CN110403197B - Application of algin oral liquid - Google Patents
Application of algin oral liquid Download PDFInfo
- Publication number
- CN110403197B CN110403197B CN201910701251.5A CN201910701251A CN110403197B CN 110403197 B CN110403197 B CN 110403197B CN 201910701251 A CN201910701251 A CN 201910701251A CN 110403197 B CN110403197 B CN 110403197B
- Authority
- CN
- China
- Prior art keywords
- algin
- oligosaccharide
- acid
- alginate
- oral liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 229940072056 alginate Drugs 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 25
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 claims abstract description 17
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Abstract
The invention provides an application of algin oral liquid, belonging to the technical field of health products, wherein algin in the algin oral liquid is degraded by enzyme, and the enzymolysis method comprises the following steps: adding 900-1100U/mL of alginate lyase enzyme solution into 24-26 times volume of 2-3% (g/mL) of alginate solution, adding 6-8mol/L of sodium p-toluenesulfonate solution, and performing enzymolysis at 29-31 deg.C for 18-20h. The effective component of the algin oral liquid provided by the invention is saturated algin oligosaccharide with the polymerization degree of 3-5, has better oxidation resistance, can up-regulate SIRT1 protein expression, and further improves the blood fat reducing capability of the algin oligosaccharide.
Description
Technical Field
The invention belongs to the technical field of health care products, and particularly relates to an application of a algin oral liquid.
Background
Algin (algin) is a filler material of brown algae cell wall, and is formed by connecting beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G) into a linear high polymer through 1 → 4 glycosidic bond, and the whole molecule forms a block structure by mannuronic acid fragment (PM), guluronic acid fragment (PG) and mannuronic acid-guluronic acid hybrid segment. The algin has wide application in the fields of chemical industry, food, medicine and the like. As a good medical material, the algin has various functions of stopping bleeding, resisting radiation, reducing blood fat, reducing blood sugar and the like; the molecular modification product has anticoagulant, antiviral, antibacterial, antiinflammatory, antitumor, and immunity enhancing effects. However, since the content, relative molecular mass (104-106), composition and M/G ratio (0.5-2) of alginate vary with different algal species, growth sites, growth periods, algal body parts and other factors, it is inconvenient to study the application of alginate, especially the relationship between its structure and function. In recent years, researches show that the algin can be degraded to obtain low molecular weight oligosaccharide with definite relative molecular mass and definite structure, namely the algin oligosaccharide is an oligomer formed by connecting 3-10 monosaccharide molecules through glycosidic bonds. Meanwhile, the alginate oligosaccharides have different functional activities due to different monomer molecular structure types and binding positions. In the field of medicine and health-care food, the brown algae oligosaccharide can be used as a curative effect food for patients with obesity, diabetes, colorectal cancer and habitual constipation. The brown alginate oligosaccharides are found to have obvious effect on mouse hyperglycemia in pharmacological experiments, and also have obvious curative effect on hereditary hyperglycemia of human. In addition, research shows that the algin oligosaccharide has higher activity in the aspects of freeness resistance and oxidation resistance.
The invention discloses a novel process for extracting algin oligosaccharide from kelp by an enzymatic hydrolysis method, aiming at the problems in the existing algin oligosaccharide extraction, and provides a production process for extracting algin oligosaccharide from kelp in the prior art, such as Chinese patent with the publication number of CN 102643882B. A novel process for extracting alginate oligosaccharides from kelp by an enzymolysis method comprises nine steps of preparing kelp powder, soaking, performing enzymolysis by cellulase, digesting, performing centrifugal filtration, bleaching, performing enzymolysis by alginate lyase, performing high-speed centrifugal fine filtration, and performing alcohol dehydration. Compared with the prior algin oligosaccharide extraction process with the yield of 14-15%, the invention has the advantages that the yield is 17-18%, the yield is improved by 3-4%, and simultaneously, the process steps are simplified, the use of chemical reagents is reduced, and the appearance of the product is more beautiful.
Disclosure of Invention
The invention aims to provide a algin oral liquid and a preparation method thereof, wherein the method comprises the step of performing enzymolysis on algin C 4 And C 5 The negative charge on the surface generates attraction with sodium ionsForming a polar covalent bond, and carrying out a beta-elimination reaction of a 4-O-glycosidic bond to obtain saturated alginate-derived oligosaccharide; the oral liquid can up-regulate SIRT1 protein expression, inhibit SREBP-1 expression, inhibit ACC and HMGCR activity, and improve blood lipid reducing ability.
