CN114214234B - Low molecular weight gellan gum production strain, screening method and application thereof - Google Patents

Low molecular weight gellan gum production strain, screening method and application thereof Download PDF

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CN114214234B
CN114214234B CN202111573521.2A CN202111573521A CN114214234B CN 114214234 B CN114214234 B CN 114214234B CN 202111573521 A CN202111573521 A CN 202111573521A CN 114214234 B CN114214234 B CN 114214234B
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孙玲
杨蕾
岳玫香
王亚珍
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Abstract

The application relates to the technical field of bioengineering, in particular to a low molecular weight gellan gum production strain, a screening method and application thereof. The application utilizes the UV-ARTP composite mutagenesis technology to carry out mutagenesis on the Sphingomonas paucimobilis, and obtains the mutant strain M155 for producing the low molecular weight gellan gum. Compared with the original strain, the yield of gellan gum produced by the strain is improved by 24%, and the molecular weight is reduced by 61%; and the mutant has stable genetic performance. The produced low molecular weight gellan gum has stable performance, low viscosity, high elasticity, low hardness and other gel characteristics, and has excellent moisturizing effect compared with high molecular weight gellan gum; can be used in the fields of cosmetics and medicine; meanwhile, the low molecular weight gellan gum has low adhesiveness, high chewing degree, no tooth sticking and chewing strength after being eaten, can endow food with better taste, and has wide application prospect in the field of food and health care product manufacturing.

Description

Low molecular weight gellan gum production strain, screening method and application thereof
Technical Field
The application relates to the technical field of bioengineering, in particular to a low molecular weight gellan gum production strain, a screening method and application thereof.
Background
Gellan gum is prepared from gram-negative bacteriaSphingomonas paucimobilis ATCC31461, which ferments to produce extracellular polysaccharide, whose molecular weight is generally 0.5-1.0X10 6 Da. The microbial extracellular polysaccharide is used as an industrial product of microbial fermentation, and has wide application prospect in the fields of food, chemical industry, 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 jams, sausage, ice cream, salad dressing, jelly, dairy products, etc. In the field of medicine, gellan gum can be applied to eye drops, slow-release medicines,Coating, tissue engineering scaffold material, etc. In the chemical industry, gellan gum can be used as adhesive, toothpaste, air freshener, etc. At present, gellan gum is the latest one in the series of food gels available in the market, and the global demand is increasing. The development and production of gellan gum have extremely high commercial economic significance and market prospect.
Molecular weight is one of the basic parameters for representing the characteristics of gellan gum and is also an important index for quality detection in industrial production. However, there are few reports on low molecular weight gellan gum, which greatly limit the precise application of low molecular weight gellan gum. The traditional preparation method of the small molecular weight gellan gum is a hydrolysis method, comprising: the physical method, the chemical method and the enzymolysis method are not controllable in most of the hydrolysis processes, the molecular weight distribution range of the obtained gellan gum is wider, and the gellan gum is randomly distributed, so that the direct use can influence the efficacy of the product, 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 step-by-step purification method is needed again, the production cost is increased, the product yield is reduced, and finally, the sales price of the gellan gum is high, so that the application range of the gellan gum is severely restricted.
Disclosure of Invention
In view of the above, the present application provides a strain for producing gellan gum with low molecular weight, and a screening method and application thereof. The mutant strain can continuously produce the low molecular weight gellan gum, and the produced low molecular weight gellan gum has uniform molecular weight distribution and lower production cost, thereby laying an application foundation for the precise application of the low molecular weight gellan gum.
In order to achieve the above purpose, the present application adopts the following technical scheme:
the application provides a low molecular weight gellan gum producing strain, which is named asSphingomonas paucimobilssM155 (simply called mutant strain M155) which is preserved in the China center for type culture collection of Wuhan, wherein the preservation number is CCTCC NO: M20211430, and the preservation unit address is: the storage date of the eight-path 299 university of Wuhan in Wuhan district of Hubei province is 2021, 11 months and 17 days.
The application uses Sphingomonas paucimobilis as raw materialSphingomonas paucimobilis) ATCC31461 as starting strain, and 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 passage ten is stable.
