CN113755403A - Exopolysaccharide-producing lactobacillus pentosus, and fermentation process and application thereof - Google Patents
Exopolysaccharide-producing lactobacillus pentosus, and fermentation process and application thereof Download PDFInfo
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- 241000186684 Lactobacillus pentosus Species 0.000 title claims abstract description 31
- 238000000855 fermentation Methods 0.000 title claims abstract description 31
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- 239000001963 growth medium Substances 0.000 claims abstract description 28
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims abstract description 20
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- YXVFQADLFFNVDS-UHFFFAOYSA-N diammonium citrate Chemical compound [NH4+].[NH4+].[O-]C(=O)CC(O)(C(=O)O)CC([O-])=O YXVFQADLFFNVDS-UHFFFAOYSA-N 0.000 description 1
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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Abstract
The invention provides lactobacillus pentosus for producing exopolysaccharides, a fermentation process and application thereof, wherein the lactobacillus pentosus YY112 is screened from the surfaces of natural fruits and vegetables, is preserved in China general microbiological culture collection management center in 2019, 09 and 16 days, and has a preservation number of CGMCC NO. 18492. The fermentation process for producing the exopolysaccharide by the lactobacillus pentosus comprises the following steps: (1) preparing a lactic acid bacteria basic culture medium, adjusting the pH value to 5.0-7.0, inoculating an activated lactobacillus pentosus seed culture solution into the culture medium, and culturing for 12-48 h at 25-40 ℃; (2) centrifuging the cultured fermentation liquor to remove thalli, adding trichloroacetic acid solution with the mass volume ratio of 80% into the supernatant until the final mass volume ratio is 4%, standing overnight at 4 ℃, centrifuging to obtain the supernatant, adding 80% ethanol solution with the volume 3 times that of the supernatant, standing for 12h at 4 ℃, centrifuging to obtain precipitates; (3) dissolving the precipitate obtained in the step (2) with water, dialyzing with deionized water at 4 ℃ for 48h, changing water every 4h, and finally performing freeze-drying treatment to obtain the extracellular polysaccharide.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus pentosus for producing extracellular polysaccharide, and a fermentation process and application thereof.
Background
Lactic acid bacteria are a general name of bacteria capable of generating lactic acid by using carbohydrate fermentation, widely exist in the nature and organisms, are known as safe microorganisms, have a series of important physiological functions of maintaining the balance of intestinal flora, enhancing immunity, reducing cholesterol, reducing blood pressure, resisting tumors, resisting oxidation, resisting mutation and the like, are important fermentation strains in the food industry, are indispensable star strains in the fields of fermented dairy products, meat products and fruit and vegetable products, can improve the flavor of products, improve the nutritional value and prolong the storage time. Lactic acid bacteria are also heavy members of the probiotic family, and the application fields thereof relate to food, feed, health products and medicines, and the application objects relate to human bodies, poultry, livestock, aquatic products and the like, and the importance of the lactic acid bacteria on the social economy is self-evident.
The extracellular polysaccharide is a general name of mucopolysaccharide and capsular polysaccharide secreted outside cell walls by microorganisms, and the lactobacillus polysaccharide has multiple functions of resisting tumors, mutation, ulcer, inflammation, immunoregulation, blood pressure reduction, cholesterol reduction and the like, and has been widely applied and paid attention to in the pharmaceutical field and the food industry. The development of novel EPS becomes one of the hot spots of industrial microorganism research, and the structure and performance of the novel EPS are also increasingly emphasized. The EPS yield of the lactic acid bacteria is related to the properties of strains and is also influenced by nutrient components and growth conditions of a culture medium, and the EPS yield of the lactic acid bacteria is generally low at present and is usually 50-425 mg.L-1How to improve the yield of the exopolysaccharide is a problem to be solved at present.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide lactobacillus pentosus for producing exopolysaccharides and a fermentation process and application thereof.
The purpose of the invention is realized by the following technical scheme:
the lactobacillus pentosus for producing the exopolysaccharides is preserved in the China general microbiological culture collection center in 2019, 09 and 16 days, and the preservation number is CGMCC 18492.