The technical scheme adopted by the invention for realizing the purpose is as follows:
providing an algin oral liquid, wherein algin is degraded by enzyme to obtain saturated algin oligosaccharide, and the algin enzymolysis method comprises the following steps: adding 900-1100U/mL of alginate lyase enzyme solution into 24-26 times volume of 2-3% (g/mL) of alginate solution, adding 6-8mol/L of sodium p-toluenesulfonate solution, and performing enzymolysis at 29-31 deg.C for 18-20h. The enzyme degradation has the advantages of specificity, high efficiency, less side reaction and the like, only degrades specific glycosidic bond without damaging other structures of the polysaccharide, and the obtained molecular weight fragment is relatively ideal. However, the oligosaccharide prepared by the enzymolysis method has a non-reducing end forming C 4,5 The unsaturated double bond destroys the natural structure, and the obtained unsaturated oligosaccharide has weaker antioxidant activity than the saturated oligosaccharide. The catalytic mechanism of the alginate lyase comprises three steps of reactions: neutralizing the negative charge on the carboxyl anion; from C 5 Thereby obtaining protons; the carboxyl group donates electrons to C 4 And C 5 Resulting in a β -elimination reaction occurring at the 4-O-glycosidic bond. When sodium p-toluenesulfonate is added during degradation of algin lyase, after carboxyl provides electrons, C 4 And C 5 The negative charge on the structure is easy to be adsorbed with sodium ions to form a polar covalent bond, which causes a beta-elimination reaction at a 4-O-glycosidic bond, and simultaneously C 4 And C 5 No double bond is formed, and the obtained saturated alginate-derived oligosaccharide has strong antioxidant activity.
In some embodiments, the alginate lyase can simultaneously degrade 1,4- β -D-mannuronic acid fragment and 1,4- α -L-guluronic acid fragment. There are mainly 3 structural fragments in the alginate molecule: β -D- (1,4) -linked polymannuronic acid fragment (PM); α -L- (1,4) -linked polyguluronic acid fragment (PG), PMG, a fragment in which G and M are alternately fused. The alginate lyase provided by the invention can degrade 1,4-beta-D-mannuronic acid fragment and 1,4-alpha-L-guluronic acid fragment simultaneously, thereby obtaining polyguluronic acid oligosaccharide and polymannuronic acid oligosaccharide simultaneously.
In some embodiments, the alginate oligosaccharide has a degree of polymerization of 3 to 5. The algin oligosaccharide components or monomers with different molecular weights or polymerization degrees have different antioxidant activities. The scavenging activity of the algin oligosaccharide component on DPPH and superoxide radical and the complexing force on ferrous ion are all increased along with the reduction of polymerization degree, the average polymerization degree of the algin oligosaccharide provided by the invention is 3-5, and the algin oligosaccharide has better antioxidant activity.
In some embodiments, the above alginate oligosaccharides include oligoguluronic acid oligosaccharides and oligomannuronic acid oligosaccharides, and the alginate oligosaccharides are fractionated at a pH =2.85-3.0 range. The solubility of the polysaccharide with high mannuronic acid content is very high when the pH value is 2.85, the solubility of the polysaccharide with high guluronic acid content is basically 0 when the pH value is lower than 3.0, when the pH value is adjusted to 2.85, the solution is layered, the upper layer dissolved part is the polysaccharide with high mannuronic acid content, and the lower layer precipitate is the polysaccharide with high guluronic acid content.
In some embodiments, the above-described alginate oligosaccharides are chemically modified; wherein the oligoguluronic acid oligosaccharide is modified by sulfation. The sulfation increases the charge quantity carried by the oligo-guluronic acid oligosaccharide molecule, so that the activity of inhibiting the generation of triglyceride of liver cells is increased, and the hypolipidemic activity of the oligo-guluronic acid sulfate ester generated after sulfation is increased.
In some embodiments, the above oligomannuronic acid oligosaccharides are modified with guanidinoacetic acid. SREBP-1 is a sterol regulatory element, and comprises a corresponding regulatory element, and phosphorylation of AMPK can directly inhibit proteolysis and nuclear migration of SREBP-1, so that the transcriptional activity of the SREBP-1 is reduced; acetyl-CoA carboxylase (ACC) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA) are target proteins of AMPK, which inhibits its activity by phosphorylating ACC and HMGCR, and are also key enzymes in the synthesis of fatty acids and cholesterol. Sirtuin-1 (SIRT 1) is an important member of the nicotinamide adenine dinucleotide (NAD +) dependent histone deacetylase family iii in mammals, and is characterized in that oligomannuronic acid oligosaccharides are modified by guanidinoacetic acid, hydroxyl groups in the oligomannuronic acid oligosaccharides react with carboxyl groups of the guanidinoacetic acid, guanidino groups are grafted to the oligomannuronic acid oligosaccharides, the modified oligomannuronic acid oligosaccharides can up-regulate SIRT1 protein expression through post-translational modification, promote expression of downstream liver kinase (LKB 1), and LKB1 can phosphorylate AMPK and inhibit dephosphorylation of the AMPK by phosphorylase, so that the modified oligomannuronic acid oligosaccharides can be activated, inhibit expression of SREBP-1, inhibit activity of ACC and HMGCR, accelerate fatty acid oxidation, inhibit synthesis of endogenous lipid, and improve blood lipid reduction effects.
In some embodiments, the method for preparing the oral liquid comprises:
extracting laminarin by an ultrasonic method;
separating and purifying the algin;
dissolving;
coarse filtration;
and (3) flavor blending: adding 0.13-0.17% (w/w) of beta-cyclodextrin, 13-17% (w/w) of xylitol, 0.13-0.17% (w/w) of citric acid and a proper amount of essence into the filtered algin solution, and uniformly stirring;
sterilizing;
and (6) filling and sealing. The flavor modulation technology provided by the invention can adjust the color and the fragrance of the product from the aspects of taste, smell, vision and the like, so that the product is sour, sweet and palatable, has pure taste and has the effects of reducing blood fat and resisting aging.