In a specific embodiment, the application also provides a breeding method of the mutant strain, wherein the breeding method is to utilize UV and ARTP to carry out composite mutagenesis, and through preliminary screening and secondary screening and verification, a mutant strain which produces low molecular weight gellan gum and is genetically stable is bred.
The breeding method of the application comprises the following steps:
(1) Preparation of bacterial suspension: inoculating the original strain into seed culture medium, shake culturing to logarithmic phase, taking out bacterial suspension, diluting with 0.9% NaCl solution to obtain bacterial solution OD 600 0.6 to 0.7; obtaining mutagenic bacterial suspension;
(2) Ultraviolet mutagenesis and ARTP mutagenesis;
(3) And (3) primary screening: and (3) selecting a large, round and smooth-surface pale yellow single colony to activate, ferment and culture to produce a crude gellan gum product, and screening the crude gellan gum product by comparing the crude gellan gum product with a starting strain.
(4) And (3) re-screening: mutant strains with significantly lower molecular weights and more stable passages were selected and identified for strain stability.
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 strain of the original strain to 250 mL conical flask containing 50 mL seed solution, shake culturing at 30deg.C and 220 r/min to logarithmic phase, taking out bacterial suspension in conical flask, diluting with 0.9% NaCl solution to obtain mutagenized bacterial suspension, and making bacterial solution OD 600 0.6-0.7. Sucking the bacterial suspension 5 mL of the mixture of the mutagenized bacterial suspension and 5% (v/v) glycerol according to the ratio of 1:1 for killingThe strain with the diameter of 9 cm is subjected to mutagenesis in a full-automatic ultraviolet mutagenesis instrument under the action of magnetic stirring, the wavelength is 254 nm, and the mutagenesis time is 150 s. Uniformly smearing 20 mu L of ultraviolet-induced bacterial suspension on a metal slide, placing the metal slide in an ARTP mutagenesis instrument, setting the power to be 120W, the ventilation amount to be 10 SLM, the treatment time to be 25 s, placing the treated metal gasket into an EP pipe filled with 980 mu L of sterile water, sufficiently vibrating and diluting according to ten times gradient, coating 100 mu L of proper gradient diluent on a solid culture medium, and culturing the solid culture medium in a 30 ℃ incubator to 3 d.
(2) Screening of mutagenized Strain
a. And (3) primary screening: performing primary screening treatment on the bacterial colony subjected to composite mutagenesis, picking a large, round and smooth-surface pale yellow single bacterial colony, inoculating the bacterial colony into seed liquid, performing shake cultivation at 30 ℃ and 220 r/min until the bacterial colony is in the late logarithmic phase, inoculating the seed liquid cultivated until the bacterial colony is in the logarithmic phase into 50 mL liquid fermentation medium according to the inoculum size, and performing constant-temperature cultivation at 30 ℃ and 220 r/min for 72 h to obtain fermentation liquor; diluting the fermentation liquor by ten times of volume, heating for 15 min in a constant temperature water bath kettle at 95 ℃, adding 2-3 times of 95% ethanol by volume while the fermentation liquor is still hot, precipitating with ethanol at 4 ℃ for overnight, centrifuging for 15 min at 4000 r/min, discarding supernatant, collecting precipitate, drying at 60 ℃ to constant weight, and obtaining a crude gellan gum product, wherein the gum yield is the gum content in the fermentation liquor per unit volume.
b. And (3) re-screening: activating and fermenting mutant strains with obviously changed gellan gum yield after primary screening, collecting gellan gum samples produced by different strains, and carrying out secondary screening; the intrinsic viscosity [ eta ] of each gellan gum sample was calculated using an Ubbelohde viscometer]According to the formula [ eta ]]=kmα, k 1.16x10 -3 Alpha is 0.67 to calculate the viscosity average molecular weight of the gellan gum; mutant strains with significantly lower molecular weights and more stable passage were selected.
