CN116135886A - Extraction method of transparent low-acyl gellan gum - Google Patents
Extraction method of transparent low-acyl gellan gum Download PDFInfo
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- 229920002148 Gellan gum Polymers 0.000 title claims abstract description 167
- 239000000216 gellan gum Substances 0.000 title claims abstract description 167
- 235000010492 gellan gum Nutrition 0.000 title claims abstract description 167
- 238000000605 extraction Methods 0.000 title claims abstract description 22
- 238000000855 fermentation Methods 0.000 claims abstract description 72
- 230000004151 fermentation Effects 0.000 claims abstract description 72
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 41
- 239000007788 liquid Substances 0.000 claims abstract description 40
- 239000012065 filter cake Substances 0.000 claims abstract description 38
- 238000005189 flocculation Methods 0.000 claims abstract description 26
- 230000016615 flocculation Effects 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 26
- 239000000047 product Substances 0.000 claims abstract description 24
- 238000001914 filtration Methods 0.000 claims abstract description 22
- 239000003085 diluting agent Substances 0.000 claims abstract description 18
- 230000020176 deacylation Effects 0.000 claims abstract description 17
- 238000005947 deacylation reaction Methods 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000003513 alkali Substances 0.000 claims abstract description 11
- 239000012535 impurity Substances 0.000 claims abstract description 10
- 238000009835 boiling Methods 0.000 claims abstract description 9
- 238000005406 washing Methods 0.000 claims abstract description 9
- 229910052784 alkaline earth metal Inorganic materials 0.000 claims abstract description 8
- 150000001342 alkaline earth metals Chemical class 0.000 claims abstract description 8
- 238000007865 diluting Methods 0.000 claims abstract description 8
- 238000010790 dilution Methods 0.000 claims abstract description 7
- 239000012895 dilution Substances 0.000 claims abstract description 7
- 238000010438 heat treatment Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 28
- 125000002252 acyl group Chemical group 0.000 claims description 22
- 238000012258 culturing Methods 0.000 claims description 15
- 238000001035 drying Methods 0.000 claims description 15
- 241000736110 Sphingomonas paucimobilis Species 0.000 claims description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- 238000001816 cooling Methods 0.000 claims description 12
- 239000004744 fabric Substances 0.000 claims description 12
- 239000002244 precipitate Substances 0.000 claims description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- 230000000813 microbial effect Effects 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 239000002253 acid Substances 0.000 claims description 7
- 238000010298 pulverizing process Methods 0.000 claims description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 claims description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 5
- 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 5
- 239000001888 Peptone Substances 0.000 claims description 5
- 108010080698 Peptones Proteins 0.000 claims description 5
- 235000019319 peptone Nutrition 0.000 claims description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- 235000011054 acetic acid Nutrition 0.000 claims description 4
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 claims description 4
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 235000015165 citric acid Nutrition 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 claims description 2
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 claims description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 claims description 2
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 claims description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 claims description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 2
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 2
- 239000001630 malic acid Substances 0.000 claims description 2
- 235000011090 malic acid Nutrition 0.000 claims description 2
- 150000007522 mineralic acids Chemical class 0.000 claims description 2
- 150000007524 organic acids Chemical class 0.000 claims description 2
- 239000011975 tartaric acid Substances 0.000 claims description 2
- 235000002906 tartaric acid Nutrition 0.000 claims description 2
- 150000008044 alkali metal hydroxides Chemical class 0.000 claims 1
- 229910001860 alkaline earth metal hydroxide Inorganic materials 0.000 claims 1
- 239000012670 alkaline solution Substances 0.000 claims 1
- 230000001376 precipitating effect Effects 0.000 claims 1
- 238000002834 transmittance Methods 0.000 abstract description 7
- 230000008569 process Effects 0.000 abstract description 5
- 230000001105 regulatory effect Effects 0.000 abstract description 5
- 238000011282 treatment Methods 0.000 abstract description 4
- 239000003960 organic solvent Substances 0.000 abstract description 3
- 238000003756 stirring Methods 0.000 description 42
- 235000019441 ethanol Nutrition 0.000 description 23
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 23
- 239000002609 medium Substances 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 238000011218 seed culture Methods 0.000 description 8
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 5