In some embodiments, the method for extracting laminarin by ultrasonic method comprises: adding dried herba Zosterae Marinae powder into distilled water at a ratio of 1. The kelp has strong swelling property, the kelp is not fully swelled when the solid-liquid ratio is too high, the extraction rate is low, and the extraction rate is reduced due to excessive swelling when the solid-liquid ratio exceeds the solid-liquid ratio with proper swelling degree; the ultrasonic wave is extracted by the aid of mechanical shearing force, the ultrasonic time is too short, the mechanical shearing action is insufficient, the extraction rate is not high, and the ultrasonic time is too long, so that the chains of macromolecular polysaccharide are broken, and the extraction rate is reduced; the increase in temperature reduces the surface tension and viscosity of the liquid medium, accelerates the diffusion of the solution, and promotes the outward diffusion of intracellular polysaccharides, but at too high a temperature, local parts of the polysaccharide structure are destroyed.
In some embodiments, the separation and purification of the algin comprises the following steps:
filtering the solution after ultrasonic extraction, and taking filter residue;
adding sodium carbonate solution into the residue, heating in water bath at 47-49 deg.C for 4.2-4.5 hr, filtering, and collecting filtrate;
adding hydrochloric acid with mass fraction of 9.5-11.5% into the filtrate until pH value is 3.8-4.2 to obtain precipitate, and freeze drying to obtain algin; the invention converts water-insoluble alginate into water-soluble alkali metal salt under the action of heating and alkaline solution, and separates from water solution under the action of inorganic calcium salt to form water-insoluble algin precipitate, and the algin yield and purity are both higher.
The invention also provides application of the algin oral liquid in reducing blood fat, resisting aging and inhibiting bacteria. The algin oral liquid provided by the invention contains the effective component of saturated algin oligosaccharide with the polymerization degree of 3-5, and has good oxidation resistance, wherein the oligomannuronic acid oligosaccharide can up-regulate SIRT1 protein expression, and further improves the blood fat reducing capability of the algin oligosaccharide.
The beneficial effects of the invention are as follows:
1) The invention adds sodium p-toluenesulfonate during the algin enzymolysis, and after the carboxyl provides electrons, C 4 And C 5 The negative charge on the structure is easy to be adsorbed with sodium ions to form a polar covalent bond, which causes a beta-elimination reaction at a 4-O-glycosidic bond, and simultaneously C 4 And C 5 Double bonds are not formed, so that saturated alginate oligosaccharides are obtained, and the antioxidant activity is strong;
2) The invention optimizes conditions of polysaccharide extraction, algin separation and purification, oral liquid preparation and the like, improves polysaccharide yield, algin yield and purity, and the obtained algin oral liquid has the functions of reducing blood fat and resisting aging and has better color, fragrance and taste;
3) The modified oligomannuronic acid oligosaccharide can up-regulate SIRT1 protein expression, promote expression of downstream liver kinase (LKB 1), phosphorylate AMPK and inhibit dephosphorylation effect of phosphorylase on AMPK, so that the modified oligomannuronic acid oligosaccharide is activated, expression of SREBP-1 is inhibited, activity of ACC and HMGCR is inhibited, fatty acid oxidation is accelerated, synthesis of endogenous lipid is inhibited, and the effect of reducing blood fat is improved.
Drawings
FIG. 1 is a schematic infrared spectrum of example 2;
FIG. 2 is a schematic infrared spectrum of comparative example 1;
FIG. 3 is a schematic representation of an immunoblot of p-AMPK, p-ACC, p-HMGCR, LKB1, SIRT1 of the present invention;
FIG. 4 is a schematic immunoblot of an inactive 125kDa fragment and an active 68kDa fragment of SREBP-1 of the present invention;
FIG. 5 shows the relative protein expression levels of p-AMPK, p-ACC, p-HMGCR, LKB1 and SIRT1 according to the present invention;
FIG. 6 shows the relative protein expression levels of the inactive 125kDa fragment and the active 68kDa fragment of SREBP-1 of the present invention;
FIG. 7 is a schematic view of test example 3 of the present invention;
FIG. 8 is a schematic view of test example 4 of the present invention.
Detailed Description
Unless otherwise indicated, all publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety as if set forth in their entirety.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.
When an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any larger range limit or preferred value and any smaller range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is described, the described range should be construed as including ranges of "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. Where numerical ranges are described herein, unless otherwise stated, the stated ranges are intended to include the endpoints of the ranges and all integers and fractions within the ranges.
In addition, the words "a" and "an" preceding an element or component of the invention are intended to mean no limitation on the number of times that the element or component appears (i.e., occurs). Thus, "a" or "an" should be understood to include one or at least one and the singular forms of the element or component also include the plural unless it is clear that the singular forms a number.