(3) Determination of the molecular weight and yield stability of Excellent mutant Strain
The excellent mutant strain is inoculated to a solid culture medium after being picked out from a solid plate, and is cultured at 30 ℃ for 2 d, and is recorded as 1 generation, and under the same conditions, the solid culture medium is subjected to plate subculture to 10 generations; the production and molecular weight of the excellent mutant strains of 1 to 10 generations were determined by the preliminary screening and re-screening methods of the mutant strains described in step (2).
The application improves mutation efficiency through UV-ARTP composite mutagenesis, and has strong mutagenesis purpose and high practicability. The mutagenesis conditions after optimization of the application are as follows: UV mutagenesis time 150 s, ARTP mutagenesis time 25 s. The mutation mortality rate of the UV and ARTP 2 rounds of mutagenesis is more than or equal to 90 percent. Under these conditions, mutagenized strains that produce low molecular weight gellan gum are more readily obtained.
The application also provides application of the mutant strain M155 in production of low molecular weight gellan gum.
The application also provides application of the mutant strain M155 in preparing foods, cosmetics, health products or medicines.
Compared with the prior art, the application has the beneficial effects that:
the application utilizes ultraviolet mutagenesis combined with normal pressure room temperature plasma composite mutagenesis technology (UV-ARTP) to carry out mutagenesis on Sphingomonas paucimobilis, thus obtaining a mutant strain M155 for producing low molecular weight gellan gum. Compared with the original strain, the gellan gum yield of the strain 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, low viscosity, high elasticity, low hardness and other gel characteristics, and has excellent moisturizing effect compared with high molecular weight gellan gum; can be used in the fields of cosmetics and medicine; meanwhile, the low molecular weight gellan gum has low adhesiveness, high chewing degree, no tooth sticking and chewing strength after being eaten, can endow food with better taste, and has wide application prospect in the field of food and health care product manufacturing. Therefore, the application lays an important theoretical and application foundation for the industrialized and accurate production of the low molecular weight gellan gum and the accurate application thereof.
Drawings
FIG. 1 is a graph of mortality; in the figure, A is an ultraviolet mutagenesis mortality curve, and B is an ARTP mortality curve;
FIG. 2 is a graph showing comparison of gellan gum yield after primary screening of mutant strains;
FIG. 3 is a graph of viscosity average molecular weight of gellan gum produced by a rescreened mutant strain;
FIG. 4 is a graph of the genetic stability of the viscosity average molecular weight and yield of mutant strain M155 over ten passages;
FIG. 5 is an elution pattern and molar mass diagram of L-GG and I-GG;
FIG. 6 is a graph showing comparison of L-GG and I-GG yields;
FIG. 7 is a graph of the rheological properties of L-GG and I-GG, wherein A is a graph of static apparent viscosity and B is a graph of dynamic rheological properties;
FIG. 8 is a texture map of L-GG and I-GG.
Detailed Description
The application is further illustrated by the following examples: those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present application. While the methods and applications of this application have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this application, without departing from the spirit or scope of the application. The experimental methods in the examples, for which specific conditions were not noted, were all according to conventional conditions; the reagents and biological materials used, unless otherwise specified, are commercially available.
Starting strain: sphingomonas paucimobilis ATCC31461 was purchased from American Type Culture Collection (ATCC) under the number ATCC@31461.
Seed culture medium: yeast powder 1 g/L, beef extract 3 g/L, tryptone 5 g/L, naCl 5 g/L, sucrose 5 g/L.
Fermentation medium: sucrose 30 g/L, yeast powder 0.2 g/L, tryptone 2 g/L, KH 2 PO 4 1 g/L、K 2 HPO 4 1.5 g/L、MgSO 4 0.6 g/L。
Example 1:
(1) UV-ARTP composite mutagenesis treatment
a. Preparation of mutagenic bacterial suspension: picking the original strain from the solid culture medium, inoculating with one-loop fungus, inoculating into 250 mL conical flask containing 50 mL seed culture medium, and shaking at 30deg.C and 220 r/minCulturing in bed until logarithmic phase, taking out bacterial suspension in conical flask, diluting bacterial suspension with 0.9% NaCl solution to obtain bacterial solution OD 600 0.6 to 0.7; the mutagenic bacterial suspension is obtained.
