A method for determining the content of alginate oligosaccharides and its applications

Abstract

The present invention discloses a method for determining the content of alginate oligosaccharides. The method is to completely convert the C6 carboxyl group of alginate oligosaccharides into a carboxyl salt form, and based on the charge conservation principle, convert it into carbonate by calcination and ashing, and then determine the amount of carbonate by an acid-base titration method, and then obtain the content of alginate oligosaccharides by conversion according to the monosaccharide residues. The method does not need to degrade alginate oligosaccharides into monosaccharides, thus avoiding the problem that the degree of degradation is difficult to control and the degradation damage rate affects the content determination. The method does not need to use monosaccharide or oligosaccharide reference substances, different mannuronic acid / guluronic acid ratios do not affect the content determination results, and there is no need to draw a standard curve. The operation is simple and convenient, and rapid quantitative analysis of the content of alginate oligosaccharides can be achieved.

Description

Technical Field

[0001] The invention belongs to the field of analytical method development, and specifically relates to a method for determining the content of alginate oligosaccharides. Background Art

[0002] Alginate oligosaccharides, also known as brown algae oligosaccharides, alginate oligosaccharides, and alginate oligosaccharides, are usually oligosaccharides prepared from alginate by physical, chemical, and enzymatic methods. At present, there is no unified description of the degree of polymerization range of oligosaccharides at home and abroad. Some scholars believe that it is 2 to 10, and some believe that it is 2 to 20. In recent years, it has also been proposed that it is 2 to 25. Structurally, alginate oligosaccharides are composed of β-D-mannuronic acid and α-L-guluronic acid through 1→4 glycosidic bonds. Alginate oligosaccharides no longer have the physical properties of alginate such as film-forming, gelling, thickening, and stability. In recent years, the biological activity of alginate oligosaccharides in improving immunity, regulating intestinal flora, improving memory, promoting growth, and anti-oxidation has attracted much attention, and it has great application prospects in the development of terminal products such as food, medicine, cosmetics, and agricultural products.

[0003] At present, there is no mature method for determining the content of alginate oligosaccharides. Since the acid precipitation and calcium precipitation of alginate oligosaccharides are far less obvious than those of macromolecular alginate, the weight method and calcium acetate method in the traditional determination method are not suitable for the determination of the content of alginate oligosaccharides. Traditional colorimetric methods, such as sulfuric acid-phenol method, sulfuric acid-carbazole method, sulfuric acid-anthrone method, and meta-hydroxybiphenyl method, can be used to a certain extent for the determination of the content of alginate oligosaccharides. For example, the local standard DB21T2598-2016 stipulates the use of meta-hydroxybiphenyl method to determine the content of alginate oligosaccharides. This method generally uses mannuronic acid monosaccharide, guluronic acid monosaccharide or a mixture of the two as standard, or uses non-composition monosaccharides such as glucuronic acid and galacturonic acid as standard to draw a standard curve. The sample to be tested is first degraded into monosaccharides by sulfuric acid and then reacts with the color developer, and the content is calculated by measuring its absorbance value. However, the acid insolubility of alginate oligosaccharides and the acid-destructive properties of monosaccharide residues pose great challenges to this type of method. Its limitations are: (1) alginate oligosaccharides cannot be completely degraded into monosaccharides under relatively mild acid conditions, but monosaccharides are easily destroyed under severe conditions (especially guluronic acid is very susceptible to acid destruction); (2) when glucuronic acid is used as a standard, its structure is different from that of alginate oligosaccharide monosaccharide residues, and there is a difference in the color development behavior of the color developer; even if mannuronic acid, guluronic acid or a mixture of the two is used as a standard, the above problems still exist because the ratio of the two in the oligosaccharides prepared from alginate from different sources is different, resulting in an inaccurate standard curve and ultimately a large error in the measurement results. There is also a method based on the acid degradation of alginate oligosaccharides followed by derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) and then using high performance liquid chromatography to determine the content. This method also cannot solve the problem that alginate oligosaccharides cannot be completely degraded by acid. The decarboxylation method can theoretically be used to determine the content of alginate oligosaccharides, but the disadvantage of this method is that the decarboxylation reaction of uronic acid is relatively slow, high concentration of acid is required, and there is a problem of incomplete decarboxylation. It is necessary to use uronic acid monosaccharide standards to correct the analysis value using the same analysis procedure. At the same time, this method is extremely demanding on the airtightness of the measurement device.