A fermentation process for producing exopolysaccharides from lactobacillus pentosus comprises the following steps:
(1) preparing a lactic acid bacteria basic culture medium, adjusting the pH value to 5.0-7.0, inoculating 3-5% of activated lactobacillus pentosus seed culture solution into the culture medium, and culturing at 25-35 ℃ for 18-30 hours;
(2) centrifuging the fermentation liquor cultured in the step (1) to remove thalli, adding a trichloroacetic acid solution with the mass volume ratio of 80% into the supernatant until the final mass volume ratio is 4%, standing overnight at 4 ℃, centrifuging to obtain the supernatant, adding an 80% ethanol solution with the volume of 3 times of that of the supernatant, standing for 12 hours at 4 ℃, centrifuging to obtain a precipitate;
(3) dissolving the precipitate obtained in the step (2) with water, dialyzing with deionized water at 4 ℃ for 48h, changing water every 4h, and finally performing freeze-drying treatment to obtain the Extracellular Polysaccharide (EPS).
Further, the lactobacillus basal culture medium is an MRS culture medium, wherein the addition amount of sucrose is 2-4%, the addition amount of soybean peptone is 0.5-1.5%, and the addition amount of yeast extract powder is 1-2%.
Furthermore, the inoculation amount of the lactobacillus pentosus seed culture solution in the step (1) is 4.31 percent, the culture time is 24.66 hours, the culture temperature is 29.61 ℃, and the initial fermentation pH value is 6.04.
Furthermore, the inoculation amount of the lactobacillus pentosus seed culture solution in the step (1) is 4%, the culture time is 24.5h, the culture temperature is 29.5 ℃, and the initial fermentation pH value is 6.0.
Another aspect of the invention:
an exopolysaccharide obtained by the fermentation process, the exopolysaccharide having a monosaccharide composition: glucose, mannose, glucosamine, galactose and rhamnose in a molar ratioIs 62.69: 85.85: 2.46: 2.92: 1.00, and has an average relative molecular weight of 5.9 × 104Da。
The application of the lactobacillus pentosus YY112, which is used for producing exopolysaccharides.
Compared with the prior art, the invention has the beneficial effects that:
the invention respectively determines the appropriate range of the related factors of different nutrient components and culture conditions by adopting a single factor method, further determines the optimum addition amount of the different nutrient components by adopting an orthogonal test analysis method, determines the appropriate level of important culture parameters by adopting a Box-Behnken response surface analysis method, and obtains the nutrient conditions and culture growth process parameters suitable for producing EPS by YY112 fermentation by combining two optimization methods. The EPS yield of the optimized YY112 strain is 380.97 mg.L-1Compared with 260 mg.L before optimization-146.52 percent is increased, the EPS yield is greatly improved, and the method has positive significance for promoting the actual production and application of the lactobacillus pentosus YY 112.
Drawings
FIG. 1 is a glucose standard curve;
FIG. 2 is a molecular weight calibration curve for polysaccharides;
FIG. 3 is the relative molecular weight distribution of EPS-2;
FIG. 4 is a standard monosaccharide chromatogram;
FIG. 5 is an EPS-2 monosaccharide chromatogram.
Biological preservation
The inventor obtains the lactobacillus pentosus from the surface screening of natural fruits and vegetables:
lactobacillus pentosus of the present invention (Lactobacillus pentosae) The strain is preserved in China general microbiological culture Collection center (CGMCC for preservation organization) in 2019, 09 and 16, and the addresses are as follows: the microbiological research institute of western road 1, 3, national academy of sciences, north-kyo, chaoyang, the postal code: 100101, preservation number is CGMCC number 18492.
Detailed Description
Reagents used in the examples of the present application: glucose, sucrose, milkSugar, maltose, fructose, sodium acetate, diammonium citrate, triammonium citrate, monopotassium phosphate, Tween-80, sulfuric acid, phenol, MgSO4 & 7H2O、MnSO4·4H2O、K2HPO4HCl, NaOH and the like are analytically pure; peptone, tryptone, soybean peptone and casein peptone are all biochemical reagents.
The culture medium comprises: MRS culture medium; SL medium; SDM medium; elliker medium; PTYG medium; skimmed milk powder culture medium.
Example 1
The embodiment provides the lactobacillus pentosus for producing the exopolysaccharides, and the lactobacillus pentosus YY112 is preserved in the China general microbiological culture collection center in 2019, 09 and 16 days, with the preservation number of CGMCC number 18492.