b. Ultraviolet mutagenesis: mixing the mutagenic bacterial suspension with 5% (v/v) glycerol according to a ratio of 1:1, shaking in a vortex oscillator under aseptic condition, taking bacterial suspension 5 mL, placing in a sterilized plate with a diameter of 9 cm, performing mutagenesis in an ultraviolet full-automatic mutagenesis instrument under the action of magnetic stirring, and setting the wavelength 254 nm, wherein the mutagenesis time is respectively 30, 60, 90, 120, 150, 180 and 210 s. Diluting the ultraviolet-induced bacteria solution with ten times of gradient, and collecting the solution with proper gradient (dilution gradient of 10 -4 The colony number of the bacterial liquid growing on the solid culture medium under the gradient is less than 300), 100 mu L of the diluted liquid is coated on the solid culture medium, and the bacterial liquid is cultured in a culture box at 30 ℃ for 3 d. The optimal mutagenesis conditions were determined from the mortality curve using non-mutagenized Sphingomonas paucimobilis as a control. Mortality (%) = (number of control group colonies-number of treated group colonies)/number of control group colonies×100%.
ARTP mutagenesis: mixing the mutagenic bacterial suspension with 5% (v/v) glycerol according to a ratio of 1:1, placing in a vortex oscillator under aseptic condition, shaking uniformly, taking 20 mu L of bacterial suspension, uniformly coating on a metal slide, placing in an ARTP mutagenic instrument, setting the power to 120W, the ventilation to 10 SLM, and the treatment time to 10, 15, 20, 25, 30 and 35 s. Placing the treated metal gasket into an EP pipe filled with 980 mu L of sterile water, sufficiently vibrating, diluting according to ten times of gradient, and taking the dilution gradient as 10 -4 100. Mu.L of the dilution of (C) was spread on a solid medium and cultured in an incubator at 30℃for 3 d. The original strain Sphingomonas paucimobilis is used as a control, and the optimal mutagenesis treatment condition is determined according to a lethality curve.
FIG. 1 is a graph of mortality; in the figure, A is an ultraviolet mutagenesis mortality curve, and B is an ARTP mortality curve; as can be seen from fig. 1, the mortality rate was 84% when the uv mutagenesis time was 150 s, and 91% when the mutagenesis time was 180 s; when the ARTP mutagenesis time is 25 s, the mortality rate is 90%; in order to ensure the survival of further strains, the UV mutagenesis time was chosen to be 150 s and the ARTP mutagenesis time was chosen to be 25 s as a suitable mutagenesis agent during the subsequent mutagenesis.
d. And (3) complex mutagenesis: mixing the mutagenic bacterial suspension with 5% (v/v) glycerol according to a ratio of 1:1, placing in a vortex oscillator for shaking uniformly under aseptic condition, taking bacterial suspension 5 mL after shaking uniformly, placing in a sterilized glass plate with the diameter of 9 cm, and under the action of magnetic stirring, carrying out mutagenesis in an ultraviolet full-automatic mutagenesis instrument with the wavelength of 254 nm and the mutagenesis time of 150 s. Uniformly smearing 20 mu L of ultraviolet-induced bacterial suspension on a metal slide, placing the metal slide in an ARTP mutagenizing instrument, setting the power to 120W, the ventilation to 10 SLM, the treatment time to 25 s, placing the treated metal gasket into an EP pipe filled with 980 mu L of sterile water, sufficiently vibrating, diluting according to ten times gradient, and taking the dilution gradient to 10 -4 100. Mu.L of the dilution of (C) was spread on a solid medium and cultured in an incubator at 30℃for 3 d.
