CN107058422B - Method for high yield of exopolysaccharide by lactobacillus plantarum - Google Patents
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Abstract
The invention discloses a method for high yield of exopolysaccharide by lactobacillus plantarum. The EPS produced by the strain used in the invention under the fermentation condition has the function of efficiently reducing cholesterol, and is embodied in that: 1) obtaining the optimal fermentation condition for producing the extracellular polysaccharide by the strain through a single-factor optimization experiment, wherein the yield can reach 2.67-3.18 g/L; 2) compared with the EPS cholesterol-reducing capability before and after optimization, the EPS cholesterol-reducing capability is obviously improved; 3) through a single-factor in-vitro qualitative experiment, the EPS has high-efficiency cholesterol removal capacity which can reach 59 percent at most; 4) the EPS which is secreted to the outside of the cell and is not extracted and purified is still found to have certain cholesterol removing capacity through experiments. The fermentation condition and the preparation method can improve the yield of the microbial EPS, and the obtained EPS has high-efficiency cholesterol-reducing capability and has wide development and application of lipid-lowering medicaments and health-care products.
Description
Technical Field
The invention belongs to the field of extracellular polysaccharide of lactic acid bacteria, and particularly relates to a method for high yield of extracellular polysaccharide by lactobacillus plantarum.
Background
Exopolysaccharides (EPS) are macromolecular polysaccharides formed by polymerization of oligosaccharides through lipid carriers, wherein the polysaccharide formed by one kind of oligosaccharides is a homotype polysaccharide, or else is a heterotypic polysaccharide, and the polysaccharide is white after extraction, purification and freeze-drying.
The physiological and biochemical functions of the exopolysaccharide are mainly embodied in food processing, for example, the exopolysaccharide can be used as a stable thickening agent, an emulsifying agent and a syneresis agent, and compared with a chemical additive, the natural non-toxicity of the exopolysaccharide has higher market value. The EPS produced by different lactic acid bacteria have different structures, namely different oligosaccharide compositions and proportions, and simultaneously, EPS with different structures can be produced by the same strain under the condition of different carbon sources. Therefore, an appropriate carbon source, fermentation conditions, strain, etc. can be selected to obtain a high-producing EPS strain.
In addition, after being transported to the outside by polymerization, the exopolysaccharide can be used as a physical protective barrier to protect thalli from being poisoned by the outside and resist outside bad information, can assist the recognition between cells and is convenient to be attached to a solid surface in special environments, for example, in environments with relatively lack of nutrition, the exopolysaccharide is fixed on a surface with relatively rich nutrition, and is dissociated from the surface when the nutrition is exhausted. EPS as a secondary metabolite protects bacteria from desiccation, predation and has a certain resistance to toxicity. The massive secretion of the bacterial EPS can make the liquid culture medium become viscous, and can be used as a good thickening agent and an emulsifier. In addition, the lactic acid bacteria EPS also have certain regulation effect on the immune system of a human body, such as enhancing the activity of macrophages, which brings research values for the food industry, the chemical industry and the pharmaceutical industry.
Intake of dietary fiber can reduce physiological risk factors of metabolic syndrome, such as hyperlipidemia and low grade inflammatory conditions. Dietary fibres are generally of plant origin, whereas microbial Exopolysaccharides (EPS) have a structure that can potentially exert similar physiological effects.
A plurality of experiments show that the EPS can inhibit the increase of blood pressure, reduce the total level of serum cholesterol, reduce the blood sugar level in animals, and induce immune response in intestinal mucosa of animals to regulate the immunity, which shows that the microorganism EPS can be used for controlling the abnormality occurring in metabolic syndrome, and the research of the EPS can provide theoretical guidance for the development of lipid-lowering medicines or health care products.
However, the production of extracellular polysaccharide of microorganism is still low, the production capacity of strains needs to be further improved when the production is required to be put into large-scale production, and the EPS structure and characteristics are different with the change of strains and fermentation conditions, such as the difference of carbon source, fermentation time and culture medium composition, and the EPS yield and performance are also different.
Further improving the yield of the microorganism EPS and exploiting the physiological and biochemical characteristics thereof, which is a breakthrough point in the field of extracellular polysaccharide research.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for high yield of exopolysaccharides by lactobacillus plantarum.
At present, in the research field of EPS production by lactic acid bacteria, the extraction and purification of EPS and the structural identification of EPS are mainly focused, the invention provides a fermentation condition of Lactobacillus plantarum high-yield extracellular polysaccharide after an optimization experiment, the extracted EPS has a function of efficiently reducing cholesterol, and the potential of reducing cholesterol by EPS is exploited.