- 239000002054 inoculum Substances 0.000 description 5
- 239000000049 pigment Substances 0.000 description 5
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000018044 dehydration Effects 0.000 description 3
- 238000006297 dehydration reaction Methods 0.000 description 3
- 150000004676 glycans Chemical class 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 125000003745 glyceroyl group Chemical group C(C(O)CO)(=O)* 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 238000010564 aerobic fermentation Methods 0.000 description 1
- PNNNRSAQSRJVSB-BXKVDMCESA-N aldehydo-L-rhamnose Chemical compound C[C@H](O)[C@H](O)[C@@H](O)[C@@H](O)C=O PNNNRSAQSRJVSB-BXKVDMCESA-N 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 150000001340 alkali metals Chemical class 0.000 description 1
- AEMOLEFTQBMNLQ-WAXACMCWSA-N alpha-D-glucuronic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-WAXACMCWSA-N 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- -1 etc. Chemical class 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical group [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
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- 235000005985 organic acids Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000003104 tissue culture media Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- 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
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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Abstract
The extraction method of the transparent low-acyl gellan gum comprises the following steps of pretreatment of fermentation liquor: diluting gellan gum fermentation liquor by 3-8 times according to volume ratio with water, and obtaining gellan gum dilution liquor at room temperature or after heating and boiling; deacylation, namely adding alkali liquor into gellan gum diluent at a high temperature of 60-80 ℃ and regulating pH to be strong alkaline to obtain low-acyl gellan gum liquid; adding alkaline earth metal flocculant and filter aid to flocculate and remove impurities; flocculation, extraction and filter cake collection; washing the filter cake with alcohol, dissolving to flocculation state, adding alkali liquor to adjust pH value to 7-8, filtering, and obtaining gellan gum filter cake again; and (5) carrying out post-treatment to obtain a powdery product. The invention has the advantages of simplified whole process, low cost, reduced organic solvent residue, good appearance, high transmittance and high gel strength.
Description
Technical Field
The invention belongs to the field of microbial fermentation processes, and particularly relates to an extraction method of low-acyl gellan gum.
Background
Gellan gum is prepared from four repeated sugar molecules in sequence of D-glucose, D-glucuronic acid, D-glucose and L-rhamnose according to the ratio of 2:1: the high molecular biological polysaccharide substance with the proportion of 1 connected by glycosidic bond can be obtained by aerobic fermentation of Sphingomonas paucimobilis (Sphingomonas paucimobilis). The gellan gum is easy to use, has good flavor release property and higher thermal stability, the time and temperature of the gel can be regulated and controlled, the gel is not easily affected by pH, and the gellan gum has various texture characteristics, thus being one of the biological gums with excellent performance and wide application at present.
Gellan gum gel characteristics are related to the acyl content of the polysaccharide molecule, and gellan gum products are generally classified into high acyl gellan gum and low acyl gellan gum based on the acyl content of the gellan gum polysaccharide molecule. The high acyl gellan gum is natural gellan gum, the total acyl content is 15% -18% (11% -13% of glyceroyl and 4% -5% of acetyl), and the gel is easy to form viscoelastic gel after hydration, has small gel strength and is easy to deform; the low acyl gellan gum has an overall acyl content of less than 2% (less than 1% glyceroyl and 1% acetyl), high gel strength, and easy brittle fracture.
The gellan gum prepared by fermentation belongs to high acyl gellan gum, and the low acyl gellan gum product is obtained by deacylation treatment in the downstream extraction stage. The downstream extraction stage is a key factor affecting the cost and quality of gellan gum, and the current common method for gellan gum purification is to add divalent metal ions into fermentation liquor or use a large amount of alcohol to perform flocculation precipitation of gellan gum, especially when deacylation treatment is performed to prepare low-acyl gellan gum, the pH of the solution is adjusted to be extremely alkaline, and the variation range is large. The traditional method for purifying gellan gum has a plurality of defects: firstly, the low-acyl transparent gellan gum is difficult to thoroughly remove protein in the gum, the light transmittance of the product is affected to a certain extent, and generally, the light transmittance can only reach more than 80%; secondly, a large amount of alcohol consumed in the pretreatment stage is washed, so that the production cost is increased, and the pretreatment stage is unsafe; the introduction of divalent metal ions makes the gellan gum product difficult to dissolve, and the formed gel is slightly whitish, which affects the quality reduction of the gellan gum.