(2) Screening of mutagenized Strain
a. And (3) primary screening: and (3) performing primary screening treatment on the bacterial colony subjected to composite mutagenesis, picking a large, round and smooth-surface pale yellow single bacterial colony, inoculating the large, round and smooth-surface pale yellow single bacterial colony into seed liquid, performing shake cultivation at 30 ℃ and 220 r/min until the later logarithmic phase, inoculating the seed liquid cultivated until the later logarithmic phase into 50 mL liquid fermentation medium according to the inoculation amount, and performing constant-temperature cultivation at 220 r/min for 72 h to obtain fermentation liquor. Diluting the fermentation liquor by ten times of volume, heating for 15 min in a constant temperature water bath kettle at 95 ℃, adding 2-3 times of 95% ethanol by volume while the fermentation liquor is still hot, precipitating with ethanol at 4 ℃ for overnight, centrifuging for 15 min at 4000 r/min, discarding supernatant, collecting precipitate, drying at 60 ℃ to constant weight, and obtaining a crude gellan gum product, wherein the gum yield is the gum content in the fermentation liquor per unit volume. After UV-ARTP mutagenesis, 210 mutant strains are obtained, wherein 54 mutant strains with high or low gum yield are preserved for subsequent verification. For comparison, crude gellan gum of the starting strain was prepared by the same method and the yield was calculated to be 4.56 g/L.
FIG. 2 is a graph showing comparison of gellan gum yield after primary screening of mutant strains; as can be seen from FIG. 2, the total gellan gum yield produced by fermentation of 54 mutant strains was significantly changed compared with the gellan gum yield of 4.56 g/L of the original strain, and was used for subsequent re-screening.
b. And (3) re-screening: and (3) activating and fermenting 54 mutant strains with obviously changed gellan gum yield 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/NaNO 3 As a solvent, a gellan gum solution having a gellan gum concentration of 1 mg/mL was prepared. Magnetically stirring 2 h in a constant temperature magnetic stirring water bath at 80 ℃ to fully dissolve gellan gum. With DMSO/NaNO 3 The solution (50 mmol/L) was used as solvent, 9.0. 9.0 mL solvent was added to a Ubbelohde viscometer, and the outflow time t was measured in a constant temperature bath at 25.+ -. 0.1 ℃ 0 The process is repeated three times, and the outflow time of each process is not different by more than 0.2 seconds, and the average value is taken. Then adding 1.0 mL of gellan gum solution with the concentration of 1 mg/mL into a Ubbelohde viscometer, diluting the gellan gum solution with the concentration of 0.1 mg/mL, and measuring the outflow time to be t by the same method 1 . The intrinsic viscosity [ eta ] of each gellan gum solution is calculated by a single-point method]According to the formula [ eta ]]=kmα, k 1.16x10 -3 Alpha is taken to be 0.67. The viscosity average molecular weight of gellan gum was calculated. Mutant strains with significantly lower molecular weights and more stable passage were selected.
FIG. 3 is a graph of viscosity average molecular weight of gellan gum produced by a rescreened mutant strain; as can be seen from FIG. 3, the mutant strain M155 has a viscosity average molecular weight of 12444.66.+ -. 350.51 Da, which is lower than most other mutant species. The yield of gellan gum combined with the strain is higher, which accords with the industrial production requirement of low molecular weight gellan gum, and the high-yield low molecular weight mutant strain M155 is selected as an excellent strain.
(3) Determination of molecular weight and yield stability of Excellent mutant Strain
Mutant strain M155 was picked from the solid plate and inoculated into solid medium, cultured at 30℃for 2 d, designated as 1 passage, and under the same conditions, the solid medium was subcultured on the plate to 10 passages. The mutant strain M155 of 1 to 10 generations was subjected to yield and molecular weight measurement according to the method for screening the mutant strain described in the above step (2), respectively. FIG. 4 is a graph of the genetic stability of the viscosity average molecular weight and yield of mutant strain M155 over ten passages; as can be seen from FIG. 4, the mutant strain M155 was relatively stable in terms of yield and molecular weight passage for less than ten generations. The obtained mutant strain M155 has a viscosity average molecular weight of 12444.66 + -350.51 Da, and an average yield of 5.68+ -0.09 g/L, which is improved by 24%.
The strain is preserved in China general culture Collection with the preservation number of CCTCC NO: M20211430, and the preservation unit address is: the storage date of the eight-path 299 university of Wuhan in Wuhan district of Hubei province is 2021, 11 months and 17 days.