Embodiments of the invention, including embodiments of the invention described in the summary section and any other embodiments described herein below, can be combined arbitrarily.
The present invention is described in detail below.
An algin oral liquid, algin is degraded by enzyme to obtain saturated algin oligose, the algin enzymolysis method is: 900-1100U/mL (preferably, e.g., 900U/mL, 920U/mL, 940U/mL, 980U/mL, 1000U/mL, 1030U/mL, 1070U/mL, 1100U/mL, etc.) of the alginate lyase enzyme solution is added to a 24-26 (preferably, e.g., 24-fold, 24.2-fold, 24.6-fold, 25.7-fold, 25.9-fold, 26-fold, etc.) volume of 2-3% (preferably, e.g., 2%, 2.1%, 2.5%, 2.7%, 3%, etc.) (g/mL) alginate solution, followed by 6-8mol/L (preferably, for example, 6mol/L, 6.5mol/L, 6.8mol/L, 6.9mol/L, 7mol/L, 7.6mol/L, 7.8mol/L, 8mol/L, etc.) sodium p-toluenesulfonate solution, carrying out enzymolysis at 29-31 ℃ (preferably, for example, 29 ℃, 29.5 ℃,30 ℃, 30.6 ℃, 31 ℃ and the like) for 18-20h (preferably, for example, 18h, 18.3h, 18.5h, 19.7h, 20h and the like), after the enzymolysis reaction is finished, centrifuging at 8000rpm for 10min, taking supernatant, concentrating under reduced pressure at 50 ℃, and freeze-drying. The enzyme degradation has the advantages of specificity, high efficiency, less side reaction and the like, and only degrades specific glycosidic bond without damaging other structures of the polysaccharide, so that the obtained molecular weight fragment is more ideal. But the oligosaccharides produced by the enzymatic hydrolysis method, the non-reducing endForm C 4,5 The unsaturated double bond destroys the natural structure, and the obtained unsaturated oligosaccharide has weaker antioxidant activity than the saturated oligosaccharide. The catalytic mechanism of the algin lyase comprises three steps of reactions: neutralizing the negative charge on the carboxyl anion; from C 5 Thereby obtaining protons; the carboxyl group donates electrons to C 4 And C 5 Resulting in a β -elimination reaction that occurs at the 4-O-glycosidic bond. When sodium p-toluenesulfonate is added during degradation of algin lyase, after carboxyl provides electrons, C 4 And C 5 The negative charge on the structure is easy to be adsorbed with sodium ions to form a polar covalent bond, which causes a beta-elimination reaction at a 4-O-glycosidic bond, and simultaneously C 4 And C 5 No double bond is formed, and the obtained saturated alginate-derived oligosaccharide has strong antioxidant activity.
In some embodiments, the alginate lyase can simultaneously degrade 1,4- β -D-mannuronic acid fragment and 1,4- α -L-guluronic acid fragment. There are mainly 3 structural fragments in the alginate molecule: β -D- (1,4) -linked polymannuronic acid fragment (PM); α -L- (1,4) -linked polyguluronic acid fragment (PG), PMG, a fragment in which G and M are alternately fused. The alginate lyase provided by the invention can degrade 1,4-beta-D-mannuronic acid fragment and 1,4-alpha-L-guluronic acid fragment simultaneously, thereby obtaining polyguluronic acid oligosaccharide and polymannuronic acid oligosaccharide simultaneously.
In some embodiments, the above alginate oligosaccharides have a degree of polymerization of 3 to 5, preferably, e.g., 3, 4, 5, 3 to 4, 4 to 5, 3 to 5. The algin oligosaccharide components or monomers with different molecular weights or polymerization degrees have different antioxidant activities. The algin oligosaccharide component has the removal activity on DPPH and superoxide radical and the complexing force on ferrous ions which are increased along with the reduction of the polymerization degree, and the algin oligosaccharide provided by the invention has the average polymerization degree of 3-5 and has better antioxidant activity.
In some embodiments, the above alginate oligosaccharides include oligoguluronic acid oligosaccharides and oligomannuronic acid oligosaccharides, which are subjected to pH =2.85-3.0 (preferably, e.g., pH =2.85, pH =2.9, pH =2.92, pH =2.97, pH =3.0, etc.) in the range of pH =2.85-3.0Grading, wherein the specific grading method comprises the following steps: alginate oligosaccharides were dissolved in 8% NaHCO 3 Adding 0.3mol/L HCl into the solution to adjust the pH value to 2.85-3.0, and centrifuging at 4000rpm for 10min to obtain supernatant a and sediment b;
taking supernatant a, adjusting pH to 7-8, adding 3 times volume of 95% ethanol, centrifuging at 4000rpm for 10min, dehydrating the precipitate with excessive anhydrous ethanol, drying, adding 8% NaHCO 3 Adjusting pH of the solution to 2.85-3.0, centrifuging at 4000rpm for 10min, collecting supernatant, and repeating the above operation for 2 times to obtain high purity oligomannuronic acid oligosaccharide.