In general, the content of gellan gum in fermentation liquor is very low, and crude gellan gum obtained by separating and extracting fermentation liquor by using organic solvents such as ethanol is only 1-2% (W/V) of fermentation liquor, and the crude gellan gum also contains a large amount of thallus fragments and proteins. Although the gellan gum content in the fermentation liquor is very low, the viscosity of the fermentation liquor is very high, and impurities in the fermentation liquor are difficult to thoroughly remove, so that the transmittance of the product is affected to a certain extent. Therefore, a series of treatments must be performed on the broth to obtain high quality gellan gum.
CN103409492a discloses a technical scheme for extracting transparent low acyl gellan gum from fermentation broth, which has the disadvantages that 2-3 times of volume of ethanol is required to be added in flocculation and reflocculation processes, the consumption of ethanol is extremely large, and the production cost is high. CN101591399B discloses a method for extracting low acyl gellan gum suitable for tissue culture medium, although the product has good appearance, high transmittance and high gel strength, the process is complicated, the ion exchange process is needed to remove impurities and decolorize, the period is long, a large amount of regenerated waste liquid is generated, and the environment is polluted. CN101724090a discloses a gellan gum production process, which is characterized in that ultrafiltration membrane is used to concentrate fermentation liquor, then pure water is used to ultrafiltrate and purify, and the fermentation liquor is sent into a drying device to be directly spray-dried to obtain a powder product, the spray-drying method requires great energy consumption, and the ultrafiltration membrane used in the extraction process is high in price and cannot be regenerated, so that the manufacturing cost is high. CN101191138A discloses a method for producing gellan gum finished products from gellan gum fermentation liquor, which is similar to a scheme of extracting gellan gum by salt flocculation precipitation, but the method has the disadvantages of high ash content and low gel strength of products due to the addition of a large amount of inorganic salt and alkali solution, especially divalent cations, in the extraction process, thus influencing the application and popularization of the products.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides an extraction method of transparent low-acyl gellan gum, which comprises the following steps:
(1) Pretreatment of fermentation broth
Diluting gellan gum fermentation liquor by 3-8 times according to volume ratio with water, and maintaining at any temperature between room temperature and heating boiling for a period of time to obtain gellan gum dilution liquor;
(2) Deacylation of
Adding alkali liquor into the fermentation liquor obtained in the step (1), regulating the pH to be strong alkaline, and deacylating under the high-temperature condition to obtain a low-acyl gellan gum solution;
(3) Flocculation impurity removal
Immediately adding alkaline earth metal flocculant solution after the step (2) is completed, adding filter aid into the reaction solution after floccule precipitation, and collecting clear gellan gum clear liquid;
(4) Flocculation extraction
Cooling the gellan gum clear liquid to room temperature, adding acid to adjust the pH to be strong acid, separating out flocculent precipitate of the gellan gum, and filtering to collect a filter cake;
(5) Alcohol washing
Adding anhydrous lower alcohol into the gellan gum filter cake, dissolving into flocculation state, adding alkali liquor to adjust pH value to 7-8, filtering, and obtaining gellan gum filter cake again;
(6) Drying and pulverizing
And drying and crushing the filter cake to obtain a powdery product.
According to an embodiment of the present invention, the method further comprises a step of microbial fermentation to obtain gellan gum fermentation broth before the step (1).
According to an embodiment of the invention, the microbial fermentation step comprises the steps of: and (3) culturing the activated Sphingomonas paucimobilis to obtain a seed solution, and inoculating the seed solution into a fermentation tank for culturing to obtain gellan gum fermentation liquor.
Wherein, the seed culture medium is: beef extract 3 g/L, peptone 5 g/L; the fermentation medium is as follows: glucose 20-30 g/L, yeast extract 1-3 g/L, peptone 2-5 g/L, KH 2 PO 4 1-5 g/L，K 2 HPO 4 1-3 g/L，MgSO 4 0.1-1 g/L; the conditions of the seed culture solution are as follows: 20-40 ℃, 150-400rpm,15-30h. The culture conditions of the fermentation tank are as follows: 20-40 ℃, pH6-8, DO15-40%,60-80h.
According to an embodiment of the present invention, in the step (1), after the gellan gum is diluted, the gellan gum is heated, boiled and stirred for 10-60min, so as to obtain a gellan gum diluted solution.