After the fermentation conditions of the lactobacillus plantarum high-yield extracellular polysaccharide disclosed by the invention are utilized, the EPS has obvious improvement on the cholesterol-reducing capacity.
The invention also aims to provide the lactobacillus plantarum exopolysaccharide prepared by the preparation method.
The invention further aims to provide application of the lactobacillus plantarum exopolysaccharide.
The purpose of the invention is realized by the following technical scheme:
a method for producing exopolysaccharide with high yield by lactobacillus plantarum comprises the following steps:
(1) standing and fermenting Lactobacillus plantarum La-10 (La-10 for short) in an improved MRS liquid culture medium, wherein the inoculation amount is 3-5% (v/v), so as to obtain a thallus fermentation liquid;
the standing fermentation condition is that the standing fermentation is carried out for 30 to 40 hours at the temperature of between 25 and 31 ℃;
the improved MRS liquid culture medium comprises: 10.0g/L of soybean peptone, 10.0g/L of beef extract, 5.0g/L of yeast extract and 30-40 g/L, K of sucrose2HPO42.0g/L, 5.0g/L sodium acetate, 2.0g/L, MgSO g/L triammonium citrate4·7H2O is 0.58g/L, MnSO4·4H20.25g/L of O, 1.0mL/L of Tween-80 and pH of 6.2-6.4, and sterilizing for later use;
(2) centrifuging the thallus fermentation liquor obtained in the step (1), and taking supernatant; adding trichloroacetic acid (TCA) to remove protein, standing at 4 deg.C, centrifuging, and collecting supernatant; precipitating polysaccharide with 95% ethanol, centrifuging, removing supernatant, precipitating, dialyzing to obtain crude polysaccharide, measuring extraction amount, and lyophilizing to obtain powder, i.e. Lactobacillus plantarum extracellular polysaccharide.
The inoculation amount in the step (1) is preferably 4-5%; most preferably 4%;
the condition of the standing fermentation in the step (1) is preferably 30-31 ℃ for 30-35 h; most preferably 30 deg.C for 30 h.
The modified MRS liquid culture medium in the step (1) is preferably: 10.0g/L of soybean peptone, 10.0g/L of beef extract, 5.0g/L of yeast extract and 30-34 g/L, K of sucrose2HPO42.0g/L, 5.0g/L sodium acetate, 2.0g/L, MgSO g/L triammonium citrate4·7H2O is 0.58g/L, MnSO4·4H20.25g/L of O, 1.0mL/L of Tween-80 and pH of 6.2-6.4, and sterilizing for later use;
most preferably, the modified MRS liquid medium described in step (1) is: soybean peptone 10.0g/L, beef extract 10.0g/L, yeast extract 5.0g/L, sucrose 30g/L, K g2HPO42.0g/L, 5.0g/L sodium acetate, 2.0g/L, MgSO g/L triammonium citrate4·7H2O is 0.58g/L, MnSO4·4H20.25g/L of O, 1.0mL/L of Tween-80 and pH of 6.2-6.4, and sterilizing for later use;
the sterilization is preferably performed at 121 ℃ for 30 minutes.
The centrifugation conditions in the step (2) are preferably 4 ℃ and 8000r/min for 15 min.
The aperture size of the dialysis bag used for dialysis in the step (2) is 3500 Da.
The extraction amount measured in the step (2) is 2.67-3.18 g/L.
A Lactobacillus plantarum exopolysaccharide is prepared by the preparation method.
The lactobacillus plantarum exopolysaccharide is applied to preparation of lipid-lowering medicines or health-care products.
Adding the lactobacillus plantarum extracellular polysaccharide into 1g/L cholesterol standard solution, wherein the final concentration of the lactobacillus plantarum extracellular polysaccharide is 0.8-1.6 g/L, and oscillating and adsorbing for 15-25 h at 30-37 ℃ under a constant temperature condition to realize efficient adsorption of cholesterol.
Preferably, the final concentration of the lactobacillus plantarum exopolysaccharide is 0.8 g/L.
Preferably, the condition of the vibration adsorption is that the vibration adsorption is carried out for 15 hours at 30 ℃ under the constant temperature condition.
The strain used by the invention is Lactobacillus plantarum La-10 (La-10 for short) which is obtained by screening by Guangdong province micro-ecological preparation engineering technology research center and has certain cholesterol reducing capability, and is currently stored in the micro-ecological preparation engineering technology research center of southern China university.