The fermentation process for producing exopolysaccharides by utilizing lactobacillus pentosus comprises the following steps:
(1) preparing a lactic acid bacteria basic culture medium, adjusting the pH value to 5.0-7.0, inoculating an activated lactobacillus pentosus seed culture solution into the culture medium, and culturing for 12-48 h at 25-40 ℃;
(2) centrifuging the fermentation liquor cultured in the step (1) to remove thalli, adding a trichloroacetic acid solution with the mass volume ratio of 80% into the supernatant until the final mass volume ratio is 4%, standing overnight at 4 ℃, centrifuging to obtain the supernatant, adding an 80% ethanol solution with the volume of 3 times of that of the supernatant, standing for 12 hours at 4 ℃, centrifuging to obtain a precipitate;
(3) dissolving the precipitate obtained in the step (2) with water, dialyzing with deionized water at 4 ℃ for 48h, changing water every 4h, and finally performing freeze-drying treatment to obtain the Extracellular Polysaccharide (EPS).
A standard curve (shown in figure 1) is drawn by using a phenol-sulfuric acid method and taking glucose as a standard substance to obtain a linear regression equation: y = 0.2606x + 0.001, R2= 0.9995. And (3) measuring the absorbance value of the EPS aqueous solution at the wavelength of 490 nm under the same condition, and calculating by using a linear regression equation to obtain the EPS content.
Comparative example 1
In order to research the influence of different culture mediums on the EPS yield, 6 lactobacillus basal culture mediums of MRS, SL, SDM, Elliker, PTYG and skim milk are respectively prepared in the comparative example, the pH value is adjusted to 6.0, 2 percent of activated seed culture solution is inoculated, the culture is carried out for 24 hours at 37 ℃, the EPS content is respectively measured, and the proper basal culture medium is determined.
On the basis of the measurements, the measurement,Lactobacillus pentosae YY112 can produce EPS in the 6 culture media, wherein the yield in the MRS culture medium is the highest and is 259.98 mg/L, and the yield in the SDM culture medium is the next and is 254.29 mg/L; the minimum value of skim milk is 227.36 mg/L, so MRS culture medium is selected as basic culture medium.
After the MRS culture medium is determined to be used as a basic culture medium, the influence of specific nutritional conditions on the EPS yield is further researched, wherein the influence comprises the addition amount of a carbon source and a carbon source, the addition amount of a nitrogen source and a yeast extract powder.
Adding 2% of glucose, fructose, sucrose, lactose and maltose respectively, preparing MRS modified culture media with different carbon sources, adjusting the pH value to 6.0, inoculating 2% of activated seed culture solution, culturing at 37 ℃ for 24h, and determining the EPS content respectively. As a result of measurement, it was found that the EPS production was highest at 274.26 mg/L when sucrose was used as a carbon source, followed by glucose (260.12 mg/L), maltose (253.09 mg/L), lactose (250.57 mg/L) and fructose (233.44 mg/L), and sucrose was selected as the carbon source. And the EPS yield is continuously increased along with the increase of the addition amount of the sucrose, when the addition amount of the sucrose reaches 4%, the EPS yield is the highest and is 292.99 mg/L, but the EPS yield is not obviously different from the EPS yield (291.42 mg/L) when the addition amount of the sucrose reaches 3%. Considering efficiency and economy comprehensively, the adding range of the sucrose is selected to be 2-4%.
Different nitrogen sources MRS improved culture media are prepared by respectively adding 1% of soybean peptone, tryptone, casein peptone and common peptone, the pH value is adjusted to 6.0, 2% of activated seed culture solution is inoculated, and the EPS content is respectively determined after the culture is carried out for 24h at 37 ℃. It was determined that all 4 kinds of peptone promoted EPS synthesis, wherein the EPS production was highest at 285.24 mg/L when soybean peptone was added as a nitrogen source, followed by casein peptone (274.46 mg/L), peptone (261.78 mg/L), and tryptone (270.80 mg/L), and soybean peptone was selected as a nitrogen source for preparation of the medium. When the addition amount of the soybean peptone is 1.0 percent, the EPS yield is the highest and is 285.22 mg/L, and 0.5 to 1.5 percent is selected as the addition amount range of the soybean peptone.
Adding yeast extract powder 0.25%, 0.50%, 0.75% and 1.00%, adjusting pH to 6.0, inoculating 2% activated seed culture solution, culturing at 37 deg.C for 24 hr, and determining EPS content. As can be seen from the measurement, the EPS yield is improved along with the increase of the addition amount of the yeast extract powder, when the addition amount of the yeast extract powder reaches 1.5 percent,Lactobacillus pentosae the YY112 EPS yield is the highest and is 276.58 mg/L, and then the yield tends to be smooth, so 1-2% of the yield is selected as the addition range of yeast extract powder.