[0004] In view of this, combined with the fact that the core structure of alginate oligosaccharides is the only one in nature with a carboxyl group on each monosaccharide residue, the present invention converts all C6 carboxyl groups on the monosaccharide residues of alginate oligosaccharides into carboxylates, calcines and ashes them to completely convert them into inorganic carbonates, and finally converts the alginate oligosaccharide content by acid-base titration. The method provided by the present invention is expected to provide a new solution for the determination of alginate oligosaccharides. However, based on the principle of this method, it is not suitable for oxidative degradation of brown algae oligosaccharides (with a diacid structure at the reducing end) and the sample contains a large amount of carboxyl-containing organic substances such as proteins. In this case, it may be used in combination with traditional methods to obtain more accurate results. Summary of the invention

[0005] The object of the present invention is to provide a method for determining the content of alginate oligosaccharides. This method does not require the use of strong acids to degrade alginate oligosaccharides into monosaccharides, thereby avoiding the problem of incomplete degradation in traditional methods and the destruction of monosaccharides during the degradation process. This method does not use various monosaccharide or oligosaccharide reference substances, and is applicable to alginate oligosaccharides with different mannuronic acid / guluronic acid ratios, and is also applicable to saturated (acid hydrolysis) and unsaturated (enzymatic hydrolysis) alginate oligosaccharides. This method does not require the drawing of a standard curve, is simple and convenient to operate, and can achieve rapid quantitative analysis of the content of alginate oligosaccharides. In order to achieve the above-mentioned purpose of the present invention, the present invention adopts the following technical scheme.

[0006] The present invention makes full use of the characteristic that the mother core structure of alginate oligosaccharide is the only one in nature in which each monosaccharide residue carries a carboxyl group, and creatively utilizes the charge conservation principle to couple the acid-base titration method to determine the content of alginate oligosaccharide.

[0007] The monosaccharide residues in the alginate oligosaccharide molecule are mannuronic acid (salt) and guluronic acid (salt), and the molecular formula is C6H7O6R (R is H or in the form of metal salt). During the calcination and ashing process of alginate oligosaccharides, the carbon, oxygen, and hydrogen (including C1, C2, C3, C4, and C5) on the monosaccharide residues will undergo a complex reaction to form CO2 and H2O and volatilize; while C6-OOR is divided into two cases. The first case is that R is H and the reaction forms CO2 and H2O and volatilizes. The second case is that R is a metal cation and non-volatile inorganic salts are formed due to the non-volatile nature of metal ions. From the point of view of the corresponding relationship of the amount of substance, if R is a univalent metal cation, the monosaccharide residue: C6-OO - :R is 1:1:1; if R is a divalent metal cation, the monosaccharide residue is: C6-OO - : R is 2:2:1; regardless of whether R is a univalent metal cation or a divalent metal cation, a monosaccharide residue always corresponds to a positive charge. Due to the non-volatile nature of metal cations, based on the charge conservation, an equal amount of negative charge needs to be formed during the ashing process to correspond to it. The negative charge is CO3 2- , the salt formed by ash is carbonate (for example, Na2CO3 when R is Na). Since the positive charge corresponding to the monosaccharide residue reacts with the CO3 formed after ash 2- The ratio is 2:1, CO3 2- When neutralizing with acid, H + The amount of substance of the monosaccharide residue is 1:1, and then through H + The amount of alginate oligosaccharides can be calculated.

[0008] In view of this, the first aspect of the present invention provides a method for determining the content of alginate oligosaccharides, comprising the following steps: S1: conversion, weighing a sample to be tested after constant weight, dissolving it with water, adding acid solution to adjust the pH to acidic, and after the pH of the solution is stable, adding alkali solution to adjust the pH to alkaline;

[0009] S2: Neutralization: After the pH of the solution obtained in step S1 is stable, acid is added dropwise to adjust the pH to neutral;

[0010] S3: carbonization, after the pH of the solution obtained in step S2 is stabilized, the solution is heated to evaporate the water, and carbonized to obtain a solid;

[0011] S4: ashing, transferring the solid obtained in step S3 into a muffle furnace for high-temperature calcination until a white or off-white calcined product is obtained;

[0012] S5: re-dissolving, dissolving the calcined product obtained in step S4 with an acid solution or water, and then making up the volume with water to obtain a constant volume solution;

[0013] S6: titration, taking an appropriate amount of the fixed volume solution obtained in step S5, adding an acid-base indicator, titrating with an alkali solution or an acid solution, and determining the titration end point based on the color change of the indicator;

[0014] S7: Calculate, convert the content of the sample to be tested according to the amount of alkali solution or acid solution used in step S6.

[0015] Furthermore, the alginate oligosaccharide comprises one or more of the following structures:

[0016]

[0017] m and g are natural numbers, 2≤g+m≤25

[0018] (Structure I)

[0019]

[0020] m and g are natural numbers, 1≤g+m≤24

[0021] (Structure II)

[0022]

[0023] m and g are natural numbers, 1≤g+m≤24

[0024] (Structure III).

[0025] Furthermore, R is H, one or more metal cations, preferably, R is H, one or more alkali metal ions.

[0026] Furthermore, the acid solution in step S1 and step S2 is a strong acid, preferably one or more of HCl, HNO3, and H2SO4; the alkali solution in step S1 is a strong base, preferably one or more of NaOH, KOH, LiOH, RbOH, CsOH, and FrOH.

[0027] Furthermore, in step S1, acid solution is added to adjust the pH value of the solution to 3-4, and alkali solution is added to adjust the pH value of the solution to 9-10.

[0028] In steps S1 and S2, the sample to be tested is first treated with acid and then with alkali. The purpose of the former is to eliminate possible carbonate interference in the sample to be tested, and the purpose of the latter is to remove the C6-OO - Completely converted to salt form.