The strain La-10 is subjected to standing fermentation in an improved MRS liquid culture medium, when the inoculation amounts are respectively 2%, 3%, 4%, 5% and 6%, the strain fermentation liquor with the inoculation amount of 4% is subjected to extraction and purification, and the EPS extraction amount detected by using a phenol-sulfuric acid method is the highest (figure 1).
The strain La-10 is respectively kept stand and fermented in modified MRS liquid culture media with the sucrose contents of 5g/L, 10g/L, 20g/L, 30g/L and 40g/L, and when the sucrose content in the culture media is 30g/L, thallus fermentation liquor is extracted and purified, and the EPS extraction amount is the highest through detection by using a phenol-sulfuric acid method (figure 2).
The strain La-10 is subjected to standing fermentation in an improved MRS liquid culture medium, the fermentation temperature is respectively 20 ℃, 25 ℃, 30 ℃, 37 ℃ and 42 ℃, and when the fermentation temperature is 30 ℃, the strain fermentation liquor is subjected to extraction and purification, and the EPS extraction amount detected by using a phenol-sulfuric acid method is the highest (figure 3).
Respectively standing and fermenting the strain La-10 in an improved MRS liquid culture medium for 15h, 25h, 30h, 35h and 40h, wherein the EPS extraction amount detected by a phenol-sulfuric acid method is the highest after 30h of thallus fermentation liquor is extracted and purified (figure 4).
The improved MRS liquid culture medium for extracellular polysaccharide fermentation of the strain comprises the following components: soybean peptone 10.0g/L, beef extract 10.0g/L, yeast extract 5.0g/L, sucrose 30g/L, K g2HPO42.0g/L, 5.0g/L sodium acetate, 2.0g/L, MgSO g/L triammonium citrate4·7H2O 0.58g/L、MnSO4·4H2O0.25 g/L, Tween-801.0 mL/L, pH6.2-6.4, and then quenching at 121 DEG CThe bacteria were used 30 minutes later.
The extraction method of lactobacillus plantarum EPS comprises the following steps: centrifuging thallus fermentation liquid at 4 deg.C and 8000r/min for 15min, collecting supernatant and discarding thallus precipitate; adding trichloroacetic acid (TCA) into the supernatant until the final mass fraction is 4%, and standing overnight at 4 ℃ to remove protein; centrifuging at 4 deg.C and 8000r/min for 15min to obtain supernatant; adding 95% ethanol with 3 times volume of the supernatant, standing overnight at 4 deg.C, and precipitating crude polysaccharide; centrifuging at 4 deg.C and 8000r/min for 15min, discarding supernatant, and dissolving precipitate in sterilized ddH2O is in; performing three-stage water dialysis (the pore size of a dialysis bag is 3500Da) for 16h, changing water every 8h, dialyzing with ultrapure water for 8h, changing water every 4h, collecting dialyzed polysaccharide solution, absorbing a small amount of the polysaccharide solution for dilution by several times, and determining the EPS content by using a phenol-sulfuric acid method according to a glucose standard curve; adding 3 times volume of 95% ethanol to precipitate residual EPS, and freeze-drying to collect polysaccharide powder.
Drawing a glucose standard curve: precisely measuring 1mL, 2mL, 4mL, 6mL and 10mL of standard glucose solution (0.6mg/mL), respectively placing in a 50mL volumetric flask, adding water to a constant volume, and shaking up; precisely measuring 2mL of the solution in each volumetric flask, respectively adding 1mL of 6% phenol solution and 6mL of 95% sulfuric acid solution, and uniformly mixing by using a sampler; water bath at 40 deg.C for 30min, taking out and cooling to room temperature; determination of light absorption value OD by spectrophotometer490(ii) a A linear regression equation was prepared using the glucose concentration as abscissa and the absorbance as ordinate, and the glucose standard curve is shown in FIG. 5.
Method for determining EPS for La-10 (phenol-sulfuric acid method): diluting the solution to be detected by multiple times, sucking 2mL of the solution into a 50mL centrifuge tube, and measuring 3 parts of the solution; respectively adding 1mL of 6% phenol solution and 6mL of 95% sulfuric acid solution, and shaking up; water bath at 40 deg.C for 30min, taking out and cooling to room temperature; determination of light absorption value OD by spectrophotometer490(ii) a The polysaccharide content was calculated according to a standard curve.
In addition, the EPS produced by the La-10 in the invention has a function of efficiently reducing cholesterol, the cholesterol clearance rate can reach 40% through in vitro optimization experiments, and the difference of the cholesterol reducing capability of the EPS before and after optimization is shown in figure 6.