According to an embodiment of the invention, in step (2), the elevated temperature is 60-80 ℃, and when step (1) is at room temperature or below this elevated temperature, the diluent is first warmed to 60-80 ℃ and maintained, preferably 68-75 ℃, before adding the lye, and the reaction is maintained for 30-45min. When the step (1) is heated to boiling or higher than the high temperature, the diluent is cooled to 60-80 ℃ and maintained, preferably 68-75 ℃ for 30-45min before adding the alkali liquor. The alkali liquor is hydroxide solution of alkali metal and alkaline earth metal, preferably one or two of sodium hydroxide and potassium hydroxide. The strong alkalinity means that the pH value is in the range of 11-14.
According to an embodiment of the present invention, in the step (3), the alkaline earth metal flocculant is one, two or more of calcium chloride, magnesium chloride, calcium sulfate, magnesium sulfate and aluminum chloride, and the addition amount thereof is 0.1% -1.5% (W/V, g/mL) of the gellan gum diluent. The filter aid is 80-150 mesh diatomite, and the addition amount of the filter aid is 0.5% -1.5% (W/V, namely g/mL) of gellan gum diluent.
According to an embodiment of the present invention, in the step (4), the acid is selected from one, two or more of inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, carbonic acid, etc., and/or organic acids such as acetic acid, citric acid, malic acid, tartaric acid, acetic acid, etc. By strong acid is meant a pH in the range of less than 2.55, for example between 1.0 and 2.5.
According to an embodiment of the present invention, in the step (5), the lower alcohol is C 1 -C 6 Alcohols such as one or two or more of ethanol, isopropanol, n-propanol, n-butanol, at a concentration of not less than 95%; the dosage is 30-70 mL/g (namely the volume of lower alcohol (mL)/mass of gellan gum filter cake (g)). The alkali liquor is the same as the alkali liquor in the step (2).
According to an embodiment of the present invention, the filtration in the steps (4) and (5) uses 150-250 mesh filter cloth.
According to an embodiment of the invention, the filter cake is dried at 80-95 ℃ for 3-6 hours.
According to an embodiment of the invention, the room temperature means a temperature in the range of 15-30 ℃.
The invention also provides a transparent low-acyl gellan gum product prepared by the method.
Drawings
FIG. 1 is an external view of a sample of low acyl gellan gum prepared in example 1 of the present application;
FIG. 2 is a graph showing the effect of the low acyl gellan gum gel prepared in example 1 of the present application;
FIG. 3 is an external view of a sample of low acyl gellan gum prepared in example 2 of the present application;
FIG. 4 is a graph showing the effect of the low acyl gellan gum gel prepared in example 2 of the present application.
Detailed Description
The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.
Example 1: (1) microbial fermentation: inoculating activated Sphingomonas paucimobilis (Sphingomonas paucimobilis) ATCC 31461 into a seed culture medium, culturing at 30 ℃ and 250 rpm for 20-24 h to obtain seed liquid, inoculating the seed liquid into a fermentation tank according to 10% (volume ratio) inoculum size, culturing at 25 ℃ and pH value of 6.8 for DO 25% for 72 h to obtain gellan gum fermentation liquor.
The seed culture medium is as follows: beef extract 3 g/L, peptone 5 g/L; the fermentation medium is as follows: glucose 25 g/L, yeast extract 1 g/L, peptone 2 g/L, KH 2 PO 4 3 g/L，K 2 HPO 4 1 g/L，MgSO 4 1 g/L。
(2) Pretreatment of fermentation broth
Taking gellan gum fermentation liquor 1000 mL, slowly adding pure water while stirring, and diluting the volume of the fermentation liquor to 5 times of the original volume to obtain gellan gum dilution liquor.
(3) Deacylation of
Heating the gellan gum diluent obtained in the step (2) to 73 ℃ and maintaining, adding 4M sodium hydroxide solution while stirring, adjusting the pH value to 13.4, and maintaining the reaction for 30 min for deacylation to obtain the low-acyl gellan gum liquid.
(4) Flocculation impurity removal
Continuously maintaining the temperature, adding 60mL of 10% calcium chloride solution into the low-acyl gellan gum liquid, stirring while adding until obvious flocculent precipitate appears in the fermentation liquor, adding 0.8% (W/V, g/mL) of 100-mesh diatom with the volume of gellan gum diluent as a reference, and filtering to remove cell fragments, proteins and pigments to obtain gellan gum clear liquid.
