CN112029807A - Preparation method of lactobacillus rhamnosus exopolysaccharide - Google Patents
Preparation method of lactobacillus rhamnosus exopolysaccharide Download PDFInfo
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- 229920002444 Exopolysaccharide Polymers 0.000 title claims abstract description 42
- 241000218588 Lactobacillus rhamnosus Species 0.000 title claims abstract description 39
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 150000004676 glycans Chemical class 0.000 claims abstract description 24
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 24
- 239000005017 polysaccharide Substances 0.000 claims abstract description 24
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000001963 growth medium Substances 0.000 claims abstract description 17
- 235000020183 skimmed milk Nutrition 0.000 claims abstract description 12
- 238000001704 evaporation Methods 0.000 claims abstract description 10
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000009835 boiling Methods 0.000 claims abstract description 6
- 238000000855 fermentation Methods 0.000 claims abstract description 5
- 230000004151 fermentation Effects 0.000 claims abstract description 5
- 230000008020 evaporation Effects 0.000 claims abstract description 4
- 239000006228 supernatant Substances 0.000 claims description 15
- 238000009630 liquid culture Methods 0.000 claims description 10
- 239000002244 precipitate Substances 0.000 claims description 10
- 238000005119 centrifugation Methods 0.000 claims description 8
- 239000008367 deionised water Substances 0.000 claims description 8
- 229910021641 deionized water Inorganic materials 0.000 claims description 8
- 238000000502 dialysis Methods 0.000 claims description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 4
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- 238000011081 inoculation Methods 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 11
- -1 DPPH free radical Chemical class 0.000 abstract description 8
- 230000003078 antioxidant effect Effects 0.000 abstract description 8
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 abstract description 7
- 229910001448 ferrous ion Inorganic materials 0.000 abstract description 7
- 230000002000 scavenging effect Effects 0.000 abstract description 6
- 235000015097 nutrients Nutrition 0.000 abstract description 2
- 238000001556 precipitation Methods 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 241001052560 Thallis Species 0.000 abstract 1
- 238000004108 freeze drying Methods 0.000 abstract 1
- 230000001376 precipitating effect Effects 0.000 abstract 1
- 238000002835 absorbance Methods 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 150000003254 radicals Chemical class 0.000 description 9
- 239000003963 antioxidant agent Substances 0.000 description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 6
- 230000002292 Radical scavenging effect Effects 0.000 description 5
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 3
- 241000186660 Lactobacillus Species 0.000 description 3
- 239000011668 ascorbic acid Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- 239000004310 lactic acid Substances 0.000 description 3
- 235000014655 lactic acid Nutrition 0.000 description 3
- 229940039696 lactobacillus Drugs 0.000 description 3
- 239000003642 reactive oxygen metabolite Substances 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 229910021577 Iron(II) chloride Inorganic materials 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 241000186000 Bifidobacterium Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000917009 Lactobacillus rhamnosus GG Species 0.000 description 1
- 241000194036 Lactococcus Species 0.000 description 1
- RXBKMJIPNDOHFR-UHFFFAOYSA-N Phenelzine sulfate Chemical compound OS(O)(=O)=O.NNCCC1=CC=CC=C1 RXBKMJIPNDOHFR-UHFFFAOYSA-N 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 229960001506 brilliant green Drugs 0.000 description 1
- HXCILVUBKWANLN-UHFFFAOYSA-N brilliant green cation Chemical compound C1=CC(N(CC)CC)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](CC)CC)C=C1 HXCILVUBKWANLN-UHFFFAOYSA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000003871 intestinal function Effects 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000005445 natural material Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 230000008855 peristalsis Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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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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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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Abstract
The invention provides a preparation method of lactobacillus rhamnosus exopolysaccharide, which comprises the steps of inoculating lactobacillus rhamnosus into a skim milk culture medium for fermentation, then centrifuging in boiling water bath to remove thalli, removing protein by trichloroacetic acid, precipitating by ethanol, and freeze-drying in vacuum to prepare the lactobacillus rhamnosus exopolysaccharide. The yield of exopolysaccharide in the method is 625 mg/L. On the basis of not changing the physical and chemical properties and nutrient components of the polysaccharide, the method of evaporation concentration is utilized, so that the dosage of trichloroacetic acid and ethanol is greatly reduced, and the efficiency of protein removal and polysaccharide precipitation is improved. The exopolysaccharide obtained by the method has antioxidant activity, has scavenging ability on DPPH free radical, hydroxyl free radical and superoxide anion free radical, and has chelating ability on ferrous ion.