[0029] Furthermore, the high temperature calcination temperature in step S4 is 400-1000°C, preferably 500-600°C.

[0030] During the implementation of the present invention, the completeness of the calcination of alginate oligosaccharides can be ensured by controlling the calcination temperature coupled with visual inspection (black→white or off-white).

[0031] Furthermore, when liquid acid is used for dissolution in step S5, the acid is a strong acid, preferably one of HCl, HNO3, and H2SO4; the acid-base indicator in step S6 is one of nitrophenol, phenolphthalein, sulfonated phenolphthalein, and azo compound indicators, preferably phenolphthalein indicator; and alkaline solution is used for titration in step S6, and the alkaline solution is a strong base, preferably one of NaOH, KOH, LiOH, RbOH, CsOH, and FrOH.

[0032] Further, the formula used for content conversion in step S7 is:

[0033]

[0034] Where:

[0035] c1——H of the acid in step S5 + Concentration, in moles per liter (mol / L);

[0036] V1——the volume of acid added in step S5, in milliliters (mL)

[0037] c2——OH of the alkali solution in step S6 - Concentration, in moles per liter (mol / L);

[0038] V2——the volume of alkali solution consumed in the titration in step S6, in milliliters (mL);

[0039] V'——the volume of the constant volume solution in step S5, in liters (L);

[0040] V” – the volume of the titrated solution in step S6, in milliliters (mL);

[0041] m——The mass of the sample to be tested in step S1, in milligrams (mg);

[0042] M R - is the molar mass of H and monovalent metal ions, or one-half of the molar mass of divalent metal ions, or one-third of the molar mass of trivalent metal ions, or the sum of the molar mass of H and / or monovalent metal ions, or / and one-half of the molar mass of divalent metal ions, or / and one-third of the molar mass of trivalent metal ions multiplied by the respective proportions;

[0043] λ——Molar mass correction number.

[0044] Furthermore, when water is used for dissolution in step S5, the acid-base indicator in step S6 is one of nitrophenol, phenolphthalein, sulfonated phenolphthalein, and azo compound indicators, preferably methyl orange indicator; and acid solution is used for titration in step S6, and the acid solution is a strong acid, preferably one of HCl, HNO3, and H2SO4.

[0045] Furthermore, the formula used for content conversion in step S7 is:

[0046]

[0047] Where:

[0048] c1——H of the acid in step S6 + Concentration, in moles per liter (mol / L);

[0049] V1——the volume of acid consumed in the titration in step S6, in milliliters (mL);

[0050] V'——the volume of the constant volume solution in step S5, in liters (L);

[0051] V” – the volume of the titrated solution in step S6, in milliliters (mL);

[0052] m——The mass of the sample to be tested in step S1, in milligrams (mg);

[0053] M R- is the molar mass of H and monovalent metal ions, or one-half of the molar mass of divalent metal ions, or one-third of the molar mass of trivalent metal ions, or is the sum of the molar mass of H and / or monovalent metal ions, or / and one-half of the molar mass of divalent metal ions, or / and one-third of the molar mass of trivalent metal ions multiplied by the respective proportions;

[0054] λ——Molar mass correction number.

[0055] Monosaccharide residues of alginate oligosaccharide C6H7O6 - The molar mass is 175. In the saturated alginate oligosaccharide structure (structure I), the non-reducing end is a saturated structure. The molar mass of the monosaccharide residue at the reducing end is 18 more than that in the middle and non-reducing ends. All monosaccharide residues need to share 18 equally, so the molar mass correction factor is introduced. The corrected molar mass of the monosaccharide residue is 175+M R +λ, the smaller the degree of polymerization, the greater the effect of λ on the molar mass of monosaccharide residues. In the unsaturated alginate oligosaccharide structure (structure II and structure III), the non-reducing end is an unsaturated structure, which has a molar mass of 18 less than that of the middle monosaccharide residue, while the reducing end has a molar mass of 18 more than that of the middle monosaccharide residue, which can be offset.

[0056] Further, the molar mass correction number λ in the formula used for content conversion in the structure I is The correction coefficient λ of the molar mass correction number in the formula used for content conversion in the structures II and III is 0.

[0057] Another aspect of the present invention provides an application of a method for determining the content of alginate oligosaccharides in an alginate oligosaccharide product.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] (1) The present invention innovatively utilizes the non-volatility of metal ions and the law of charge conservation to completely salt alginate oligosaccharides, and then converts the carboxylate into carbonate by calcination. The amount of monosaccharide residues is indirectly determined by measuring the amount of carbonate, providing a new method and new idea for the determination of alginate oligosaccharide content;

[0060] (2) Compared with the colorimetric method and the PMP derivatization HPLC method, the method provided by the present invention does not need to degrade alginate oligosaccharides into monosaccharides, thus avoiding the situation that alginate oligosaccharides cannot be completely degraded into monosaccharides under relatively mild acid conditions, while monosaccharides are easily destroyed under severe acid conditions. The method does not require drawing a standard curve, is simple, fast, and easy to operate;

[0061] (3) Compared with the decarboxylation method, the method provided by the present invention quantifies the carbonate obtained by calcination and ashing. During the calcination process, it is very easy to determine whether the ashing is sufficient by visual inspection (black carbide → white or off-white ashing), thereby overcoming the problems of slow decarboxylation reaction of uronic acid, difficulty in monitoring the degree of decarboxylation, and extremely strict requirements on the airtightness of the measuring device in the decarboxylation method. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the diagrams and charts required to be used in the embodiments or the description of the prior art.