(5) Flocculation extraction
Cooling the gellan gum clear liquid to room temperature, adding 6M hydrochloric acid while stirring, continuously slowly stirring for 10 minutes to adjust the pH of the gellan gum clear liquid to 1.5, separating out gellan gum flocculent precipitate, filtering with 200-mesh filter cloth, squeezing, dehydrating, and collecting filter cakes.
(6) Alcohol washing
Adding 70 mL/g (namely lower alcohol volume (mL)/mass (g)) of absolute ethyl alcohol into the obtained gellan gum filter cake, stirring until the absolute ethyl alcohol is dissolved into a flocculation state, adding 4M of sodium hydroxide solution, stirring uniformly for 1 h, adjusting the pH value to 7-8, filtering with 200-mesh filter cloth, squeezing for dehydration, and collecting the filter cake.
(7) Drying and pulverizing
Drying the gellan gum filter cake at 90 ℃ for 4 h, and crushing to obtain the low-acyl transparent gellan gum powdery product.
Example 2: (1) microbial fermentation: inoculating activated Sphingomonas paucimobilis (Sphingomonas paucimobilis) ATCC 31461 into a seed culture medium, culturing at 30 ℃ and 250 rpm for 20-24 h to obtain seed liquid, inoculating the seed liquid into a fermentation tank according to 10% (volume ratio) inoculum size, culturing at 25 ℃ and pH value of 6.8 for DO 25% for 72 h to obtain gellan gum fermentation liquor. The seed medium and fermentation medium were the same as in example 1.
(2) Pretreatment of fermentation broth
Taking gellan gum fermentation liquor 1000 mL, slowly adding pure water while stirring, diluting the volume of the fermentation liquor to 5 times of the original volume to obtain gellan gum dilution liquor, boiling and slowly stirring for 30 min.
(3) Deacylation of
Cooling the gellan gum diluent obtained in the step (2) to 72 ℃ and maintaining, adding 4M sodium hydroxide solution while stirring, adjusting the pH value to 13.2, and maintaining the reaction for 30 min for deacylation to obtain the low-acyl gellan gum liquid.
(4) Flocculation impurity removal
Continuously maintaining the temperature, adding 60mL of 10% calcium chloride solution into the low-acyl gellan gum liquid, stirring while adding until obvious flocculent precipitate appears in the fermentation liquor, adding 0.8% (W/V, g/mL) of 100-mesh diatom with the volume of gellan gum diluent as a reference, and filtering to remove cell fragments, proteins and pigments to obtain gellan gum clear liquid.
(5) Flocculation extraction
Cooling the gellan gum clear liquid to room temperature, adding 6M hydrochloric acid while stirring, continuously slowly stirring for 10 minutes to adjust the pH of the gellan gum clear liquid to 1.5, separating out gellan gum flocculent precipitate, filtering with 200-mesh filter cloth, squeezing, dehydrating, and collecting filter cakes.
(6) Alcohol washing
Adding 70 mL/g (namely lower alcohol volume (mL)/mass (g)) of absolute ethyl alcohol into the obtained gellan gum filter cake, stirring until the absolute ethyl alcohol is dissolved into a flocculation state, adding 4M of sodium hydroxide solution, stirring uniformly for 1 h, adjusting the pH value to 7-8, filtering with 200-mesh filter cloth, squeezing for dehydration, and collecting the filter cake.
(7) Drying and pulverizing
Drying the gellan gum filter cake at 90 ℃ for 4 h, and crushing to obtain the low-acyl transparent gellan gum powdery product.
Example 3: (1) microbial fermentation: inoculating activated Sphingomonas paucimobilis (Sphingomonas paucimobilis) ATCC 31461 into a seed culture medium, culturing at 30 ℃ and 250 rpm for 20-24 h to obtain seed liquid, inoculating the seed liquid into a fermentation tank according to 10% inoculum size, culturing at 25 ℃, pH value of 6.8 and DO of 25%, and culturing for 72 h to obtain gellan gum fermentation liquor. The seed medium and fermentation medium were the same as in example 1.