And (3) carrying out suction filtration on the precipitate b, adding water to dissolve the precipitate b to 6%, adjusting the pH value to 7.0-8.0 by using 10% NaOH to dissolve the precipitate, filtering to remove impurities, taking filtrate, adjusting the pH value to 2.85-3.0 by using 0.3mol/L HCl, carrying out suction filtration to obtain a precipitate, and repeating the operation for 2 times to obtain the high-purity oligoguluronic acid oligosaccharide. The solubility of the polysaccharide with high mannuronic acid content is very high when the pH value is 2.85, the solubility of the polysaccharide with high guluronic acid content is basically 0 when the pH value is lower than 3.0, when the pH value is adjusted to 2.85, the solution is layered, the upper layer dissolved part is the polysaccharide with high mannuronic acid content, and the lower layer precipitate is the polysaccharide with high guluronic acid content.
In some embodiments, the above alginate oligosaccharides are chemically modified; wherein the oligoguluronic acid oligosaccharide is modified by sulfation. The specific modification method comprises the following steps: the oligoguluronic acid oligosaccharides were suspended in molecular sieve-dehydrated Dimethylformamide (DMF) in the ratio of 1 3 Pyridine, heating and stirring, reacting at 60 ℃ for 4h, centrifuging, collecting precipitate, washing with 95% ethanol for 3 times, adding water for redissolution, adjusting pH to 7.0-8.0 with 10% NaOH, dialyzing, purifying, and freeze drying. The sulfation increases the charge quantity carried by the oligo-guluronic acid oligosaccharide molecule, so that the activity of inhibiting the generation of triglyceride of liver cells is increased, and the hypolipidemic activity of the oligo-guluronic acid sulfate ester generated after sulfation is increased.
In some embodiments, the oligomannuronic acid oligosaccharide described above is modified with guanidinoacetic acid. More preferably, the specific modification method is: the oligoguluronic acid oligosaccharide is suspended in Dimethylformamide (DMF) after dehydration treatment with molecular sieve according to the ratio of 1. SREBP-1 is a sterol regulatory element and comprises a corresponding regulatory element, and phosphorylation of AMPK can directly inhibit proteolysis and nuclear migration of SREBP-1, so that the transcriptional activity of the SREBP-1 is reduced; acetyl-CoA carboxylase (ACC) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA) are target proteins of AMPK, which inhibits its activity by phosphorylating ACC and HMGCR, and are also key enzymes in the synthesis of fatty acids and cholesterol. Sirtuin-1 (SIRT 1) is an important member of the nicotinamide adenine dinucleotide (NAD +) dependent histone deacetylase family iii in mammals, and is characterized in that oligomannuronic acid oligosaccharides are modified by guanidinoacetic acid, hydroxyl groups in the oligomannuronic acid oligosaccharides react with carboxyl groups of the guanidinoacetic acid, guanidino groups are grafted to the oligomannuronic acid oligosaccharides, the modified oligomannuronic acid oligosaccharides can up-regulate SIRT1 protein expression through post-translational modification, promote expression of downstream liver kinase (LKB 1), and LKB1 can phosphorylate AMPK and inhibit dephosphorylation of the AMPK by phosphorylase, so that the modified oligomannuronic acid oligosaccharides can be activated, inhibit expression of SREBP-1, inhibit activity of ACC and HMGCR, accelerate fatty acid oxidation, inhibit synthesis of endogenous lipid, and improve blood lipid reduction effects.
In some embodiments, the method for preparing the oral liquid comprises:
extracting laminarin by an ultrasonic method;
separating and purifying the algin;
dissolving;
coarse filtration;
and (3) flavor blending: adding 0.13-0.17% (preferably, for example, 0.13%, 0.14%, 0.15%, 0.16%, 0.168%, 0.17%, etc.) (w/w) of beta-cyclodextrin, 13-17% (preferably, for example, 13%, 13.5%, 14%, 15%, 15.5%, 16%, 16.8%, 17%, etc.) (w/w) of xylitol, 0.13-0.17% (preferably, for example, 0.13%, 0.14%, 0.15%, 0.16%, 0.168%, 0.17%, etc.) (w/w) of citric acid and appropriate amount of essence into the filtered algin solution, and stirring uniformly;
and (3) sterilization: sterilizing with high pressure steam at 121 deg.C for 30min;
filling and sealing: the filling adopts a 20mL brown oral liquid glass bottle, and the air bottle and the bottle cap need to be cleaned and disinfected before the filling. The flavor modulation technology provided by the invention adjusts the color and the fragrance of the product from the aspects of taste, smell, vision and the like, so that the product is sweet and sour, has pure taste and has the effects of reducing blood fat and resisting aging.
In some embodiments, the method for extracting laminarin by the ultrasonic method comprises: dry kelp powder is added to distilled water in a ratio of 1 to 70 (preferably, e.g., 1. The kelp has strong swelling property, the kelp is not fully swelled when the solid-liquid ratio is too high, the extraction rate is low, and the extraction rate is reduced due to excessive swelling when the solid-liquid ratio exceeds the solid-liquid ratio with proper swelling degree; the ultrasonic wave is extracted by the assistance of mechanical shearing force, the ultrasonic time is too short, the mechanical shearing action is insufficient, the extraction rate is not high, the ultrasonic time is too long, the macromolecular polysaccharide is broken, and the extraction rate is reduced; the increase in temperature reduces the surface tension and viscosity of the liquid medium, accelerates the diffusion of the solution, and promotes the outward diffusion of intracellular polysaccharides, but at too high a temperature, local parts of the polysaccharide structure are destroyed.