The original MRS liquid culture medium before optimization comprises the following components: 10.0g/L bacteriological peptone, 10.0g/L beef extract, 5g/L yeast powder, 2.0g/L dipotassium phosphate, 2.0g/L diammonium hydrogen citrate, 20.0g/L glucose, 801.0 mL/L Tween-sodium, 5.0g/L sodium acetate, 0.58g/L magnesium sulfate and 0.25g/L, pH 6.5.5 manganese sulfate.
La-10 is fermented in the original MRS liquid culture medium under the optimal fermentation conditions respectively, the EPS cholesterol-reducing rate obtained by extracting and purifying the fermentation liquor is 17.12% +/-2.9%, and the EPS cholesterol-reducing rate obtained by extracting and purifying the fermentation liquor is 40.60% +/-3.2% after fermenting in the improved MRS liquid culture medium.
The EPS extracted and purified by the La-10 under the fermentation conditions of the optimal carbon source content, fermentation time, fermentation temperature, inoculation amount and culture medium composition in the invention has the advantages of improved yield and cholesterol-reducing performance.
Extracellular polysaccharide powders (0.1g/L, 0.4g/L, 0.8g/L, 1.2g/L and 1.6g/L) extracted from La-10 fermentation liquor are respectively and accurately weighed and placed in a 50mL centrifuge tube, and 10mL cholesterol standard solution (1mg/mL) is added, with no extracellular polysaccharide added as a control. Oscillating and adsorbing for 12h at 37 ℃ under the constant temperature condition, centrifuging for 6 minutes at 10000rpm, taking the supernatant, and measuring the cholesterol content of the supernatant by an o-phthalaldehyde method, wherein the optimal EPS mass concentration is 0.8g/L, and the cholesterol adsorption rate is up to 43.79% +/-1.36% (figure 7).
Accurately weighing corresponding proper amount of extracellular polysaccharide powder extracted from La-10 fermentation liquor, respectively, placing the extracellular polysaccharide powder into a 50mL centrifuge tube, adding 10mL cholesterol standard solution, and taking no extracellular polysaccharide added as a control. Oscillating and adsorbing for a certain time (5h, 10h, 15h, 20h and 25h) at 37 ℃ under a constant temperature condition, centrifuging for 6 minutes at 10000rpm, taking supernate, measuring the cholesterol content of the supernate by an o-phthalaldehyde method, and calculating the cholesterol adsorption rate of the La-10 extracellular polysaccharide by using the formula, wherein the optimal adsorption time is 15h, and the cholesterol adsorption rate is 43.85% +/-1.32% (figure 8).
Accurately weighing corresponding proper amount of extracellular polysaccharide powder extracted from La-10 fermentation liquor, respectively, placing the extracellular polysaccharide powder into a 50mL centrifuge tube, adding 10mL cholesterol standard solution, and taking no extracellular polysaccharide added as a control. Oscillating and adsorbing for several hours at constant temperature (15 ℃, 25 ℃, 30 ℃, 37 ℃ and 42 ℃), centrifuging for 2 minutes at 12000rpm, taking the supernatant, measuring the cholesterol content of the supernatant by an o-phthalaldehyde method, and calculating the cholesterol adsorption rate of the La-10 extracellular polysaccharide by using the formula. The optimum adsorption temperature was 30 ℃ and cholesterol clearance was 33.78% + -0.4% (FIG. 9).
The standard curve for cholesterol was plotted, see FIG. 10.
TABLE 1 Standard Curve sample application System
When preparing the mixing system, adding concentrated sulfuric acid slowly, mixing with a sampler, standing at room temperature for 10min, and measuring OD with a spectrophotometer550。
The method for quantifying cholesterol in the supernate (the terephthalaldehyde method) comprises the following steps: diluting the solution to be detected by multiple times, sucking 400 mu L of the solution into a 15mL centrifuge tube, and measuring 3 parts; 2mL of the terephthalaldehyde reagent and 1mL of 95% sulfuric acid solution are respectively added, and the materials are uniformly mixed by a sampler; standing at room temperature for 10 min; determination of light absorption value OD by spectrophotometer550(ii) a And calculating the cholesterol content according to the standard curve, and calculating the cholesterol clearance rate according to a formula.