(2) Pretreatment of fermentation broth
Taking gellan gum fermentation liquor 2000 mL, slowly adding pure water while stirring, diluting the volume of the fermentation liquor to 5 times of the original volume to obtain gellan gum dilution liquor, boiling and slowly stirring for 30 min.
(3) Deacylation of
Cooling the gellan gum diluent obtained in the step (2) to 70 ℃ and maintaining, adding 4M sodium hydroxide solution while stirring, adjusting the pH value to 12.8, and maintaining the reaction for 35 min for deacylation to obtain the low-acyl gellan gum liquid.
(4) Flocculation impurity removal
Maintaining the temperature and adding 130 mL of 10% magnesium chloride solution into the low-acyl gellan gum liquid, stirring while adding until obvious flocculent precipitate appears in the fermentation liquor, adding 100-mesh diatom of 1% (W/V, g/mL) gellan gum diluent, and filtering to remove cell fragments, proteins and pigments to obtain gellan gum clear liquid.
(5) Flocculation extraction
Cooling the gellan gum clear liquid to room temperature, adding 30% citric acid solution with stirring, adjusting pH of the gellan gum clear liquid to 2.0, slowly stirring for 10 min, separating out flocculent precipitate of gellan gum, filtering with 200 mesh filter cloth, squeezing, dehydrating, and collecting filter cake.
(6) Alcohol washing
Adding 50 mL/g (namely lower alcohol volume (mL)/mass (g) of the gellan gum filter cake) isopropanol into the obtained gellan gum filter cake, stirring until the mixture is dissolved into a flocculation state, adding a sodium hydroxide solution with the concentration of 4M, adjusting the pH value to 7-8, uniformly stirring for 0.5 h, filtering with 200-mesh filter cloth, squeezing for dehydration, and collecting the filter cake.
(7) Drying and pulverizing
Drying the gellan gum filter cake at 90 ℃ for 4 h, and crushing to obtain the low-acyl transparent gellan gum powdery product.
Example 4: (1) microbial fermentation: inoculating activated Sphingomonas paucimobilis (Sphingomonas paucimobilis) ATCC 31461 into a seed culture medium, culturing at 30 ℃ and 250 rpm for 20-24 h to obtain seed liquid, inoculating the seed liquid into a fermentation tank according to 10% (volume ratio) inoculum size, culturing at 25 ℃ and pH value of 6.8 for DO 25% for 72 h to obtain gellan gum fermentation liquor. The seed medium and fermentation medium were the same as in example 1.
(2) Pretreatment of fermentation broth
Taking gellan gum fermentation liquor 1000 mL, slowly adding pure water while stirring, diluting the volume of the fermentation liquor to 5 times of the original volume to obtain gellan gum dilution liquor, boiling and slowly stirring for 30 min.
(3) Deacylation of
Cooling the gellan gum diluent obtained in the step (2) to 75 ℃ and maintaining, adding 4M potassium hydroxide solution while stirring, adjusting the pH value to 11.8, and maintaining the reaction for 30 min for deacylation to obtain low-acyl gellan gum liquid.
(4) Flocculation impurity removal
Continuously maintaining the temperature, adding 80mL of 10% calcium sulfate solution into the low-acyl gellan gum liquid, stirring while adding until obvious flocculent precipitate appears in the fermentation liquor, adding 1.2% (W/V, g/mL) of gellan gum diluent, and filtering with 100-mesh diatom to remove cell debris, protein and pigment to obtain gellan gum filtrate.
(5) Flocculation extraction
Cooling the gellan gum clear liquid to room temperature, adding 6M sulfuric acid while stirring, continuously and slowly stirring for 10 minutes to adjust the pH of the gellan gum clear liquid to 2.2, separating out flocculent precipitate of gellan gum, filtering with 200-mesh filter cloth, squeezing, dehydrating, and collecting filter cakes.
(6) Alcohol washing
Adding 60 mL/g (namely lower alcohol volume (mL)/mass (g) of the gellan gum filter cake) of n-butyl alcohol into the obtained gellan gum filter cake, stirring until the mixture is dissolved into a flocculation state, adding a potassium hydroxide solution with the concentration of 4M, regulating the pH value to 7-8, continuously stirring the mixture uniformly for 0.5 h, filtering with 200-mesh filter cloth, squeezing and dehydrating the mixture, and collecting the filter cake.