With the addition of carbon source, nitrogen source and yeast powder as factors and EPS yield as an index, L9 (3) is designed3) And (4) performing orthogonal test to determine the nutritional condition of high-yield EPS. Finally, the optimal combination is determined to be that the adding amount of sucrose is 3%, the adding amount of soybean peptone is 1% and the adding amount of yeast extract powder is 2%.
Comparative example 2
In order to explore the influence of the culture conditions on the EPS yield, the optimal factor level is determined in the comparative example by designing an experiment by adopting a Box-Behnken response surface method with the initial pH value, the inoculation amount, the culture temperature and the culture time as factors and the EPS as an index.
Single factor experiments:
adjusting the initial pH value of the fermentation medium to 5.0, 6.0, 7.0 and 8.0 respectively, inoculating 2% activated seed culture solution, culturing at 37 deg.C for 24 hr, and determining EPS content respectively. Initial pH value pairLactobacillus pentosaeThe effect of YY112 EPS production was: when the pH value is 6, the EPS yield reaches the maximum of 320.93 mg/L, so that 5-7 is selected as the initial pH value range of fermentation.
Adjusting the pH value of the culture medium to 6.0, respectively inoculating the activated seed culture solution into a fermentation culture medium according to the volume ratio of 1%, 2%, 3%, 4% and 5%, culturing at 37 ℃ for 24h, and respectively determining the EPS content. Different inoculation amount pairsLactobacillus pentosaeThe effect of YY112 EPS production was: the EPS production increased with increasing inoculum size, and was highest at 363.07 mg/L when the inoculum size was 4%. When the inoculation amount is 5%, the EPS yield slightly decreases, so that 3% -5% is selected as the optimal inoculation amount range.
Adjusting the pH value of the culture medium to 6.0, inoculating 2% activated seedsThe culture solution was cultured at 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, and 40 deg.C for 24h, respectively, to determine EPS content. Temperature pair for cultivationLactobacillus pentosae The effect of YY112 EPS production was: when the culture temperature reaches 30 ℃, the EPS yield is the highest and is 345.51 mg/L. When the culture temperature is low or high, the EPS production tends to decrease. This is consistent with the literature reporting that the EPS production is facilitated when the culture temperature is lower than the optimal growth temperature, so that 25-35 ℃ is selected as the optimal culture temperature range.
Adjusting the pH value of the culture medium to 6.0, inoculating 2% activated seed culture solution, culturing at 37 deg.C for 18h, 24h, 30h, 36h and 42h, and determining EPS content. Pair of cultivation timeLactobacillus pentosae The effect of YY112 EPS production was: when the culture time reaches 24h, the EPS yield reaches the maximum, and is 321.57 mg/L. When the cultivation time is prolonged, EPS gradually decreases, probably due to the decrease of nutrients in the medium or hydrolysis of polysaccharides. Therefore, the optimum culture time range is selected to be 18-30 h.
Finally, the optimal culture conditions are obtained through software analysis: the inoculation amount is 4.31 percent, the culture time is 24.66 hours, the culture temperature is 29.61 ℃, the initial fermentation pH value is 6.04 under the culture conditionLactobacillus pentosaeThe theoretical yield of YY112 EPS is 382.886 mg/L. In view of the actual operability, the parameters are optimized as: the inoculation amount is 4 percent, the culture time is 24.5 hours, the culture temperature is 29.5 ℃, and the initial fermentation pH value is 6.0.
Example 2
The exopolysaccharide obtained in example 1 isLactobacillus pentosae YY112 crude polysaccharide, which was then purified using DEAE-Sepharose Fast Flow ion exchange column. A chromatographic column (D2.6X 30 cm) was packed with DEAE-Sepharose Fast Flow gel, the ethanol in the filler was washed away with degassed ultrapure water, and the filler was equilibrated by adding 0.1 mol/L NaCl solution to the washed-away filler. Will be provided withLactobacillus pentosaeThe YY112 crude polysaccharide sample was dissolved in ultrapure water (50 mg/mL) and loaded at 100 mg. Respectively eluting with ultrapure water, 0.1 mol/L NaCl solution and 0.2 mol/L NaCl solution at a flow rate of 3 mL/min. Collecting each fraction of polysaccharide solution with an automatic fraction collector for 6 mL/tube at a collection time of 4 min/tubeAnd (4) liquid.