In some embodiments, the separation and purification of the algin comprises the following steps:
filtering the solution after ultrasonic extraction, and taking filter residue;
adding sodium carbonate solution into the residue, heating in water bath at 47-49 deg.C (preferably 47 deg.C, 47.3 deg.C, 48 deg.C, 48.2 deg.C, 48.9 deg.C, 49 deg.C, etc.) for 4.2-4.5 hr (preferably 4.2 hr, 4.3 hr, 4.4 hr, 4.5 hr, etc.), filtering, and collecting filtrate;
adding 9.5-11.5% (preferably, such as 9.5%, 9.8%, 10.2%, 10.8%, 11%, 11.3%, etc.) hydrochloric acid to the filtrate to pH of 3.8-4.2 (preferably, such as 3.8, 3.9, 4..0, 4.1, 4.2, etc.) to obtain precipitate, and freeze drying to obtain algin. The invention converts water-insoluble alginate into water-soluble alkali metal salt under the action of heating and alkaline solution, and separates from water solution under the action of inorganic calcium salt to form water-insoluble algin precipitate, and the algin yield and purity are both higher
The invention also provides an application of any one of the algin oral liquid in reducing blood fat, resisting aging and inhibiting bacteria. The active ingredient in the algin oral liquid provided by the invention is saturated algin oligosaccharide with the polymerization degree of 3-5, and the algin oral liquid has good oxidation resistance, wherein the oligomannuronic acid oligosaccharide can up-regulate SIRT1 protein expression, and further improves the blood fat reducing capability of the algin oligosaccharide.
The present invention is further described in detail with reference to the following examples:
example 1:
a preparation method of algin comprises the following steps:
extracting laminarin by an ultrasonic method: 5g of dry kelp powder was added to 362.5mL of distilled water and sonicated at 65 ℃ for 60min.
And (3) separation and purification of algin: filtering the solution after ultrasonic extraction, and taking filter residue; adding 2.2g/mL sodium carbonate solution 20mL into the filter residue, heating at 48 ℃ for 4.3h in a water bath kettle, and filtering to obtain filtrate; and adding hydrochloric acid with the mass fraction of 10% into the filtrate until the pH value is 4 to obtain a precipitate, and freeze-drying to obtain the algin.
Example 2:
a preparation method of algin comprises the following steps:
degrading the algin by adopting an enzyme method: adding 5mL of 1000U/mL of alginate lyase enzyme solution into 125mL of 2% (g/mL) alginate solution, adding 12mL of 6mol/L sodium p-toluenesulfonate solution, carrying out enzymolysis for 20h at 30 ℃, adding 50% of absolute ethyl alcohol to stop the enzymolysis reaction, centrifuging at 8000rpm for 10min, taking supernatant, concentrating at 50 ℃ under reduced pressure, and carrying out freeze drying to obtain the alginate oligosaccharide.
Example 3:
a preparation method of algin comprises the following steps:
the alginate oligosaccharide obtained in example 2 was dissolved in 8% NaHCO 3 Adding 0.3mol/L HCl into the solution to adjust the pH value to 2.85, and centrifuging for 10min at 4000rpm to obtain supernatant a and sediment b;
taking supernatant a, adjusting pH to 7-8, adding 3 times volume of 95% ethanol, centrifuging at 4000rpm for 10min, dehydrating the precipitate with excessive anhydrous ethanol, drying, adding 8% NaHCO 3 Adjusting pH of the solution to 2.85, centrifuging at 4000rpm for 10min, collecting supernatant, and repeating the above operation for 2 times to obtain high purity oligomannuronic acid oligosaccharide.
And (3) carrying out suction filtration on the precipitate b, adding water to dissolve the precipitate b to 6%, adjusting the pH value to 7.0 by using 10% NaOH to dissolve the precipitate, filtering to remove impurities, taking filtrate, adjusting the pH value to 2.85 by using 0.3mol/L HCl, carrying out suction filtration to obtain a precipitate, and repeating the operation for 2 times to obtain the high-purity oligosaccharide guluronic acid oligosaccharide.
Modifying oligomannuronic acid oligosaccharide with guanidinoacetic acid: the oligoguluronic acid oligosaccharides were suspended in Dimethylformamide (DMF) dehydrated by molecular sieve at a ratio of 1.
Carrying out sulfation modification on oligoguluronic acid oligosaccharide: the oligoguluronic acid oligosaccharides were suspended in molecular sieve-dehydrated Dimethylformamide (DMF) in a ratio of 1 3 Pyridine, heating and stirring, reacting at 60 ℃ for 4h, centrifuging, taking precipitate, washing with 95% ethanol for 3 times, adding water for redissolving, adjusting pH to 7.0 with 10% NaOH, dialyzing, purifying, and freeze-drying.