Besides, the function of the lactobacillus plantarum La-10 exopolysaccharide for lowering cholesterol is also embodied in that:
according to the fermentation method of the high-yield exopolysaccharide, about 3.4mL of thallus fermentation liquor capable of extracting 10mg of exopolysaccharide is sucked, the thallus is boiled in hot water for 15 minutes to inactivate the thallus, the inactivated thallus is centrifugally taken out and placed in a sterilized centrifuge tube, another 3.4mL of turbid fermentation liquor is taken and placed in the sterilized centrifuge tube, the hot water boiling is carried out for 15 minutes to inactivate the thallus, then 10mg of EPS solid powder is taken and placed in another clean centrifuge tube, 10mL of improved MRS liquid culture medium containing 1% of cholesterol solution (10mg/mL) is respectively added, the mixture is placed still for 12 hours at 30 ℃, the cholesterol reduction capability comparison is carried out, the cholesterol content is calculated by respectively taking the freshly prepared improved MRS liquid culture medium without the thallus and 3.4mL as a reference, the cholesterol reduction rate of the EPS, the inactivated thallus and the bacteria liquid is respectively calculated by referring to the formula.
The method for measuring the content of cholesterol in the bacterial liquid comprises the following steps: centrifuging at 10000rpm for 6min to obtain 0.5mL of supernatant, and adding 3.0mL of absolute ethanol; adding 2.0mL of 50% KOH, and uniformly mixing by using an oscillator; water bath at 60 deg.C for 15 min; cooling, adding 5.0mL of n-hexane, uniformly mixing, adding 3.0mL of first-stage water, uniformly mixing, and standing at room temperature for 15 min; after the solution is layered, taking 400 mu L of solution of the n-hexane layer (the uppermost layer) and evaporating in a boiling water bath to dryness; adding 2.0mL of a terephthalaldehyde solution and 1.0mL of a 95% sulfuric acid solution, and uniformly mixing by using a sampler; standing at room temperature for 10min, and measuring light absorption value (OD) with spectrophotometer550) (ii) a And (4) calculating the cholesterol content according to the standard curve, and comparing the cholesterol reducing capability of the thalli, the EPS and the bacterial liquid.
The results show that: the cholesterol-reducing rate of EPS is up to 59%, and the cholesterol-reducing rates of the heat-inactivated thallus and the zymocyte liquid are respectively 6.45% and 9.99% (figure 11).
The heat inactivated bacteria and the zymophyte liquid have certain removal effect on cholesterol in a culture medium, the zymophyte liquid has higher adsorption rate compared with the inactivated bacteria, which indicates that chemical substances in the bacteria liquid or metabolites secreted by the bacteria have the cholesterol reduction capability, such as certain organic acids, colloidal exopolysaccharides and the like, lactobacillus plantarum EPS is secreted to the outside of cells and is in a mucus shape or a colloid shape, and the freeze-dried powder is shown in figure 12.
The invention not only provides a fermentation condition and an extraction and purification method for lactobacillus plantarum high-yield extracellular polysaccharide, but also finds that the extracted EPS has a high-efficiency cholesterol removal function through experiments, and provides a new idea for development of modern lipid-lowering medicines and health-care products.
The invention further researches the cholesterol-reducing function of the EPS in different states, and provides guidance for developing different market applications of the EPS.
Compared with the prior art, the invention has the following advantages and effects:
the strain used by the invention has the characteristic of efficiently producing extracellular polysaccharide, and EPS produced by the strain under the fermentation condition in the invention has the function of efficiently lowering cholesterol, which is specifically shown in the following steps: 1) obtaining the optimal fermentation condition for producing the extracellular polysaccharide by the strain through a single-factor optimization experiment, wherein the yield can reach 2.67-3.18 g/L; 2) compared with the EPS cholesterol-reducing capability before and after optimization, the EPS cholesterol-reducing capability is obviously improved; 3) through a single-factor in-vitro qualitative experiment, the EPS has high-efficiency cholesterol removal capacity which can reach 59 percent at most; 4) the EPS which is secreted to the outside of the cell and is not extracted and purified is still found to have certain cholesterol removing capacity through experiments. The fermentation condition and the preparation method can improve the yield of the microbial EPS, and the obtained EPS has high-efficiency cholesterol-reducing capability and has wide development and application of lipid-lowering medicaments and health-care products.
Drawings
FIG. 1 shows the result of the determination of the optimal inoculation amount of the high-yield EPS of Lactobacillus plantarum La-10.
FIG. 2 shows the determination result of the optimum sucrose content of Lactobacillus plantarum La-10 for high yield EPS.
FIG. 3 is the result of measuring the optimal fermentation temperature for high yield of EPS of Lactobacillus plantarum La-10.