The collected polysaccharide is subjected to a phenol-sulfuric acid method, and the polysaccharide content is measured by using an enzyme-labeling instrument. Drawing an elution curve, collecting single peak polysaccharide solution according to the distribution of each component, dialyzing with ionized water for 3d, changing water every 8h, concentrating the dialyzed sample solution, and freeze-drying to obtain the exopolysaccharide.
The monosaccharide components and molecular weights of the purified exopolysaccharides of this example were determined.
Different Dextran standards were tested using High Performance Gel Filtration Chromatography (HPGFC) and standard curves were plotted based on the results, as shown in FIG. 2.
The regression equation can be obtained from the data in the graph as: log M =12.706-0.4818RT (R = 0.9918). EPS-2 was injected under the same conditions, and the results are shown in FIG. 3. As can be seen from the figure, EPS-2 shows a single symmetrical peak pattern. The retention time of the elution curve was 17.02 min, and the retention time was substituted into the regression equation, and the average relative molecular weight was calculated to be 5.9X 104 Da.
Hydrolyzing different monosaccharide standards with TFA, and injecting sample to obtain standard monosaccharide chromatogram as shown in FIG. 4. EPS-2 was injected under the same conditions, and its monosaccharide chromatogram was determined as shown in FIG. 5. Their monosaccharide composition can be inferred by comparison with standard monosaccharide chromatograms.
As can be seen from FIG. 5, EPS-2 is composed of five monosaccharides, i.e., glucose, mannose, glucosamine, galactose and rhamnose, and the molar ratio is calculated as follows: 62.69: 85.85: 2.46: 2.92: 1.00.
finally, it should be noted that the above only illustrates the technical solution of the present invention, but not limited thereto, and although the present invention has been described in detail with reference to the preferred arrangement, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made thereto without departing from the spirit and scope of the technical solution of the present invention.
Claims (8)
1. The lactobacillus pentosus for producing the exopolysaccharides is characterized in that the lactobacillus pentosus YY112 is preserved in the China general microbiological culture collection management center in 2019, 09 and 16 months with the preservation number of CGMCC number 18492.
2. A fermentation process for exopolysaccharide production by lactobacillus pentosus according to claim 1, wherein the fermentation process comprises the following steps:
(1) preparing a lactic acid bacteria basic culture medium, adjusting the pH value to 5.0-7.0, inoculating 3-5% of activated lactobacillus pentosus seed culture solution into the culture medium, and culturing at 25-35 ℃ for 18-30 h;
(2) centrifuging the fermentation liquor cultured in the step (1) to remove thalli, adding a trichloroacetic acid solution with the mass volume ratio of 80% into the supernatant until the final mass volume ratio is 4%, standing overnight at 4 ℃, centrifuging to obtain the supernatant, adding an 80% ethanol solution with the volume of 3 times of that of the supernatant, standing for 12 hours at 4 ℃, centrifuging to obtain a precipitate;
(3) dissolving the precipitate obtained in the step (2) with water, dialyzing with deionized water at 4 ℃ for 48h, changing water every 4h, and finally performing freeze-drying treatment to obtain the Extracellular Polysaccharide (EPS).
3. The fermentation process of lactobacillus pentosus for producing exopolysaccharides according to claim 2, wherein the inoculation amount of the lactobacillus pentosus seed culture solution in step (1) is 4.31%, the culture time is 24.66h, the culture temperature is 29.61 ℃, and the initial fermentation pH value is 6.04.
4. The fermentation process for producing exopolysaccharides of lactobacillus pentosus according to claim 2, wherein the inoculation amount of the lactobacillus pentosus seed culture solution in step (1) is 4%, the culture time is 24.5h, the culture temperature is 29.5 ℃, and the initial fermentation pH value is 6.0.
5. The fermentation process for producing exopolysaccharides by lactobacillus pentosus according to claim 2, wherein the lactobacillus basal medium is an MRS medium, wherein the addition amount of sucrose is 2-4%, the addition amount of soybean peptone is 1%, and the addition amount of yeast extract powder is 2%.
6. Exopolysaccharide, obtainable by a fermentation process according to any one of claims 2 to 4.
7. Exopolysaccharide according to claim 6, characterized in that its monosaccharide composition is: glucose, mannose, glucosamine, galactose and rhamnose with a molar ratio of 62.69: 85.85: 2.46: 2.92: 1.00 and an average relative molecular weight of 5.9 × 104Da。
8. Use of lactobacillus pentosus according to claim 1 for the production of exopolysaccharides.
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