Mixing the modified oligomannuronic acid oligosaccharide and the modified oligoguluronic acid oligosaccharide to obtain the modified algin oligosaccharide.
Example 4:
a preparation method of algin oral liquid comprises:
dissolving: dissolving algin in water to obtain 10mg/mL algin solution;
coarse filtration: removing insoluble impurities;
and (3) flavor blending: adding 0.15% (w/w) of beta-cyclodextrin, 15% (w/w) of xylitol, 0.15% (w/w) of citric acid and a proper amount of essence into the filtered algin oligosaccharide solution, and uniformly stirring;
and (3) sterilization: sterilizing with high pressure steam at 121 deg.C for 30min;
filling and sealing: the filling adopts a 20mL brown oral liquid glass bottle, and the air bottle and the bottle cap need to be cleaned and disinfected before the filling.
The prepared oral liquid kelp is light in fishy smell and gelatin, dark brown or tan in color, moderate in sour and sweet taste, fine and smooth in taste, free of peculiar smell, clear and transparent in appearance, free of visible impurities, allowed to have a small amount of turbidity or precipitation, and evaluated for 98 minutes in sensory evaluation.
Comparative example 1:
sodium p-toluenesulfonate was not added, and the remainder was identical to that of example 2.
Comparative example 2:
the oligomannuronic acid oligosaccharides were not modified and the rest was identical to example 3.
Test example 1:
infrared spectrum analysis:
by P 2 O 5 And (3) drying the sample in a vacuum drying oven for 48 hours, and performing infrared spectrum determination on the sample by adopting a potassium bromide tabletting method. The infrared spectra are shown in fig. 1 and fig. 2.
As can be seen from FIGS. 1 and 2, the absorption peaks of example 2 and comparative example 1 appeared at substantially the same positions, and the structures of the functional groups were the same, wherein 3443cm -1 Is hydroxyl absorption peak, 2921.52cm -1 ，2918.13cm -1 Is the C-H vibration peak of saccharide, 1628.71cm -1 ，1628.43cm -1 Characteristic absorption peak of asymmetric stretching vibration of carboxyl group C = O, 1406.95cm -1 、1405.82cm -1 、1302.33cm -1 、1301.91cm -1 One group of peaks is carboxylate radical absorption peak, 1089.12cm -1 ，1088.94cm -1 Is represented by two sugar rings C 1 Characteristic absorption Peak of Acetal in position, 1039.37cm -1 ，1038.93cm -1 Is a C-O telescopic vibration characteristic absorption peak, 948.70cm -1 ，947.68cm -1 Is an asymmetric stretching vibration characteristic absorption peak of pyranose ring, 889.11cm -1 ，888.20cm -1 Is C 2 And C 4 C-H variable angle vibration in the level of the flat bond configuration, 818.53cm -1 ，817.35cm -1 Is a characteristic absorption peak of mannuronic acid, and a characteristic absorption peak of non-reducing terminal carboxylic acid and double bond conjugation is also included in the middle of the carboxylate absorption peak in comparative example 1, and is 869.74cm -1 An asymmetric vibration characteristic absorption peak of the double bond at the non-reducing end appears. While no absorption peak of non-reducing terminal double bond and double bond conjugation appears in example 2, which indicates that the alginate oligosaccharide after enzymolysis in example 2 is not in C 4 And C 5 Double bonds are formed between the two groups, and the obtained algin oligosaccharide is saturated algin oligosaccharide.
Test example 2:
the blood fat reducing capability of the oligomannuronic acid oligosaccharide is analyzed by an immunoblotting method:
HepG2 cell culture: thawing and recovering the frozen HepG2 cells, inoculating the thawed and recovered HepG2 cells into a cell culture bottle of a DMEM HG culture medium containing 10% fresh fetal calf serum, putting the cell culture bottle into a CO2 incubator at 37 ℃ and 5%, changing the solution every 3d, digesting and carrying out passage when the cells grow over 85% of the culture bottle, wherein the passage ratio is 1:2. And selecting a cell algebra with better activity to perform an experiment.
Immunoblotting to detect intracellular phosphorylated AMPK (p-AMPK), phosphorylated acetyl-coa carboxylase (p-ACC), phosphorylated 3-hydroxy-3-methylglutaryl-coa reductase (p-HMGCR), sterol regulatory element 1 (SREBP-1), liver kinase (LKB 1) and sirtuin regulator 1 (SIRT 1): inoculating HepG2 cells into a 6-well plate for culture, dividing the cell into a control group and a sample group according to requirements, adding 100 mu g/mL oligomannuronic acid oligosaccharide into the sample group for intervention, extracting the total cell protein by using a protein lysate on ice after the intervention is finished, detecting the protein concentration by using a BCA protein quantitative kit, and finally adding 1/4 volume of Loading buffer for boiling denaturation treatment. And then sequentially carrying out electrophoresis, membrane conversion and sealing treatment, adding p-AMPK, p-ACC, p-HMGCR, SREBP-1, LKB1 and SIRT1 primary antibody, incubating overnight in a refrigerator at 4 ℃, adding corresponding secondary antibody, continuing incubating for 2h, finally incubating the PVDF membrane and ECL luminous liquid for 1min, exposing, developing and washing the film in a dark room, carrying out optical density scanning on the immunoblotting target strip by using a scanner, and analyzing the result by adopting imagePro plus software. The immunoblotting maps of p-AMPK, p-ACC, p-HMGCR, SREBP-1, LKB1 and SIRT1 are shown in figure 3, figure 4, and the relative protein expression is shown in figure 5 and figure 6.