Example 2: preparation of low molecular weight gellan gum
(1) The method for activating and culturing the seed liquid comprises the following steps: and (3) taking a loop of bacteria from the solid culture medium at the temperature of 4 ℃, streaking the loop of bacteria on the solid culture medium, culturing at the temperature of 30 ℃, culturing at the temperature of 2 d, and then taking a loop of single colony, inoculating the single colony into the seed culture medium, and culturing by a shaking table at the temperature of 30 ℃ and at the speed of 200 r/min until the single colony is in the late logarithmic phase.
(2) The liquid fermentation culture method comprises the following steps: the seed solution was transferred to a fermentation medium at 10% (V/V) and shake-cultured at 30℃and 220 r/min for 3: 3 d.
(3) Culturing mutant strain M155 by using activated seed liquid, performing liquid shaking fermentation to obtain fermentation liquor, diluting the fermentation liquor by ten times of volume, heating in a constant-temperature water bath at 95 ℃ for 15 min, adding 2-3 times of 95% ethanol by volume while the fermentation liquor is hot, precipitating with ethanol at 4 ℃ for overnight, centrifuging at 4000 r/min for 15 min, discarding supernatant, collecting precipitate, and drying at 60 ℃ to constant weight to obtain a gellan gum crude product. Weighing a certain amount of crude gellan gum, preparing 2 mg/mL solution, deproteinizing by trichloroacetic acid method, dialyzing with dialysis bag with molecular weight cut-off of 6000-8000 Da, changing water every 4 h, dialyzing for 48 h, collecting gellan gum polysaccharide solution in the dialysis bag, and vacuum drying to obtain low molecular weight gellan gum (L-GG).
In contrast, gellan gum (I-GG) was produced by fermentation using the starting strain ATCC31461 as a starting material by the same production method as the L-GG production process.
Example 3: determination of gellan gum molecular weight and yield
(1) Determination of viscosity average molecular weight (Da): weighing a certain amount of crude gellan gum, and using 50 mmol/L DMSO/NaNO 3 As solvent, gellan gum with final concentration of 1 mg/mL was preparedAnd magnetically stirring the gellan gum solution in a constant-temperature magnetic stirring water bath kettle at 80 ℃ for 2 h to fully dissolve the gellan gum for later use. 9.0 mL DMSO/NaNO was added to an Ubbelohde viscometer 3 Solution (50 mmol/L), flow time t was measured in a constant temperature bath at 25.+ -. 0.1 ℃ C 0 The process is repeated three times, and the outflow time of each process is not different by more than 0.2 seconds, and the average value is taken. Adding 1.0 mL gellan gum solution into Ubbelohde viscometer, diluting to 0.1 mg/mL gellan gum solution, and measuring with the same method to give a flow-out time t 1 . Measurement of the intrinsic viscosity [ eta ] of gellan gum solution by single-point method]According to the formula [ eta ]]The viscosity average molecular weight of gellan gum was calculated =kmα and the results are shown in table 1. k is 1.16X10 -3 Alpha is taken to be 0.67.
(2) Weight average molecular weightMwNumber average molecular weightMnIs determined by: two gellan gum measurements were performed separately using size exclusion chromatography combined with multi-angle laser scattering (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 ℃.
Table 1 shows the comparison of the molecular weight measurements of I-GG and L-GG. As shown in Table 1, the mutant strain M155 was fermented to give gellan gum (L-GG) having a viscosity average molecular weight,MwAndMnthe molecular weight of gellan gum (I-GG) produced by the strain is greatly reduced by 37.8%, 44.6% and 61.0% respectively. As can be seen, the gellan gum produced by mutant strain M155 has a lower molecular weight, and the present application results in a low molecular weight gellan gum.
TABLE 1 molecular weights of I-GG and L-GG
Sample of I-GG L-GG
Viscosity average molecular weight (Da) 20014±401.97 12444.66±350.51
Mw (g/mol) 2.079×10 5 1.151×10 5
Mn (g/mol) 2.074×10 5 8.092×10 4
Mw/Mn 1.003 1.423
The molecular weights of the two gellan gums were analyzed using Size Exclusion Chromatography (SEC). L-GG and I-GG were placed in 0.1. 0.1M aqueous sodium chloride solution, respectively, and SEC measurements were performed in an environment at 25 ℃. FIG. 5 is a diagram showing elution patterns and molar masses of L-GG and I-GG. As shown in fig. 5, a monomodal near normal distribution without shoulders was observed in the SEC elution mode, indicating that they had relatively high uniformity over a defined molar mass range, as well as a relatively uniform molecular weight distribution.