FIG. 4 is the result of the determination of the optimum fermentation time for the Lactobacillus plantarum La-10 to produce EPS with high yield.
FIG. 5 is a glucose standard curve.
FIG. 6 is the difference in cholesterol lowering ability of EPS before and after fermentation optimization.
FIG. 7 shows the results of determination of the best mass concentration of EPS for lowering cholesterol.
FIG. 8 shows the results of determination of the optimum adsorption time for EPS to lower cholesterol.
FIG. 9 shows the measurement results of the optimum adsorption temperature for lowering cholesterol by EPS.
FIG. 10 is a standard curve for cholesterol.
FIG. 11 shows the results of the comparative measurement of cholesterol-lowering ability of the bacterial suspension, the inactivated bacterial cells and EPS.
FIG. 12 shows the EPS solid powder obtained after extraction, purification and freeze-drying of Lactobacillus plantarum La-10.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1 fermentation and extraction purification of Lactobacillus plantarum La-10 exopolysaccharides
Inoculating lactobacillus plantarum La-10 in an inoculation amount of 4% volume fraction in an improved MRS liquid culture medium, performing static culture at a fermentation temperature of 30 ℃ for 30 hours, wherein the sucrose content in the improved MRS liquid culture medium is 30 g/L.
TABLE 2 maximum extraction of the individual factors
| Single factor | EPS(g/L) |
| Amount of inoculation | 3.1756±0.02b |
| Sucrose content | 2.8255±0.03a |
| Temperature of fermentation | 2.9417±0.09a |
| Time of fermentation | 2.6694±0.001a |
Improved MRS liquid medium composition: soybean peptone 10.0g/L, beef extract 10.0g/L, yeast extract 5.0g/L, sucrose 30g/L, K g2PO42.0g/L, 5.0g/L sodium acetate, 2.0g/L, MgSO g/L triammonium citrate4·7H2O is 0.58g/L, MnSO4·4H2O is 0.25g/L, Tween-80 is 1.0mL/L, and pH is 6.2-6.4.
The EPS extraction method comprises the following steps:
(1) centrifuging at 4 deg.C and 8000r/min for 15min, collecting supernatant, and removing thallus precipitate.
(2) Trichloroacetic acid (TCA) was added to the supernatant to a final mass fraction of 4%, and the mixture was allowed to stand overnight at 4 ℃ to remove proteins.
(3) Centrifuging at 4 deg.C and 8000r/min for 15min to obtain supernatant.
(4) Adding 95% ethanol 3 times the volume of the supernatant, standing overnight at 4 deg.C, and precipitating crude polysaccharide.
(5) Centrifuging at 4 deg.C and 8000r/min for 15min, discarding supernatant, and dissolving precipitate in sterilized ddH2And (4) in O.
(6) Performing three-stage water dialysis (pore size of the dialysis bag is 3500Da) for 16h, changing water every 8h, dialyzing with ultrapure water for 8h, changing water every 4h, collecting dialyzed polysaccharide solution, absorbing a small amount of the polysaccharide solution for dilution, and determining EPS content to be 2.664g/L by phenol-sulfuric acid method.
(7) Adding 3 times volume of 95% ethanol solution, standing overnight at 4 deg.C, collecting polysaccharide, and lyophilizing.
Drawing a glucose standard curve: precisely measuring 1mL, 2mL, 4mL, 6mL and 10mL of standard glucose solution (0.6mg/mL), respectively placing in a 50mL volumetric flask, adding water to a constant volume, and shaking up; precisely measuring 2mL of the solution in each volumetric flask, respectively adding 1mL of 6% phenol solution and 6mL of 95% sulfuric acid solution, and uniformly mixing by using a sampler; water bath at 40 deg.C for 30min, taking out and cooling to room temperature; determination of light absorption value OD by spectrophotometer490(ii) a A linear regression equation was prepared using the glucose concentration as abscissa and the absorbance as ordinate, and the glucose standard curve is shown in FIG. 5.
Phenol-sulfuric acid process: sucking 300 mu L of supernatant, diluting by 30 times, sucking 2mL of supernatant into a 50mL centrifuge tube, and measuring 3 parts; respectively adding 1mL of 6% phenol solution and 6mL of 95% sulfuric acid solution, and shaking up; water bath at 40 deg.C for 30min, taking out and cooling to room temperature; determination of light absorption value OD by spectrophotometer490(ii) a The polysaccharide content was calculated according to a standard curve.