(7) Drying and pulverizing
Drying the gellan gum filter cake at 90 ℃ for 4 h, and crushing to obtain the low-acyl transparent gellan gum powdery product.
Example 5: (1) microbial fermentation: inoculating activated Sphingomonas paucimobilis (Sphingomonas paucimobilis) ATCC 31461 into a seed culture medium, culturing at 30 ℃ and 250 rpm for 20-24 h to obtain seed liquid, inoculating the seed liquid into a fermentation tank according to 10% (volume ratio) inoculum size, culturing at 25 ℃ and pH value of 6.8 for DO 25% for 72 h to obtain gellan gum fermentation liquor. The seed medium and fermentation medium were the same as in example 1.
(2) Pretreatment of fermentation broth
Taking gellan gum fermentation liquor 1000 mL, slowly adding pure water while stirring, diluting the volume of the fermentation liquor to 5 times of the original volume, boiling and slowly stirring for 30 min.
(3) Deacylation of
Cooling the gellan gum diluent obtained in the step (2) to 69 ℃ and maintaining, adding 4M potassium hydroxide solution while stirring, adjusting the pH value to 13.5, and maintaining the reaction for 30 min for deacylation to obtain low-acyl gellan gum liquid.
(4) Flocculation impurity removal
Continuously maintaining the temperature, adding 65 mL of 10% calcium chloride solution into the low-acyl gellan gum liquid, stirring while adding until obvious flocculent precipitate appears in the fermentation liquor, adding 1% (W/V, g/mL) of gellan gum diluent, and filtering with 100 mesh diatom to remove cell debris, protein and pigment to obtain gellan gum filtrate.
(5) Flocculation extraction
Cooling the gellan gum clear liquid to room temperature, adding 6M hydrochloric acid while stirring, continuously and slowly stirring for 10 minutes to adjust the pH of the gellan gum clear liquid to 2.5, separating out flocculent precipitate of gellan gum, filtering with 200-mesh filter cloth, squeezing, dehydrating, and collecting filter cakes.
(6) Alcohol washing
Adding 65 mL/g (namely lower alcohol volume (mL)/mass (g) of the gellan gum filter cake) absolute ethyl alcohol into the obtained gellan gum filter cake, stirring until the absolute ethyl alcohol is dissolved into a flocculation state, adding a potassium hydroxide solution with the concentration of 4M, regulating the pH value to 7-8, uniformly stirring for 0.5 h, filtering with 200-mesh filter cloth, squeezing and dehydrating, and collecting the filter cake.
(7) Drying and pulverizing
Drying the gellan gum filter cake at 90 ℃ for 4 h, and crushing to obtain the low-acyl transparent gellan gum powdery product.
Example 6: gel experiments were performed on the low acyl transparent gellan gum powder products obtained in examples 1-5, the gel procedure being as follows:
taking gellan gum 0.5 g, adding distilled water to 100 mL, stirring 2.2 h, adding 0.5 g sodium chloride, heating the solution to 80deg.C, continuously stirring and maintaining at 80deg.C for 1 min, supplementing the evaporated water to original volume, stopping heating and stirring, and cooling to room temperature to form gel.
FIGS. 1 and 2 are graphs showing the effects of the low acyl gellan gum prepared in example 1; fig. 3 and 4 are graphs showing the effect of the low acyl gellan gum prepared in example 2 on the gel.
Gel strength measurements were performed using a Brookfield AnalyserCT-3 texture analyzer. The light transmittance was measured using a spectrophotometer at 497 nm using distilled water control. Colloidal purity determination is referred to the method in the GB25535-2010 standard.
Table 1 qualitative test data for low acyl clear gellan gum
TABLE 2 Low acyl transparent gellan gum detection results
Compared with the existing extraction method of low-acyl gellan gum, the process provided by the invention has the advantages that:
(1) The pretreatment stage of gellan gum fermentation liquor is preferably high-temperature boiling sterilization for 30 min, which is helpful for removing thalli and proteins in the fermentation liquor on the premise of not introducing protease, so that the quality of gellan gum is greatly improved, and meanwhile, deacylation is carried out by utilizing the temperature, so that the whole process is simplified and the cost is reduced.