(3) Determination of gellan gum yield: weighing a proper amount of gellan gum fermentation liquor, diluting by ten times of volume, heating in a constant-temperature water bath kettle at 95 ℃ for 15 min, adding 2-3 times of 95% ethanol for ethanol precipitation of gellan gum while the gellan gum is still hot, centrifuging at 4 ℃ for 15 min at 4000 r/min, discarding supernatant, collecting precipitate, and drying at 60 ℃ to constant weight to obtain crude gellan gum product with gum yield being the gum content in unit volume of fermentation liquor. FIG. 6 is a graph showing comparison of L-GG and I-GG yields; as shown in FIG. 6, the crude gum yield of 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 for producing Low molecular weight gellan gum mutant Strain M155
Using rDNA 16s primers 27F and 1492R, the sequences are set forth in SEQ ID NOs: 1. SEQ ID NO: 2, namely: 5 '-agagttttgatcctggctcag-3' and 5 '-ggttacttgttacgacttt-3'; PCR amplification was performed on mutant strain M155 producing low molecular weight gellan gum, reaction conditions: pre-denaturation at 94℃for 10 min, denaturation at 94℃for 30 s, annealing at 58℃for 30 s, extension at 72℃for 40 s, and a total of 34 cycles. And (3) after the PCR product is qualified through agarose gel electrophoresis, sending the PCR product to Shanghai Jie Li biotechnology Co. Sequencing results were analyzed by blast software alignment in NCBI. The nucleotide sequence of the mutant strain M155 is shown in SEQ ID NO: 3, mutant strain M155 and Sphingomonas paucimobilis @Sphingomonas elodeaATCC 31461) was 100% compared to the 16S DNA sequence, indicating that the mutant strain was still sphingomonas paucimobilis.
Example 4: rheological Properties of Low molecular weight gellan gum produced by mutant Strain M155
The original gellan gum (I-GG) obtained by shake flask fermentation of the original strain and the low molecular weight gellan gum (L-GG) obtained by shake flask fermentation of the mutant strain M155 are taken as samples respectively. Accurately weighing gellan gum sample 0.3 g, adding deionized water 100 mL, swelling at room temperature for 24 h, placing in 80 deg.C constant temperature magnetic stirring water bath for magnetic stirring for 2 h until gellan gum sample is completely dissolved, and heating to 5% CaCl when it is hot 2 Solution 1 mL and 80 ℃ deionized water was added to make up for the water lost by evaporation. The prepared samples were placed on rheometer test benches respectively, and the rheometer test was performed in parallel plate mode with a plate diameter of 40 mm for static rheometry and dynamic viscoelasticity measurement.
FIG. 7 is a graph of rheological properties of L-GG and I-GG; wherein A is a static apparent viscosity map and B is a dynamic rheological property map; as shown in FIG. 7, compared with the original gellan gum, the low molecular weight gellan gum prepared by the application has overall reduced apparent viscosity, gradually exhibits the characteristic of fluid, and forms gel with strong fluidity. G ' of both I-GG and L-GG is greater than G ', G ' of L-GG is lower than I-GG, and it is seen that the internal water content of L-GG is higher.
Gellan gum with higher apparent viscosity has the influence of difficult stirring, difficult filtering, prolonged production period and the like in the cosmetic production application. The humectant prepared from the high molecular gellan gum only forms a layer of breathable film on the surface of the skin, so that the skin is smooth and moist, but is not easy to permeate the skin and be absorbed by the skin. The gellan gum with low molecular weight has small molecular weight and low apparent viscosity, can well permeate the skin, has better skin absorption, has better moisturizing effect and has better internal water content. The low molecular gellan gum prepared by the application can improve the convenience of industrial operation, is more suitable for industrial production, and has wide application prospect in the field of medical cosmetology.