Description
Technical Field
The invention relates to the field of lactobacillus exopolysaccharide, in particular to a preparation method of lactobacillus rhamnosus exopolysaccharide.
Background
Lactic acid bacteria are a general term for microorganisms that can ferment carbohydrates and produce large amounts of lactic acid. The method mainly comprises the following steps: lactobacillus, Bifidobacterium, lactococcus andstreptococcus. It is generally recognized as a safe microorganism (GRAS) because of its long history of safe use in food production. In an organism, the LAB can regulate intestinal flora, promote intestinal peristalsis, protect intestinal ecological balance, improve intestinal functions, improve organism immunity, inhibit putrefying bacteria breeding and the like. The exopolysaccharide is water soluble long chain polysaccharide produced and secreted by lactobacillus during its growth and metabolism, and has molecular weight of 4.0 × 104～6.0×106u is between. Exopolysaccharides produced by lactic acid bacteria have a variety of potential biological activities, including antioxidant, anti-inflammatory, gut flora regulating, anti-tumor, immune regulating, and antibacterial, among others.
Under normal conditions, reactive oxygen species in the body are byproducts of aerobic metabolism including hydroxyl radicals, superoxide anions, hydrogen peroxide, and the like, and the production and elimination of reactive oxygen species maintain the oxidation-antioxidant balance and play an important role in regulating the conduction of signal pathways and cell proliferation. When the balance is disrupted, the level of reactive oxygen species increases, resulting in the production of free radicals which may have deleterious effects on proteins, lipids and DNA, causing the body to develop oxidative stress, causing oxidative damage to cells and developing multisystem diseases. Although synthetic antioxidants can effectively slow down the oxidation process, they also have many side effects on the body, and thus, the attention on safe antioxidants in natural substances is increasing. Exopolysaccharides have high antioxidant activity and low cytotoxicity, and thus have received extensive attention from researchers. The existing method for culturing and fermenting exopolysaccharide by lactobacillus rhamnosus has low yield and cannot achieve the application of exopolysaccharide in production.
Disclosure of Invention
The invention aims to provide a preparation method of lactobacillus rhamnosus exopolysaccharide, which solves the problem of low yield of the existing method for culturing and fermenting the exopolysaccharide by lactobacillus rhamnosus, and the yield of the exopolysaccharide is 625 mg/L.
1. The preparation method of the extracellular polysaccharide provided by the invention specifically comprises the following steps: a preparation method of lactobacillus rhamnosus exopolysaccharide is characterized by comprising the following steps:
(1) inoculating lactobacillus rhamnosus into a skim milk liquid culture medium containing glucose, and standing and fermenting in a constant-temperature incubator;
(2) carrying out boiling water bath on the fermented culture medium to inactivate thallus and enzyme, denaturalizing protein, taking out, cooling to room temperature, centrifuging at low temperature and taking supernatant;
(3) evaporating and concentrating the supernatant to 1/3 of the original volume to obtain a first concentrated solution, then adding trichloroacetic acid with the mass concentration of 80% into the first concentrated solution, wherein the final mass concentration of the trichloroacetic acid is 4%, standing to fully precipitate the protein, and centrifuging at low temperature to obtain the supernatant;
(4) evaporating and concentrating the supernatant to 1/3 of the original volume to obtain a second concentrated solution, then adding pre-cooled 95% ethanol with mass concentration, wherein the volume ratio of the ethanol to the second concentrated solution is 3:1, standing to fully precipitate polysaccharide, then centrifuging at low temperature, removing the supernatant, and taking the precipitate;
(5) transferring the precipitate to a dialysis bag, dialyzing with deionized water at 4 ℃ for 3 days, and changing water every 12 hours;
(6) and carrying out vacuum freeze drying on the dialyzed extracellular polysaccharide to finally obtain white floccule, namely the crude polysaccharide.