[0063] Attached Figure 1 is the structure of alginate oligosaccharide in Example 1;

[0064] Attached Figure 2 is the alginate oligosaccharide in Example 1 1 H-NMR spectrum;

[0065] Attached Figure 3 is the infrared spectrum of alginate oligosaccharide in Example 1;

[0066] Attached Figure 4 This is a high performance liquid chromatography analysis diagram of alginate oligosaccharides in Example 1;

[0067] Attached Figure 5 This is a diagram showing the degree of polymerization of alginate oligosaccharides in Example 1;

[0068] Attached Figure 6 is the infrared spectrum of the calcined product of alginate oligosaccharide in Example 1;

[0069] Attached Figure 7 This is the structure of alginate oligosaccharide in Example 3. DETAILED DESCRIPTION

[0070] Embodiments of the present invention are described in detail below in conjunction with specific examples. However, it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. In the examples, if specific conditions are not specified, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be purchased commercially.

[0071] The following is an explanation of a method for determining the content of alginate oligosaccharides provided by the present invention.

[0072] Example 1 Identification of calcined alginate oligosaccharide products

[0073] The alginic acid oligosaccharide used in this example is an acid-hydrolyzed saturated oligosaccharide, which is obtained by high-pressure degradation of sodium alginate by hydrochloric acid, neutralization by sodium hydroxide, ultrafiltration classification, nanofiltration desalination, and spray drying. Its structure is shown in the attached figure. Figure 1 The sodium alginate raw material used is from Ascophyllum nodosum, which is food additive grade, with a viscosity of 35mPa.s / 20℃, and all quality indicators meet the national standards for food additives (GB 1886.243-2016).

[0074] Attached Figure 2 Alginate oligosaccharide 1 H-NMR diagram, the spectrum signal is consistent with the literature report (Preparation of a series of alginate oligosaccharides and research on their antioxidant-related biological activities, doctoral dissertation of Ocean University of China, 2016). Figure 3 This is the infrared spectrum of alginate oligosaccharide, 1610cm -1 COO - Asymmetric stretching vibration peak (COOH asymmetric stretching vibration peak should be 1690cm -1 The peak was not found in the spectrum), and the preparation process and the component analysis results in Table 1 indicated that alginate oligosaccharides were mainly in the form of Na salt. Figure 4 HPLC analysis of alginate oligosaccharides (chromatographic column: TSKgelG3000PW XL , mobile phase: Na2SO4, detector: RID), the results show that in addition to the main peak of alginate oligosaccharides, there is no obvious inorganic salt residue in the sample (small molecule inorganic salts such as NaCl generally appear after the Na2SO4 system peak). In addition, the results in Table 1 show that alginate oligosaccharides contain a small amount of fat and very little protein. Figure 5 This is a polymerization degree analysis diagram of alginate oligosaccharides, and its average polymerization degree is about 4.

[0075] Table 1 Component analysis of alginate oligosaccharides

[0076] Element content Fat% 1.8 protein% 0.11 K mg / kg 457 Ca mg / kg <![CDATA[2.33×10 3 ]]> Na mg / kg <![CDATA[1.00×10 5 ]]> Mg mg / kg 106 As mg / kg 0.0352 Hg mg / kg 0.00185 Zn mg / kg 2.85 Pb mg / kg 0.113 Cd mg / kg Not detected (<0.003) I mg / kg Not detected (<1.4)

[0077] The above-mentioned alginate oligosaccharides were calcined and ashed under the conditions disclosed in the present invention. Table 2 shows the ashing conditions at different calcination temperatures. Figure 6 The infrared spectrum of the completely ash-treated alginate oligosaccharide is shown in Figure 1. Figure 3 The characteristic absorption of alginate oligosaccharides in Figure 6 This indicates that the alginate oligosaccharide was calcined and ash-treated thoroughly. Figure 6 Compared with the standard infrared spectrum of Na2CO3 (CAS: 497-19-8) in the CAS database Figure 1 1436.83cm -1 The broad infrared absorption peak at asCO3 , 879.88cm -1 The infrared absorption band at is attributed to sodium carbonate.CO3 (Chang Ming, Yun Haili, Dong Sisi, Zhang Liying, Zhen Miaomiao, Jiao Yanyan, Yu Hongwei. Study on infrared spectrum of sodium carbonate [J]. Infrared Technology, 2016, 38(9): 803-810). The above analysis shows that the calcination product of alginate oligosaccharide is sodium carbonate.

[0078] Table 2 Ashing of alginate oligosaccharides at different calcination temperatures

[0079]

[0080]

[0081] Example 2 Comparison of different methods for determining the content of alginate oligosaccharides

[0082] The alginate oligosaccharides used in Example 1 were used to compare different content determination methods.