Example 2 Cholesterol lowering qualitative method of Lactobacillus plantarum La-10 exopolysaccharide
Collecting EPS powder which is extracted, purified and freeze-dried after La-10 is subjected to static culture in an improved MRS liquid culture medium when the inoculation amount is 4%, the sucrose content is 30g/L, the fermentation time is 30h and the fermentation temperature is 30 ℃.
0.8g/L EPS solid powder was weighed into a 15mL clean centrifuge tube and 10mL cholesterol standard (1mg/mL) was added, with no exopolysaccharide added as a control. Oscillating and adsorbing for 15h at the constant temperature of 30 ℃, centrifuging for 6 minutes at 10000rpm, taking the supernatant, measuring the cholesterol content of the supernatant by an o-phthalaldehyde method, and calculating the cholesterol adsorption rate of the La-10 extracellular polysaccharide to be 41.45 +/-1.3 percent by utilizing the formula.
The method of terephthalaldehyde: sucking 500 mu L of supernatant, diluting the solution to be detected by 5 times, sucking 400 mu L of supernatant into a 15mL centrifuge tube, and measuring 3 parts; 2mL of the terephthalaldehyde reagent and 1mL of 95% sulfuric acid solution are respectively added, and the materials are uniformly mixed by a sampler; standing at room temperature for 10 min; determination of light absorption value OD by spectrophotometer550(ii) a And calculating the cholesterol content according to the cholesterol standard curve, and calculating the cholesterol clearance according to a formula.
The standard curve for cholesterol was plotted as shown in table 1 and fig. 10.
Example 3 production of EPS by Lactobacillus plantarum La-10 liquid stationary fermentation
Inoculating lactobacillus plantarum La-10 in an inoculation amount of 5% volume fraction into an improved MRS liquid culture medium, performing static culture at a fermentation temperature of 31 ℃ for 35 hours, wherein the sucrose content in the improved MRS liquid culture medium is 34 g/L.
MRS liquid medium composition: 10.0g/L of soybean peptone, 10.0g/L of beef extract, 5.0g/L of yeast extract and 34g/L, K of sucrose2PO42.0g/L, 5.0g/L sodium acetate, 2.0g/L, MgSO g/L triammonium citrate4·7H2O is 0.58g/L, MnSO4·4H2O is 0.25g/L, Tween-80 is 1.0mL/L, and pH is 6.2-6.4.
The EPS extraction method comprises the following steps:
(1) centrifuging at 4 deg.C and 8000r/min for 15min, collecting supernatant, and removing thallus precipitate.
(2) Trichloroacetic acid (TCA) was added to the supernatant to a final mass fraction of 5%, and the mixture was allowed to stand overnight at 4 ℃ to remove proteins.
(3) Centrifuging at 4 deg.C and 8000r/min for 10min to obtain supernatant.
(4) The supernatant was added with 95% ethanol in an amount of 4 times the volume of the supernatant, and the mixture was allowed to stand overnight at 4 ℃ to precipitate crude polysaccharide.
(5) Centrifuging at 4 deg.C and 8000r/min for 10min, discarding supernatant, and dissolving precipitate in sterilized ddH2And (4) in O.
(6) Performing three-stage water dialysis (pore size of dialysis bag is 3500Da) for 16h, changing water every 8h, dialyzing with ultrapure water for 8h, changing water every 4h, collecting dialyzed polysaccharide solution, absorbing a small amount of diluted polysaccharide solution, and determining EPS content to be 2.955 + -0.039 g/L by phenol-sulfuric acid method.
(7) Adding 3 times volume of 95% ethanol solution, standing overnight at 4 deg.C, collecting polysaccharide, and lyophilizing.
Drawing a glucose standard curve: precisely measuring 1mL, 2mL, 4mL, 6mL and 10mL of standard glucose solution (0.6mg/mL), respectively placing in a 50mL volumetric flask, adding water to a constant volume, and shaking up; precisely measuring 2mL of the solution in each volumetric flask, respectively adding 1mL of 6% phenol solution and 6mL of 95% sulfuric acid solution, and uniformly mixing by using a sampler; water bath at 40 deg.C for 30min, taking out and cooling to room temperature; determination of light absorption value OD by spectrophotometer490(ii) a A linear regression equation was prepared using the glucose concentration as abscissa and the absorbance as ordinate, and the glucose standard curve is shown in FIG. 5.