Example 5: this example shows the texture characteristics of low molecular weight gellan gum produced by mutant strain M155
The original gellan gum (I-GG) obtained by shake flask fermentation of the original strain and the low molecular weight gellan gum (L-GG) obtained by shake flask fermentation of the mutant strain M155 are taken as samples. Weighing gellan gum samples 0.3 and g respectively, adding deionized water 100 mL, swelling at room temperature for 24 h, then placing in a 80 ℃ constant temperature magnetic stirring water bath kettle for magnetic stirring for 2 h until the gellan gum samples are completely dissolved, adjusting pH to 10 by using 0.1 mol/L NaOH solution, preserving heat for 20 min at 80 ℃, adding 80 ℃ deionized water to supplement the water lost by evaporation, subpackaging in plates of 30 mm multiplied by 30 mm, placing in a 4 ℃ refrigerator for 48 h, and demolding for texture measurement.
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 testing mode is simple TPA. The pre-measurement speed was 2.0 mm/s, the measurement speed was 1.0 mm/s, the post-measurement speed was 1 mm/s, the deflection was 2 mm, the dwell time between two compressions was 2 s, and the trigger force was 0.049N. Samples were equilibrated at room temperature for 0.5. 0.5 h prior to performance testing. FIG. 8 is a texture map of L-GG and I-GG; as shown in FIG. 8, the gel texture of the gellan gum is greatly affected by the low molecular weight gellan gum L-GG. Table 2 is a comparison of texture properties of I-GG and L-GG.
TABLE 2 comparison of texture Properties of I-GG and L-GG
Sample of I-GG L-GG
Hardness of 182.611±5.51 a 135.231±6.15 b
Viscosity of the adhesive -6.319±0.35 a -12.078±0.56 b
Cohesive force 0.431±0.08 b 0.651±0.12 a
Elasticity of 81.297±0.57 b 88.529±0.63 a
Tackiness of the adhesive 78.731±0.89 b 88.033±1.01 a
Degree of mastication 64.005±1.23 b 77.934±0.93 a
Stiffness can be used to characterize the strength properties of a gel structure in a compressed state, generally the greater the stiffness the higher the gel strength and the denser the gel network structure. Cohesive forces may reflect the ease of disruption of the internal structure of the gel. As is clear from Table 2, L-GG has higher elasticity and lower hardness than I-GG, indicating that L-GG has higher water content in the gel network structure, and higher cohesive force than I-GG in moisturizing effect, and higher capability of maintaining gel integrity, and is not easy to break. The L-GG has low viscosity and high chewing degree, and has better palatability when being used as an additive in the fields of food, health care products, medicines and the like, has excellent mouthfeel and has wide application prospect in the field of food.
The foregoing has shown and described the basic principles, main features and advantages of the present application. It will be understood by those skilled in the art that the present application is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present application, and various changes and modifications may be made without departing from the spirit and scope of the application, which is defined in the appended claims. The scope of the application is defined by the appended claims and equivalents thereof.
Sequence listing
<110> university of Jiangsu
<120> a low molecular weight gellan gum producing strain, screening method and application thereof
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<170> SIPOSequenceListing 1.0
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<212> DNA
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agagtttgat cctggctcag 20
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ggttaccttg ttacgactt 19
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<213> Artificial sequence (Artificial Sequence)
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gaacgagatc cttcggggtc tagtggcgca cgggtgcgta acgcgtggga atctgccttg 60
gggttcggaa taactccccg aaaggggtgc taataccgga tgatgtcgaa agaccaaaga 120
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 (4)

1. A low molecular weight gellan gum producing strain is characterized in that the strain is preserved in China Center for Type Culture Collection (CCTCC) No. M20211430, the preservation date is 2021, 11 and 17, and the strain is named asSphingomonas paucimobilis M155。
2. The strain according to claim 1, wherein the mutant strain is obtained by UV mutagenesis and ARTP mutagenesis.
3. Use of the strain of claim 1 for the production of low molecular weight gellan gum.
4. The use of the strain according to claim 1 for the preparation of food, cosmetics, health products or medicines.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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)

* Cited by examiner, † Cited by third party
Title
多糖裂合酶及其应用前景;唐志红,鞠宝,林剑,任育红,王长海;生命的化学(第02期);摘要 *

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