As can be seen from fig. 3, fig. 4, fig. 5 and fig. 6, compared with comparative example 2, the protein expression levels of p-AMPK, p-ACC, p-HMGCR, SREBP-1, LKB1 and SIRT1 in example 2 are up-regulated, which indicates that the modified oligomannuronic acid oligosaccharide can increase the SIRT1 protein expression level, promote the expression of LKB1 downstream thereof, increase the phosphorylation degree of AMPK, activate AMPK, and inhibit the expression levels of inactive 125kDa precursor and active 68kDa fragment of SREBP-1, and the activated AMPK inhibits ACC and HMGCR activities through phosphorylation, thereby reducing the synthesis of cholesterol and fatty acid.
Test example 3:
determination of antioxidant Activity:
60 male Kunming mice of 18-22g are randomly divided into a control group, a model group and an administration group, and each group comprises 20 mice. Adaptive feeding for 3-5 days, wherein the mice freely feed common feed and drink water during the experiment period, the administration group is administered by gastric lavage (alginate-derived oligosaccharide 100 mg/kg), after 2h, the administration group is administered with alcohol 4.8g/kg once, after 16h, the eyes are bled, centrifuged at 3000rpm for 15min, and serum is prepared by separation. The levels of GSH, T-AOC, SOD and MDA in serum were determined according to the kit instructions of Glutathione (GSH), total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and Malondialdehyde (MDA), and the results are shown in FIG. 7.
As can be seen from FIG. 7, both example 2 and comparative example 1 can effectively increase serum T-AOC, GSH level and SOD level in serum, and reduce MDA level in serum, which indicates that both example 2 and comparative example 1 can effectively increase in vivo antioxidant system; the serum T-AOC in example 2 has obviously higher GSH level and SOD level in serum than those in comparative example 1, and has obviously lower MDA level than that in comparative example 1, which shows that the antioxidant capacity of example 2 is higher.
Test example 4:
and (3) measuring the blood fat reducing capacity:
establishing a model: 60 male Kunming mice of 18-22g are randomly divided into a control group, a model group and an administration group, and each group comprises 20 mice. The control group was fed with normal pellet diet, and the other groups were fed with high-sugar, high-fat diet for 2 months. At 7 weeks of high-sugar and high-fat diet, rats were injected with streptozotocin 15mg/kg in tail vein, and the control group was injected with citric acid buffer only.
Gavage was administered on week 10. The administration group was administered with 100mg/kg of alginate-derived oligosaccharides once a day for 4 weeks. At weeks 10 and 14, blood was collected from the eyeball, centrifuged at 2000rpm for 30min, serum was collected, and the levels of Triglyceride (TG), total Cholesterol (TC), high density lipoprotein (HDL-C) and low density lipoprotein (LDL-C) were measured by a full-automatic biochemical analyzer, and the results are shown in FIG. 8.
As can be seen from FIG. 8, the TG, TC and LDL-C levels of rats with diabetes and hyperlipidemia can be obviously reduced and the HDL-C level can be increased in both the example 3 and the comparative example 2, which shows that the TG, TC and LDL-C levels can be obviously reduced and the HDL-C level can be increased more obviously in the example 2 than in the comparative example 2, and this shows that the blood fat reducing capability of the algin oligosaccharide can be further improved after the oligomannuronic acid oligosaccharide is modified.
Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.
The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.
Claims (5)
1. An algin oral liquid, wherein algin is degraded by enzyme to obtain saturated algin oligose, which is characterized in that: the enzymolysis method of the algin comprises the following steps: adding 900-1100U/mL of alginate lyase enzyme solution into 24-26 times of volume of 2-3% (g/mL) of alginate solution, adding 6-8mol/L of sodium p-toluenesulfonate solution, and performing enzymolysis at 29-31 deg.C for 18-20h;
the alginate oligosaccharides comprise oligoguluronic acid oligosaccharides and oligomannuronic acid oligosaccharides, and the alginate oligosaccharides are classified in the range of pH =2.85-3.0, and the oligomannuronic acid oligosaccharides are modified by guanidinoacetic acid.
2. The alginate oral liquid of claim 1, wherein: the alginate lyase can degrade 1,4-beta-D-mannuronic acid fragment and 1,4-alpha-L-guluronic acid fragment simultaneously.
3. The algin oral liquid of claim 1, wherein: the polymerization degree of the algin oligosaccharide is 3-5.
4. The algin oral liquid of claim 1, wherein: chemically modifying the algin oligosaccharide; wherein the oligoguluronic acid oligosaccharide is modified by sulfation.
5. Use of the oral liquid as claimed in any one of claims 1 to 4 for the preparation of a hypolipidemic and anti-ageing medicament.
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