The invention also has the following features:
1. the inoculation amount of lactobacillus rhamnosus is 3%.
2. In the step (1), the skim milk liquid culture medium contains per liter: 100g of skimmed milk powder and 10g of glucose.
3. In the step (1), the standing fermentation is carried out at 37 ℃ for 36 hours.
4. In the steps (2) and (3), the low-temperature centrifugation conditions are as follows: centrifugation was carried out at 10000 Xg for 15 minutes at 4 ℃.
5. In the steps (3) and (4), the evaporation and concentration conditions are as follows: concentrating under reduced pressure at 60-65 ℃.
6. In steps (3) and (4), the mixture was allowed to stand at 4 ℃ for 12 hours.
7. In the step (4), the low-temperature centrifugation conditions are as follows: centrifugation was carried out at 10000 Xg for 20 minutes at 4 ℃.
8. In the step (5), the dialysis bag is boiled in boiling water for 10 minutes before use, and the specification is 8000-14000 Da.
9. In step (5), the dialysis was performed with deionized water at 4 ℃ for 3 days, and the water was changed every 12 hours.
The invention has the advantages and beneficial effects that: according to the preparation method of the exopolysaccharide, glucose is used as a carbon source, and the lactobacillus rhamnosus is fermented by using the skim milk culture medium, so that the exopolysaccharide yield is increased and is 625 mg/L. On the basis of not changing the physical and chemical properties and nutrient components of the polysaccharide, the method of evaporation concentration is utilized, so that the dosage of trichloroacetic acid and ethanol is greatly reduced, and the efficiency of protein removal and polysaccharide precipitation is improved. By verification, the extracellular polysaccharide obtained by the method has antioxidant activity, has scavenging capacity on DPPH free radicals, hydroxyl free radicals and superoxide anion free radicals, has chelating capacity on ferrous ions, and provides reference for application of the extracellular polysaccharide in industrial production.
Drawings
FIG. 1 shows the DPPH radical scavenging ability of extracellular polysaccharide of Lactobacillus rhamnosus;
FIG. 2 shows the hydroxyl radical scavenging ability of extracellular polysaccharide of Lactobacillus rhamnosus;
FIG. 3 shows the scavenging ability of extracellular polysaccharide of Lactobacillus rhamnosus to superoxide anion radical;
FIG. 4 shows the chelating ability of extracellular polysaccharide of Lactobacillus rhamnosus to ferrous ions.
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.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified
Example 1: fermentation culture of lactobacillus rhamnosus
1. Lactobacillus rhamnosus activation
The lactobacillus rhamnosus (L.rhamnosus ATCC53103) required by the invention is streaked from a glycerol tube preserved at the temperature of-80 ℃ to an MRS plate for activation, the MRS plate is placed in a constant temperature incubator at the temperature of 37 ℃ for culture for 24 hours, a single colony is picked by an inoculating loop to 5mL of MRS liquid culture medium, the MRS liquid culture medium is transferred to 100mL of MRS liquid culture medium after the MRS liquid culture medium is placed in the constant temperature incubator at the temperature of 37 ℃ for culture for 12 hours, and the MRS liquid culture medium is reserved.
2. Preparation of skim milk culture medium
200g of skimmed milk powder, 20g of glucose and 2000mL of deionized water, autoclaving at 115 ℃ for 15min, cooling and placing at normal temperature for later use.
3. Fermentation culture of lactobacillus rhamnosus
Inoculating activated lactobacillus rhamnosus in MRS liquid culture medium into 2L skim milk culture medium at an inoculation amount of 3% (v/v), and standing and culturing in a constant temperature incubator at 37 deg.C for 36 h.
Example 2: extraction of extracellular polysaccharide of lactobacillus rhamnosus
1. Placing the fermented skim milk culture medium in boiling water bath for 10min to inactivate thallus and enzyme, and denature protein. Then taking out and cooling to room temperature, centrifuging for 15min at 4 ℃ at 10000 Xg, and taking the supernatant.