Phenol-sulfuric acid process: sucking 300 mu L of supernatant, diluting by 30 times, sucking 2mL of supernatant into a 50mL centrifuge tube, and measuring 3 parts; respectively adding 1mL of 6% phenol solution and 6mL of 95% sulfuric acid solution, and shaking up; water bath at 40 deg.C for 30min, taking out and cooling to room temperature; determination of light absorption value OD by spectrophotometer490(ii) a The polysaccharide content was calculated according to a standard curve.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for producing exopolysaccharide with high yield by lactobacillus plantarum is characterized by comprising the following steps:
(1) lactobacillus plantarum (A)Lactobacillus plantarum) Standing and fermenting the La-10 in an improved MRS liquid culture medium, wherein the inoculation amount is 3-5 percent, and obtaining thallus fermentation liquor;
the standing fermentation condition is that the standing fermentation is carried out for 30 to 40 hours at the temperature of between 25 and 31 ℃;
the improved MRS liquid culture medium comprises: 10.0g/L of soybean peptone, 10.0g/L of beef extract, 5.0g/L of yeast extract and 30-40 g/L, K of sucrose2HPO42.0g/L, 5.0g/L sodium acetate, 2.0g/L, MgSO g/L triammonium citrate4·7H2O is 0.58g/L, MnSO4·4H20.25g/L of O, 1.0mL/L of Tween-80 and pH of 6.2-6.4, and sterilizing for later use;
(2) centrifuging the thallus fermentation liquor obtained in the step (1), and taking supernatant; adding trichloroacetic acid to remove protein, standing at 4 deg.C, centrifuging, and collecting supernatant; precipitating polysaccharide with 95% ethanol, centrifuging, removing supernatant, precipitating, dialyzing to obtain crude polysaccharide, measuring extraction amount, and lyophilizing to obtain powder, i.e. Lactobacillus plantarum extracellular polysaccharide.
2. The method for high yield of exopolysaccharides from lactobacillus plantarum according to claim 1, characterized in that:
the inoculation amount in the step (1) is 4-5%.
3. The method for high yield of exopolysaccharides from lactobacillus plantarum according to claim 1, characterized in that:
the standing fermentation condition in the step (1) is 30-31 ℃ for 30-35 h.
4. The method for high yield of exopolysaccharides from lactobacillus plantarum according to claim 1, characterized in that:
the improved MRS liquid culture medium in the step (1) is as follows: 10.0g/L of soybean peptone, 10.0g/L of beef extract, 5.0g/L of yeast extract and 30-34 of sucroseg/L、K2HPO42.0g/L, 5.0g/L sodium acetate, 2.0g/L, MgSO g/L triammonium citrate4·7H2O is 0.58g/L, MnSO4·4H20.25g/L of O, 1.0mL/L of Tween-80 and pH of 6.2-6.4, and sterilizing for later use.
5. A method for high yield of exopolysaccharides from Lactobacillus plantarum according to claim 4, characterized in that:
the improved MRS liquid culture medium in the step (1) is as follows: soybean peptone 10.0g/L, beef extract 10.0g/L, yeast extract 5.0g/L, sucrose 30g/L, K g2HPO42.0g/L, 5.0g/L sodium acetate, 2.0g/L, MgSO g/L triammonium citrate4·7H2O is 0.58g/L, MnSO4·4H20.25g/L of O, 1.0mL/L of Tween-80 and pH of 6.2-6.4, and sterilizing for later use.
6. The method for high yield of exopolysaccharides from lactobacillus plantarum according to claim 1, characterized in that:
the centrifugation conditions in the step (2) are all centrifugation for 15min at 8000r/min and 4 ℃;
the aperture size of the dialysis bag used for dialysis in the step (2) is 3500 Da;
the extraction amount measured in the step (2) is 2.67-3.18 g/L.
7. A Lactobacillus plantarum exopolysaccharide characterized by being prepared by the preparation method according to any one of claims 1 to 6.
8. The use of the extracellular polysaccharide of lactobacillus plantarum as defined in claim 7 for the preparation of lipid-lowering drugs or health products.
9. Use according to claim 8, characterized in that:
adding the lactobacillus plantarum extracellular polysaccharide into 1g/L cholesterol standard solution, wherein the final concentration of the lactobacillus plantarum extracellular polysaccharide is 0.8-1.6 g/L, and oscillating and adsorbing for 15-25 h at 30-37 ℃ under a constant temperature condition.
10. Use according to claim 9, characterized in that:
the final concentration of the lactobacillus plantarum extracellular polysaccharide is 0.8 g/L;
the oscillating adsorption condition is oscillating adsorption for 15 hours at 30 ℃ under the constant temperature condition.
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| CN105400725A (en) * | 2015-12-18 | 2016-03-16 | 上海理工大学 | Lactobacillus plantarum strain, and applications thereof |
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