynamic rheological profile; as shown in fig. 7, the low molecular weight gellan gum prepared by the present invention has an overall reduced apparent viscosity, gradually exhibits fluid characteristics, and forms a gel having a higher fluidity than the virgin gellan gum. The G ' of both I-GG and L-GG is greater than G ", and the G ' and G ' of L-GG are both lower than that of I-GG, and it can be seen that the water content in L-GG is higher.
Gellan gum with higher apparent viscosity has the effects of difficult stirring, difficult filtration, prolonged production cycle, etc. in cosmetic production applications. And the humectant made of the high-molecular gellan gum only forms a breathable film on the surface of the skin, so that the skin is smooth and moist, but the humectant is not easy to permeate the skin and is absorbed by the skin. The low molecular weight gellan gum can well permeate skin due to small molecular weight and low apparent viscosity, has better skin absorption, better moisturizing effect and better internal water content. The low-molecular gellan gum prepared by the method can improve the convenience of industrial operation, is more suitable for industrial production, and has wide application prospect in the field of medicine and beauty.
Example 5: this example shows the texture characteristics of low molecular weight gellan gum produced by mutant strain M155
Original gellan gum (I-GG) obtained by shake flask fermentation of an original strain and low molecular weight gellan gum (L-GG) obtained by shake flask fermentation of a mutant strain M155 are taken as samples. Respectively weighing 0.3 g of gellan gum sample, adding 100 mL of deionized water, swelling for 24 h at room temperature, placing in a 80 ℃ constant-temperature magnetic stirring water bath kettle, magnetically stirring for 2 h until the gellan gum sample is completely dissolved, adjusting the pH to 10 by using 0.1 mol/L NaOH solution, preserving the heat at 80 ℃ for 20 min, adding 80 ℃ deionized water to supplement water lost due to evaporation, subpackaging in a 30 mm x 30 mm flat dish, placing in a 4 ℃ refrigerator for 48 h, demolding, and carrying out texture determination.
The food physical property instrument is adopted for testing, the selected clamp is SMSP/25 (a cylindrical probe with the diameter of 25 mm), the test mode is Simplified TPA. PRJ, and the specific test conditions are as follows: the speed before measurement is 2.0 mm/s, the test speed is 1.0 mm/s, the speed after measurement is 1 mm/s, the deformation is 2 mm, the residence time between two times of compression is 2 s, and the trigger force is 0.049N. The samples were allowed to equilibrate at room temperature for 0.5 h before the relevant performance tests were performed. FIG. 8 is a qualitative graph of L-GG and I-GG; as shown in FIG. 8, low molecular weight gellan gum L-GG has a large influence on the gel texture of the gellan gum. Table 2 is a table comparing the texture characteristics of I-GG and L-GG.
TABLE 2 comparison table of I-GG and L-GG texture characteristics
| Sample (I) | I-GG | L-GG |
| Hardness of | 182.611±5.51a | 135.231±6.15b |
| Viscosity of | -6.319±0.35a | -12.078±0.56b |
| Cohesion force | 0.431±0.08b | 0.651±0.12a |
| Elasticity | 81.297±0.57b | 88.529±0.63a |
| Adhesiveness | 78.731±0.89b | 88.033±1.01a |
| Degree of mastication | 64.005±1.23b | 77.934±0.93a |
Hardness can be used to characterize the strength properties of the gel structure in the compressed state, generally the greater the hardness the higher the gel strength and the denser the gel network structure. Cohesion may reflect the ease with which the internal structure of the gel is broken. As can be seen from Table 2, compared with I-GG, L-GG has high elasticity and low hardness, which shows that the gel network structure of L-GG has high water molecule content and stronger moisturizing effect than I-GG, and L-GG has higher cohesive force, higher capability of keeping gel integrity and difficult cracking. The L-GG has low viscosity and high chewiness, has better palatability when being used as an additive in the fields of food, health products, medicines and the like, has excellent mouthfeel, and has wide application prospect in the field of food.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
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tttatcgccc tgagatgagc ccgcgtagga ttagctagtt ggtgtggtaa aggcgcacca 180
aggcgacgat ccttagctgg tctgagagga tgatcagcca cactgggact gagacacggc 240
ccagactcct acgggaggca gcagtgggga atattggaca atgggcgaaa gcctgatcca 300
gcaatgccgc gtgagtgatg aaggccttag ggttgtaaag ctcttttacc cgggaagata 360
atgactgtac cgggagaata agccccggct aactccgtgc cagcagccgc ggtaatacgg 420
agggggctag cgttgttcgg aattactggg cgtaaagcgc acgtaggcgg ctttgtaagt 480
cagaggtgaa agcctggagc tcaactccag aactgccttt gagactgcat cgcttgaatc 540
caggagaggt gagtggaatt ccgagtgtag aggtgaaatt cgtagatatt cggaagaaca 600
ccagtggcga aggcggctca ctggactggt attgacgctg aggtgcgaaa gcgtggggag 660
caaacaggat tagataccct ggtagtccac gccgtaaacg atgataacta gctgtccggg 720
tgcttggcac ttgggtggcg cagctaacgc attaagttat ccgcctgggg agtacggccg 780
Claims (9)
1. The low molecular weight gellan gum producing strain is preserved in the China Wuhan type culture collection center with the preservation number of CCTCC NO: M20211430 and the preservation date of 2021 year, 11 months and 17 days, and is named asSphingomonas paucimobilss M155。