[0083] (1) The method developed by the present invention

[0084] Accurately weigh 0.5g of alginate oligosaccharide sample after constant weight in a crucible and dissolve it with 20mL pure water; adjust the pH to 3-4 with 0.5mol / L hydrochloric acid solution, stir thoroughly until the pH is stable, then adjust the pH to 9-10 with 0.5mol / L sodium hydroxide solution, stir thoroughly until the pH is stable, then adjust the pH to about 7 with 0.5mol / L hydrochloric acid solution, stir thoroughly until the pH is stable. Heat the solution after adjusting the pH on an electric furnace to evaporate the water and carbonize to obtain a black solid. Transfer the crucible containing the solid to a muffle furnace and calcine and ash at 600℃ for 4h; after the first calcination, use a medicine spoon to gently scrape off the residue attached to the crucible wall after cooling, and calcine again at 600℃ for 2h to obtain a white calcined product. Dissolve the powder obtained after calcination and ashing with an appropriate amount of pure water, and then dilute to 1L with pure water. Accurately measure 50.0mL of the constant volume solution in a 250mL conical flask, add 4-5 drops of methyl orange indicator, and titrate with 0.05mol / L HCl standard solution until the solution changes from yellow to orange, and record the volume of HCl standard solution consumed. The alginate oligosaccharide measured in this example is Na salt, M R is 23, the degree of polymerization is 4, and λ is 4.5. The content calculation is shown as follows:

[0085]

[0086] Where:

[0087] c1——concentration of HCl standard solution, in moles per liter (mol / L);

[0088] V1——volume consumed by titration of HCl standard solution, in milliliters (mL);

[0089] V'——fixed volume of calcined product, in liters (L);

[0090] V” – the sampling volume of the calcined product constant volume solution for analysis, in milliliters (mL);

[0091] m——the sampling volume of the sample to be tested, in milligrams (mg);

[0092] M R --twenty three;

[0093] λ——4.5.

[0094] The content of alginate oligosaccharides in this example was calculated according to the above calculation formula, and the results are shown in Table 3:

[0095] Table 3 Content determination results of the method developed by the present invention

[0096]

[0097] (2) Sulfuric acid-carbazole method

[0098] Preparation of standard solution: Accurately weigh a mixed standard of mannuronic acid / guluronic acid monosaccharide (m / m=1 / 1) and prepare a 50μg / mL standard solution with pure water. Preparation of test sample: Accurately weigh 10mg of alginate oligosaccharide and prepare a 40μg / mL solution with pure water. Preparation of carbazole solution: Weigh 0.25g, add anhydrous ethanol to dissolve and make up to 250mL. Preparation of sulfuric acid-borax reagent: Weigh 3.6g of borax, add 40mL of pure water and 360mL of concentrated sulfuric acid, and let stand overnight before use.

[0099] Standard curve drawing: Accurately pipette 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5mL of standard solution into stoppered test tubes, fill each with pure water to 0.5mL, add 3mL of sulfuric acid-borax reagent, shake evenly, react in a 100℃ water bath for 10min, cool to room temperature, add 0.1mL of carbazole reagent, shake evenly, react in a 100℃ water bath for 10min, cool to room temperature. Adjust the blank with pure water, measure the absorbance at 530nm, and make three parallels for each standard sample. Draw a standard curve with the standard concentration (C) as the horizontal axis and the absorbance (A) as the vertical axis.

[0100] Sample determination: Pipette 0.5 mL of the sample solution to be tested and perform the determination according to the standard curve determination method described above. Make three parallel samples and calculate the content based on the standard curve.

[0101] The content of alginate oligosaccharides in this example was calculated according to the standard curve, and the results are shown in Table 4:

[0102] Table 4 Sulfuric acid-carbazole method content determination results

[0103]

[0104] (3) Decarboxylation method

[0105] Accurately weigh 0.25g of alginate oligosaccharide and add it to the decomposition bottle, add 100mL of sulfuric acid solution. Build the experimental device according to the requirements of the decarboxylation method, then pass nitrogen into the decomposition bottle to ensure a certain flow rate, and then use soapy water to check the tightness of the entire system until the tightness is intact. After checking the tightness, pass nitrogen for 15 minutes, then add 10 drops of n-butanol, 25mL of 0.25mol / L sodium hydroxide solution, and 40mL of distilled water to each of the two absorption bottles. First pass water through the condenser tube, then use the heating jacket to heat until the acid in the decomposition bottle boils, and keep the acid boiling for 2.5h. Stop heating, pass nitrogen through the device for 10 minutes, first remove the two absorption bottles, and then turn off the nitrogen. Combine the solutions in the two wash bottles, rinse them with distilled water, then add 10mL of 10% barium hydroxide solution and stir gently for 1min, then seal and place for 5min, and filter into a conical flask. Add 3 drops of 1% phenolphthalein indicator to the conical flask, and titrate with hydrochloric acid standard solution until the red color disappears, which is the end point, and record the consumption of hydrochloric acid standard solution. Perform a blank experiment according to the above steps, and record the consumption of hydrochloric acid standard solution. The content calculation is shown as follows:

[0106]

[0107] Where:

[0108] c——Concentration of HCl standard solution, in moles per liter (mol / L);

[0109] V1——the volume consumed by the titration of the HCl standard solution in the blank experiment, in milliliters (mL);

[0110] V2——the volume consumed by the titration of the HCl standard solution in the sample experiment, in milliliters (mL);

[0111] m——the sampling volume of the sample to be tested, in milligrams (mg);

[0112] M R --twenty three;

[0113] λ——4.5.