2. And (3) evaporating and concentrating the supernatant to 1/3 of the original volume at 60-65 ℃, adding 80% by mass of trichloroacetic acid until the final concentration is 4% by mass, standing in a 4 ℃ refrigerator for 12h, centrifuging at 4 ℃ by 10000 Xg for 15min, and taking the supernatant.
3. And (3) evaporating and concentrating the supernatant to 1/3 of the original volume at 60-65 ℃, adding precooled 95% ethanol into the concentrated supernatant, fully oscillating until part of floccules are separated out, and standing in a refrigerator at 4 ℃ for 12 hours. After the polysaccharide is fully separated out in the form of floccule, centrifuging at 4 ℃ 10000 Xg for 20min and collecting polysaccharide precipitate.
4. The collected polysaccharide was transferred to dialysis bag (8000-. And after the dialysis is finished, carrying out vacuum freeze drying on the polysaccharide to finally obtain the dry white flocculent fluffy crude polysaccharide.
Example 3: determination of antioxidant activity of lactobacillus rhamnosus exopolysaccharide
1. Scavenging ability for DPPH free radical
1.0mL exopolysaccharide solution (0.25-3.0mg/mL) was mixed with 2.0mL of 0.2mM freshly prepared ethanol solution of DPPH, shaken vigorously and mixed well, and incubated at room temperature in the dark for 1 h. The absorbance value A of the mixture was then measured at 517nm1Ascorbic acid was used as a positive control. DPPH radical scavenging capacity was calculated by the following formula:
capacity to clear (%) - (1- (A)1-Ai)/A0]×100
Wherein A isiIs the absorbance value of the sample mixed with alcohol, A0Is the absorbance value of the mixed solution without the sample.
2. Scavenging ability for hydroxyl radicals;
1.0mL exopolysaccharide sample solution (0.25-3.0mg/mL), 1.0mL brilliant green solution (0.435mM), 2.0mL FeSO4(0.5mM) and 1.5mL H2O2(3.0%, w/v) was added to the tube and mixed well. The tubes were incubated at 37 ℃ for 30 min. Then, the absorbance value A was measured at 624nm1Ascorbic acid was used as a positive control. The hydroxyl radical scavenging ability was calculated by the following formula:
clearance (%) - (A)0–A1)/(A0-A)]×100
Wherein A is0Is the absorbance value of the mixed solution of the equal volume deionized water substitute sample, A is the equal volume deionized water substitute sample and H2O2The absorbance value of the mixed solution of (1).
3. Scavenging ability for superoxide anion radicals;
the exopolysaccharide solution (0.25-3.0mg/mL), 20. mu.M PMS, 156. mu.M NaDH and 52. mu.M NBT each 1.0mL were mixed well and the mixture was incubated at room temperature for 10 min. The absorbance value A of the mixture was then measured at 560nm1Ascorbic acid was used as a positive control. The superoxide anion radical scavenging capacity was calculated by the following formula:
clearance (%) ═ a0–A1)/A0×100
WhereinA0Is the absorbance value of the mixed solution without the sample.
4. Chelating ability for ferrous ions.
2.75mL of deionized water, 1mL of exopolysaccharide solution (0.25-3.0mg/mL), 0.2mL of felazine solution (5mM) and 0.05mL of FeCl2The solution (4mM) was mixed well, shaken vigorously and incubated at room temperature for 10 min. The absorbance value A of the mixture was then measured at 562nm1Ethylenediaminetetraacetic acid was used as a positive control. The chelating ability of ferrous ions was calculated by the following formula.
Chelating ability (%) ═ a0-(A1-Ai)/A0]×100
Wherein A is0Is the absorbance value of the sample-free mixed solution, AiIs free of FeCl2The absorbance value of the mixed solution of (1).