(2) In the method in the prior art, the gellan gum fiber is washed by using lower alcohol before deacylation, but the method omits the step, only involves a small amount of alcohol washing in the later period, greatly reduces the production cost, ensures that the residue of the organic solvent of the produced product is far lower than the standard, and expands the application range of the low-acyl transparent gellan gum product.
(3) The quality of the gellan gum prepared by the method is greatly improved, and the advanced level in China is reached. The product has good appearance, light transmittance of more than 90%, and gel strength of more than 1000 g/cm 2 Is superior to the current commercial products.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The extraction method of the transparent low-acyl gellan gum is characterized by comprising the following steps of:
(1) Pretreatment of fermentation broth
Diluting gellan gum fermentation liquor by 3-8 times according to volume ratio with water, and maintaining at any temperature between room temperature and heating boiling for a period of time to obtain gellan gum dilution liquor;
(2) Deacylation of
Adding alkali liquor into the gellan gum diluent obtained in the step (1) at a high temperature of 60-80 ℃, and adjusting the pH to be strong alkaline to obtain low-acyl gellan gum liquid;
(3) Flocculation impurity removal
Maintaining the temperature after the step (2) is completed, adding an alkaline earth metal flocculant solution, precipitating floccules, adding a filter aid into the reaction solution, and collecting clear gellan gum clear liquid;
(4) Flocculation extraction
Cooling the gellan gum clear liquid to room temperature, adding acid to adjust the pH to be strong acid, separating out flocculent precipitate of the gellan gum, and filtering to collect a filter cake;
(5) Alcohol washing
Adding anhydrous lower alcohol into the gellan gum filter cake, dissolving into flocculation state, adding alkali liquor to adjust pH value to 7-8, filtering, and obtaining gellan gum filter cake again;
(6) Drying and pulverizing
And drying and crushing the filter cake to obtain a powdery product.
2. The extraction method according to claim 1, further comprising the step of microbial fermentation to obtain gellan gum broth before step (1), comprising the steps of: and (3) culturing the activated Sphingomonas paucimobilis to obtain a seed solution, and inoculating the seed solution into a fermentation tank for culturing to obtain gellan gum fermentation liquor.
3. The extraction method according to claim 2, wherein the medium for the fermenter culture is: glucose 20-30 g/L, yeast extract 1-3 g/L, peptone 2-5 g/L, KH 2 PO 4 1-5 g/L，K 2 HPO 4 1-3 g/L，MgSO 4 0.1-1 g/L。
4. The method according to claim 1, wherein in the step (1), the gellan gum is diluted, and the gellan gum is heated, boiled and stirred for 10 to 60 minutes to obtain a gellan gum diluted solution.
5. The method according to claim 1, wherein in the step (2), the alkaline solution is an alkali metal hydroxide solution and alkaline earth metal hydroxide solution, and the strong basicity is a pH value in a range of 11-14.
6. The method according to claim 1, wherein in the step (3), the alkaline earth metal flocculant in the alkaline earth metal flocculant solution is one or two or more of calcium chloride, magnesium chloride, calcium sulfate, magnesium sulfate and aluminum chloride, the addition amount of the alkaline earth metal flocculant is 0.1% -1.5% g/mL of gellan gum diluent, the filter aid is 80-150 mesh diatomite, and the addition amount of the filter aid is 0.5% -1.5% g/mL of gellan gum diluent.
7. The method according to claim 1, wherein in the step (4), the acid is one, two or more selected from inorganic acid selected from hydrochloric acid, sulfuric acid, phosphoric acid, carbonic acid, and/or organic acid selected from acetic acid, citric acid, malic acid, tartaric acid, acetic acid, and the strong acidity means that the pH value range is less than or equal to 2.5.
8. The method according to claim 1, wherein in the step (5), the lower alcohol is C 1 -C 6 Alcohol, the concentration of which is not less than 95%; the dosage of the method is 30-70 mL/g of the gellan gum filter cake obtained in the step (4).
9. The method according to claim 1, wherein the filtration in the steps (4) and (5) is performed by using 150-250 mesh filter cloth, and the filter cake is dried at 80-95 ℃ for 3-6 hours in the step (6).
10. A transparent low acyl gellan gum prepared by the extraction method of any one of claims 1-9.
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| CN116655821A (en) * | 2023-06-13 | 2023-08-29 | 嘉保生物科技(保山)有限公司 | Post-extraction method of low-acyl transparent gellan gum |
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