[0114] The content of alginate oligosaccharides in this example was calculated according to the above calculation formula, and the results are shown in Table 5:

[0115] Table 5 Decarboxylation method content determination results

[0116]

[0117] Example 3 Determination of the content of enzymatically hydrolyzed alginate oligosaccharides

[0118] The alginic acid oligosaccharide used is enzymatically unsaturated alginic acid oligosaccharide, which is obtained by degradation of sodium alginate by alginate lyase, nanofiltration desalination, and spray drying. Its degree of polymerization distribution is 2, 3, 4, 5 sugars, and its structure is as shown in the attached Figure 7 The sodium alginate raw material used is from Ascophyllum nodosum, which is food additive grade, with a viscosity of 35mPa.s / 20℃, and all quality indicators meet the national standards for food additives (GB 1886.243-2016).

[0119] Accurately weigh 0.5g of alginate oligosaccharide after constant weight in a crucible and dissolve it with 20mL pure water; adjust the pH to 3-4 with 0.5mol / L hydrochloric acid solution, stir thoroughly until the pH is stable, and then adjust the pH to 9-10 with 0.5mol / L sodium hydroxide solution. After stirring thoroughly until the pH is stable, adjust the pH to about 7 with 0.5mol / L hydrochloric acid solution. Heat the solution after adjusting the pH on an electric furnace to evaporate the water and carbonize to obtain a solid. Transfer the crucible containing the solid to a muffle furnace and calcine and ash at 550℃ for 4h; after the first calcination, use a medicine spoon to gently scrape off the residue attached to the crucible wall after cooling, and calcine again at 550℃ for 2h to obtain a white calcined product. Dissolve the powder obtained after calcination with an appropriate amount of pure water, and then dilute to 1L with pure water. Accurately measure 50.0mL of the constant volume solution in a 250mL conical flask, add 4-5 drops of methyl orange indicator, and titrate with 0.05mol / L HCl standard solution until the solution changes from yellow to orange, and record the volume of HCl standard solution consumed. The alginate oligosaccharide measured in this example is Na salt, M R =23, the content calculation steps are as follows:

[0120]

[0121] Where:

[0122] c1——concentration of HCl standard solution, in moles per liter (mol / L);

[0123] V1——volume consumed by titration of HCl standard solution, in milliliters (mL);

[0124] V'——fixed volume of calcined product, in liters (L);

[0125] V” – the sampling volume of the calcined product constant volume solution for analysis, in milliliters (mL);

[0126] m——the sampling volume of the sample to be tested, in milligrams (mg);

[0127] M R ——is 23.

[0128] λ——0.

[0129] The content of enzymatically hydrolyzed alginate oligosaccharides was calculated according to the above calculation formula, and the results are shown in Table 6:

[0130] Table 6 Determination results of enzymatic hydrolysis of alginate oligosaccharide content

[0131]

[0132] Example 4 Determination of the content of alginate oligosaccharide zinc

[0133] The alginate oligosaccharide zinc used in this embodiment is obtained by acid hydrolyzing sodium alginate, neutralizing it with zinc oxide, and then filtering and ethanol precipitation to remove salt. The sodium alginate raw material used is from Ascophyllum nodosum, which is food additive grade, with a viscosity of 35mPa.s / 20°C, and various quality indicators meet the national standards for food additives (GB 1886.243-2016). Through polymerization degree analysis, its average polymerization degree is 6. Calculated by elemental analysis, the molar ratio of sodium to zinc in the alginate oligosaccharide zinc is 7.3:1.

[0134] Accurately weigh 0.5g of alginate oligosaccharide zinc after constant weight in a crucible and dissolve it with 20mL pure water; adjust the pH to 3-4 with 0.5mol / L hydrochloric acid solution, stir thoroughly until the pH is stable, and then adjust the pH to 9-10 with 0.5mol / L sodium hydroxide solution. After stirring thoroughly until the pH is stable, adjust the pH to about 7 with 0.5mol / L hydrochloric acid solution. Heat the solution after adjusting the pH on an electric furnace to evaporate the water and carbonize to obtain a solid. Transfer the crucible containing the solid to a muffle furnace and calcine and ash at 600℃ for 4h; after the first calcination, gently scrape off the residue attached to the crucible wall with a medicine spoon after cooling, and calcine again at 600℃ for 4h to obtain an off-white calcined product. Add 20mL of 0.5mol / L sulfuric acid to the powder obtained after calcination and ashing, heat and dissolve, and then dilute to 1L with pure water. Accurately measure 50.0 mL of the constant volume solution in a 250 mL conical flask, add 4-5 drops of phenolphthalein indicator, and titrate with 0.05 mol / L NaOH standard solution until the solution changes from colorless to red, and record the volume of NaOH standard solution consumed. R It is 23×(7.3 / 8.3)+65 / 2×(1 / 8.3), which is calculated to be 24.14. λ is 3. The steps for calculating its content are as follows:

[0135]

[0136] Where:

[0137] c1——acid H used to dissolve the calcined product + Concentration, in moles per liter (mol / L);

[0138] V1——The volume of acid used to dissolve the calcined product, in milliliters (mL)

[0139] C2——OH of titrated alkali solution - Concentration, in moles per liter (mol / L);

[0140] V2——volume of alkali solution consumed in titration, in milliliters (mL);

[0141] V'——fixed volume of calcined product, in liters (L);

[0142] V” – the sampling volume of the calcined product constant volume solution for analysis, in milliliters (mL);

[0143] m——the sampling volume of the sample to be tested, in milligrams (mg);

[0144] M R ——24.14;

[0145] λ——3.

[0146] The content of alginate oligosaccharide zinc was calculated according to the above calculation formula, and the results are shown in Table 7:

[0147] Table 7 Alginate oligosaccharide zinc determination results

[0148]

[0149] Example 5 Determination of the content of brown algae oligosaccharides in enzymatically hydrolyzed kelp powder

[0150] The enzymatically hydrolyzed kelp powder used in this embodiment is obtained by homogenizing kelp, extracting with sodium carbonate, neutralizing with hydrochloric acid, enzymolyzing with alginate lyase and cellulase, filtering and removing residues, and spray drying. The enzymatically hydrolyzed kelp powder contains sugar substances such as alginate oligosaccharides, fucoidan, brown algae starch, inorganic salts, a small amount of protein and other small molecular substances.

[0151] Accurately weigh 0.5g of the enzymatic hydrolyzed kelp powder sample after constant weight in a crucible and dissolve it with 20mL of pure water; adjust the pH to 3-4 with 0.5mol / L hydrochloric acid solution, stir thoroughly until the pH is stable, and then adjust the pH to 9-10 with 0.5mol / L sodium hydroxide solution. After stirring thoroughly until the pH is stable, adjust the pH to about 7 with 0.5mol / L hydrochloric acid solution. Heat the solution after adjusting the pH on an electric furnace to evaporate the water and carbonize to obtain a solid. Transfer the crucible containing the solid to a muffle furnace and calcine and ash at 550℃ for 4h; after the first calcination, gently scrape off the residue attached to the crucible wall with a medicine spoon after cooling, and calcine again at 550℃ for 2h to obtain an off-white calcined product. Dissolve the powder obtained after calcination and ashing with an appropriate amount of pure water, and then dilute to 1 with pure water. Accurately measure 50.0mL of the constant volume solution in a 250mL conical flask, add 4-5 drops of methyl orange indicator, and titrate with 0.05mol / L HCl standard solution until the solution changes from yellow to orange, and record the volume of HCl standard solution consumed. R =23, the content calculation steps are as follows:

[0152]

[0153] Where:

[0154] c1——concentration of HCl standard solution, in moles per liter (mol / L);

[0155] V1——volume consumed by titration of HCl standard solution, in milliliters (mL);

[0156] V'——fixed volume of calcined product, in liters (L);

[0157] V” – the sampling volume of the calcined product constant volume solution for analysis, in milliliters (mL);

[0158] m——the sampling volume of the sample to be tested, in milligrams (mg);

[0159] M R ——is 23;

[0160] λ——0.

[0161] The alginate oligosaccharide content in the enzymatically hydrolyzed kelp powder was calculated according to the above calculation formula, and the results are shown in Table 8:

[0162] Table 8 Determination results of alginate oligosaccharide content in enzymatic kelp powder

[0163]

[0164] Although the present invention has been illustrated and described with specific embodiments, it will be appreciated that many other changes and modifications may be made without departing from the spirit and scope of the present invention. Therefore, it is intended to include all such changes and modifications within the scope of the present invention in the appended claims.