The in-vitro antioxidant activity analysis of the lactobacillus rhamnosus exopolysaccharide obtained by the method shows that the exopolysaccharide has the capability of removing DPPH free radicals, hydroxyl free radicals, superoxide anion free radicals and chelating ferrous ions, when the concentration of the exopolysaccharide is 3.0mg/mL, the clearance rate of the exopolysaccharide to DPPH free radicals is 66.83%, the clearance rate of the exopolysaccharide to hydroxyl free radicals is 83.93%, the chelating capacity of the ferrous ions is up to 98.62%, and when the concentration of the exopolysaccharide is 2.0mg/mL, the clearance rate of the superoxide anion is 50.70%.
Therefore, the lactobacillus rhamnosus exopolysaccharide can be used as a potential antioxidant, has wide application prospect in the fields of medicine, food and livestock husbandry, and can provide reference for the application of the lactobacillus rhamnosus exopolysaccharide in antioxidant food, medicine and feed additives.
Claims (10)
1. A preparation method of lactobacillus rhamnosus exopolysaccharide is characterized by comprising the following steps:
(1) inoculating lactobacillus rhamnosus into a skim milk liquid culture medium containing glucose, and standing and fermenting in a constant-temperature incubator;
(2) carrying out boiling water bath on the fermented culture medium to inactivate thallus and enzyme, denaturalizing protein, taking out, cooling to room temperature, centrifuging at low temperature and taking supernatant;
(3) evaporating and concentrating the supernatant to 1/3 of the original volume to obtain a first concentrated solution, then adding trichloroacetic acid with the mass concentration of 80% into the first concentrated solution, wherein the final mass concentration of the trichloroacetic acid is 4%, standing to fully precipitate the protein, and centrifuging at low temperature to obtain the supernatant;
(4) evaporating and concentrating the supernatant to 1/3 of the original volume to obtain a second concentrated solution, then adding pre-cooled 95% ethanol with mass concentration, wherein the volume ratio of the ethanol to the second concentrated solution is 3:1, standing to fully precipitate polysaccharide, then centrifuging at low temperature, removing the supernatant, and taking the precipitate;
(5) transferring the precipitate to a dialysis bag, and dialyzing with deionized water;
(6) and carrying out vacuum freeze drying on the dialyzed extracellular polysaccharide to finally obtain white floccule, namely the crude polysaccharide.
2. The preparation method of exopolysaccharide of lactobacillus rhamnosus according to claim 1, characterized in that: the inoculation amount of lactobacillus rhamnosus is 3%.
3. The preparation method of exopolysaccharide of lactobacillus rhamnosus according to claim 1 or 2, characterized in that: in the step (1), the skim milk liquid culture medium contains per liter: 100g of skimmed milk powder and 10g of glucose.
4. The preparation method of exopolysaccharide of lactobacillus rhamnosus according to claim 3, characterized in that: in the step (1), the standing fermentation is carried out at 37 ℃ for 36 hours.
5. The preparation method of exopolysaccharide of lactobacillus rhamnosus according to claim 3, characterized in that: in the steps (2) and (3), the low-temperature centrifugation conditions are as follows: centrifugation was carried out at 10000 Xg for 15 minutes at 4 ℃.
6. The preparation method of exopolysaccharide of lactobacillus rhamnosus according to claim 3, characterized in that: in the steps (3) and (4), the evaporation and concentration conditions are as follows: concentrating under reduced pressure at 60-65 ℃.
7. The preparation method of exopolysaccharide of lactobacillus rhamnosus according to claim 3, characterized in that: in steps (3) and (4), the mixture was allowed to stand at 4 ℃ for 12 hours.
8. The preparation method of exopolysaccharide of lactobacillus rhamnosus according to claim 3, characterized in that: in the step (4), the low-temperature centrifugation conditions are as follows: centrifugation was carried out at 10000 Xg for 20 minutes at 4 ℃.
9. The preparation method of exopolysaccharide of lactobacillus rhamnosus according to claim 3, characterized in that: in the step (5), the dialysis bag is boiled in boiling water for 10 minutes before use, and the specification is 8000-14000 Da.
10. The preparation method of exopolysaccharide of lactobacillus rhamnosus according to claim 3, characterized in that: in step (5), the precipitate was transferred to a dialysis bag and dialyzed against deionized water at 4 ℃ for 3 days, with water being changed every 12 hours.
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