2. The mutant strain according to claim 1, which is obtained by UV mutagenesis and ARTP mutagenesis.
3. A screening method of a low molecular weight gellan gum producing strain is characterized in that sphingomonas paucimobilis is used as an initial strain, ultraviolet mutagenesis is carried out on a bacterial suspension of the low molecular weight gellan gum producing strain, ARTP mutagenesis is carried out after ultraviolet irradiation, and a mutant strain for producing the low molecular weight gellan gum is screened.
4. The screening method according to claim 3, comprising the steps of:
(1) preparation of bacterial suspension: inoculating the starting strain into seed culture medium, shake culturing to logarithmic phase, taking out bacterial suspension, diluting with 0.9% NaCl solution to obtain bacterial liquid OD600In the range of 0.6-0.7; obtaining mutagenic bacteria suspension;
(2) performing UV-ARTP composite mutagenesis;
(3) primary screening: selecting large, round and smooth-surfaced faint yellow single bacterial colony, activating, fermenting and culturing to produce a crude product of gellan gum, and screening according to the gum yield compared with the original strain;
(4) re-screening: selecting mutant strains with lower molecular weight and more stable passage, and identifying the stability of the strains.
5. The screening method according to claim 3, wherein the UV mutagenesis is carried out for 150 seconds, and the ARTP mutagenesis is carried out for 25 seconds.
6. The screening method according to claim 4, wherein the UV-ARTP complex mutagenesis is specifically: inoculating a starting strain from a solid culture medium to a 250 mL conical flask filled with 50 mL of seed liquid, performing shake culture at 30 ℃ and 220 r/min to logarithmic phase, taking out the bacterial suspension in the conical flask, diluting the bacterial suspension with 0.9% NaCl solution to obtain a mutant bacterial suspension, and making the OD of the bacterial solution of the mutant bacterial suspension600In the range of 0.6-0.7; sucking 5 mL of bacterial suspension mixed by mutagenic bacterial suspension and 5% (v/v) glycerol according to the proportion of 1:1 into a sterilized glass plate with the diameter of 9 cm, and carrying out mutagenesis in an ultraviolet full-automatic mutagenic instrument under the action of magnetic stirring, wherein the wavelength is 254 nm, and the mutagenesis time is 150 s; uniformly coating 20 mu L of the bacterial suspension subjected to ultraviolet mutagenesis on a metal slide, placing the metal slide in an ARTP mutagenesis instrument, setting the power to be 120W, the ventilation capacity to be 10 SLM and the processing time to be 25 s, placing the processed metal gasket in an EP tube filled with 980 mu L of sterile water, diluting according to ten times of gradient after fully shaking, coating 100 mu L of proper gradient diluent on a solid culture medium, and culturing for 3 d in an incubator at 30 ℃.
7. The screening method according to claim 4, wherein the lower molecular weight is an intrinsic viscosity [. eta. ] of gellan gum produced by each of the rescreened strains calculated by Ubbelohde viscometer]According to the formula [ eta]K is 1.16 × 10 = kM α, k-3And taking alpha as 0.67, calculating the viscosity-average molecular weight of each gellan gum, and screening.
8. Use of the mutant strain of claim 1 for the production of low molecular weight gellan gum.
9. Use of the mutant strain according to claim 1 in the preparation of a food product, a cosmetic product, a health product or a pharmaceutical product.
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| CN101665778A (en) * | 2009-09-25 | 2010-03-10 | 浙江大学 | Uranidin generation deficiency sphingolipid sphingomonas paucimobilis and application thereof in gellan gum production |
| CN110144318A (en) * | 2019-04-22 | 2019-08-20 | 南京工业大学 | Non-pigment low molecular weight Weilan gum produces bacterial strain and its construction method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101665778A (en) * | 2009-09-25 | 2010-03-10 | 浙江大学 | Uranidin generation deficiency sphingolipid sphingomonas paucimobilis and application thereof in gellan gum production |
| CN110144318A (en) * | 2019-04-22 | 2019-08-20 | 南京工业大学 | Non-pigment low molecular weight Weilan gum produces bacterial strain and its construction method and application |
Non-Patent Citations (1)
| Title |
|---|
| 唐志红,鞠宝,林剑,任育红,王长海: "多糖裂合酶及其应用前景", 生命的化学, no. 02 * |
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