Claims

1. A method for determining the content of alginate oligosaccharides, characterized in that: The method comprises the following steps: S1: Conversion, weigh the sample to be tested after constant weight, dissolve it in water, add acid solution to adjust the pH to acidic, and after the pH of the solution is stable, add alkali solution to adjust the pH to alkaline; S2: Neutralization: After the pH of the solution obtained in step S1 is stable, acid is added dropwise to adjust the pH to neutral; S3: carbonization, after the pH of the solution obtained in step S2 is stabilized, the solution is heated to evaporate the water, and carbonized to obtain a solid; S4: ashing, transferring the solid obtained in step S3 into a muffle furnace for high-temperature calcination until a white or off-white calcined product is obtained; S5: re-dissolving, dissolving the calcined product obtained in step S4 with an acid solution or water, and then making up the volume with water to obtain a constant volume solution; S6: titration, taking an appropriate amount of the fixed volume solution obtained in step S5, adding an acid-base indicator, titrating with an alkali solution or an acid solution, and determining the titration end point based on the color change of the indicator; S7: Calculate, convert the content of the sample to be tested according to the amount of alkali solution or acid solution used in step S6, 1) When acid solution is used for dissolution in step S5, the formula used for converting the alginate oligosaccharide content is: Where: c1——H of the acid in step S5 + Concentration, in moles per liter (mol / L), V1——the volume of acid added in step S5, in milliliters (mL), c2——OH of the alkali solution in step S6 - Concentration, in moles per liter (mol / L), V2——the volume of alkali solution consumed in the titration in step S6, in milliliters (mL), V'——The volume of the constant volume solution in step S5, in liters (L), V” - the volume of the titrated solution in step S6, in milliliters (mL). m——The mass of the sample to be tested in step S1, in milligrams (mg), M R ——For H + , the molar mass of a monovalent metal ion, or one-half the molar mass of a divalent metal ion, or one-third the molar mass of a trivalent metal ion, or H + or / and the sum of the molar mass of the monovalent metal ion or / and one-half the molar mass of the divalent metal ion or / and one-third the molar mass of the trivalent metal ion multiplied by the respective proportions, λ——molar mass correction number; 2) When the alginate oligosaccharide is dissolved in water in step S5, the formula used for converting the alginate oligosaccharide content is: Where: c1——H of the acid in step S6 + Concentration, in moles per liter (mol / L), V1——the volume of acid consumed in the titration in step S6, in milliliters (mL), V'——The volume of the constant volume solution in step S5, in liters (L), V” - the volume of the titrated solution in step S6, in milliliters (mL). m——The mass of the sample to be tested in step S1, in milligrams (mg), M R ——For H + , the molar mass of the monovalent metal ion, or one-half the molar mass of the divalent metal ion, or one-third the molar mass of the trivalent metal ion, or H + or / and the sum of the molar mass of the monovalent metal ion or / and one-half the molar mass of the divalent metal ion or / and one-third the molar mass of the trivalent metal ion multiplied by the respective proportions, λ——Molar mass correction number.

2. The method for determining the content of alginate oligosaccharides according to claim 1, characterized in that: The alginate oligosaccharide comprises one or more of the following structures:

3. The method for determining the content of alginate oligosaccharides according to claim 2, characterized in that: The R is H, one or more metal cations.

4. The method for determining the content of alginate oligosaccharides according to claim 3, characterized in that: The R is H, one or more alkali metal ions.

5. The method for determining the content of alginate oligosaccharides according to claim 1, characterized in that: The acid solution in step S1 and step S2 is a strong acid, and the alkali solution in step S1 is a strong base.

6. The method for determining the content of alginate oligosaccharides according to claim 5, characterized in that: The strong acid is one or more of HCl, HNO3, and H2SO4; the strong base is one or more of NaOH, KOH, LiOH, RbOH, CsOH, and FrOH.

7. The method for determining the content of alginate oligosaccharides according to claim 1, characterized in that: In the step S1, an acid solution is added to adjust the pH value of the solution to 3-4, and an alkali solution is added to adjust the pH value of the solution to 9-10.

8. The method for determining the content of alginate oligosaccharides according to claim 1, characterized in that: The high temperature calcination temperature in the step S4 is 400-1000°C.

9. The method for determining the content of alginate oligosaccharides according to claim 8, characterized in that: The high temperature calcination temperature is 500-600°C.

10. The method for determining the content of alginate oligosaccharides according to claim 1, characterized in that: When acid solution is used for dissolution in step S5, the acid solution is a strong acid; the acid-base indicator in step S6 is one of nitrophenol, phenolphthalein, sulfonated phenolphthalein, and azo compound indicators; and alkaline solution is used for titration in step S6, and the alkaline solution is a strong base.

11. A method for determining the content of alginate oligosaccharides according to claim 10, characterized in that: The strong acid is one of HCl, HNO3, and H2SO4.

12. The method for determining the content of alginate oligosaccharides according to claim 10, characterized in that: In step S6, the acid-base indicator is phenolphthalein indicator.

13. The method for determining the content of alginate oligosaccharides according to claim 10, characterized in that: The alkali solution is one of NaOH, KOH, LiOH, RbOH, CsOH, and FrOH.

14. The method for determining the content of alginate oligosaccharides according to claim 1, characterized in that: When water is used for dissolution in step S5, the acid-base indicator in step S6 is one of nitrophenol, phenolphthalein, sulfonated phenolphthalein, and azo compound indicators; and acid solution is used for titration in step S6, and the acid solution is a strong acid.

15. A method for determining the content of alginate oligosaccharides according to claim 14, characterized in that: In the step S6, the acid-base indicator is methyl orange indicator.

16. A method for determining the content of alginate oligosaccharides according to claim 14, characterized in that: The strong acid is one of HCl, HNO3, and H2SO4.

17. A method for determining the content of alginate oligosaccharides according to claim 2, characterized in that: The molar mass correction number λ in the formula used for content conversion for the structure I is: The correction coefficient λ of the molar mass correction number in the formula used for content conversion in the structures II and III is 0.

18. Use of the method for determining the content of alginate oligosaccharides according to claim 1 in determining the content of alginate oligosaccharides in alginate oligosaccharides products.

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