Sulfated yellow water polysaccharide, its preparation method and application

Abstract

The present invention discloses a sulfated yellow water polysaccharide, a preparation method and an application thereof, belonging to the technical field of reuse of liquor by-products. In the present invention, a chlorosulfonic acid-pyridine sulfation reagent is added to a homogeneous solution of yellow water polysaccharide for sulfation reaction, and finally, after neutralization, alcohol precipitation, redissolution, dialysis and freeze-drying, the sulfated yellow water polysaccharide is obtained. The present invention introduces sulfate groups into the yellow water polysaccharide, improves the water solubility of the polysaccharide, increases the utilization rate of the organism, and at the same time, the introduction of sulfate groups endows the sulfated modified yellow water polysaccharide with better antioxidant properties, realizing the high-value utilization of yellow water; at the same time, the present invention can extract yellow water polysaccharide with higher purity from yellow water through fractional alcohol precipitation.

Description

Technical Field

[0001] The present invention belongs to the technical field of reuse of by-products of Chinese liquor, and specifically relates to a yellow water polysaccharide, a method for extracting the same, a sulfated yellow water polysaccharide, a method for preparing the same, and an application thereof. Background Art

[0002] The excessive accumulation of free radicals will affect the physiological functions of normal cells and tissues. The excessive accumulation of free radicals in living organisms will disrupt the redox balance, and the excess free radicals will cause cross-linking and breakage of biological macromolecules such as proteins and nucleic acids, destroy the protein conformation or affect the biological membrane function, and then cause cell damage and degeneration, and ultimately lead to death. Therefore, it has always been a trend to find safe and highly efficient antioxidant active substances.

[0003] Yellow water is a by-product in the process of Chinese liquor brewing, and the discharge amount is huge. A large amount of yellow water is directly discarded after treatment, resulting in serious waste of resources and environmental pollution. Therefore, realizing the full development and utilization of yellow water will provide an important basis for the high-value utilization of yellow water. Sulfation modification can improve various biological activities of polysaccharides by changing the structure and conformation of polysaccharides. The polysaccharides in yellow water have certain antioxidant activities, but the sulfation modification thereof has not been studied. Summary of the Invention

[0004] In order to realize the high-value recycling of yellow water, a by-product of liquor brewing, the present invention obtains yellow water polysaccharide from yellow water by fractional alcohol precipitation, and further improves the activity of yellow water polysaccharide by chemical modification methods, and improves the antioxidant activity, water solubility and other properties of yellow water polysaccharide.

[0005] The present invention first provides a sulfated yellow water polysaccharide, which is a sulfated derivative of yellow water polysaccharide and is obtained by sulfation reaction of yellow water polysaccharide.

[0006] The present invention also provides a method for preparing a sulfated yellow water polysaccharide, which comprises the following steps:

[0007] A. Prepare a sulfation reagent: Mix pyridine and chlorosulfonic acid to obtain a sulfation reagent;

[0008] B. Prepare a homogeneous solution of yellow water polysaccharide: Dissolve yellow water polysaccharide in an organic solvent to obtain a homogeneous solution of yellow water polysaccharide;

[0009] C. Mix the sulfation reagent obtained in step A and the homogeneous solution of yellow water polysaccharide obtained in step B, heat for sulfation reaction, after the reaction is completed, neutralize the system to neutrality, then carry out alcohol precipitation to obtain a precipitate, and the obtained precipitate is successively redissolved in water, dialyzed and freeze-dried to obtain a sulfated yellow water polysaccharide.

[0010] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, in step A, the volume ratio of the pyridine to the chlorosulfonic acid is 1-6:1.

[0011] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, in step B, the concentration of the homogeneous solution of the yellow water polysaccharide is 5-20 mg / mL.

[0012] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, in step B, the organic solvent is at least one of formamide, N,N-dimethylformamide, dimethyl sulfoxide or dimethylacetamide.

[0013] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, in step C, the volume ratio of the sulfuric acid esterification reagent to the homogeneous solution of the yellow water polysaccharide is controlled to be 1:0.5-2.

[0014] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, in step C, the sulfuric acid esterification reagent is added dropwise to the homogeneous solution of the yellow water polysaccharide.

[0015] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, in step C, the temperature of the sulfuric acid esterification reaction is 60-80 °C.

[0016] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, in step C, the time of the sulfuric acid esterification reaction is 2-4 h.

[0017] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, in step C, a 2-5 M NaOH solution is used to neutralize the system to neutrality.

[0018] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, in step C, the operation of alcohol precipitation is: mixing the neutralized system with ethanol 4-6 times the volume of the neutralized system for precipitation.

[0019] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, in step C, the time of alcohol precipitation is 12-15 h.

[0020] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, in step C, the operation of water re-dissolution is: dissolving the precipitate obtained by alcohol precipitation with water 2-4 times the volume of the precipitate.

[0021] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, in step C, the operation of dialysis is: dialyzing the re-dissolved system with a 3500 kd dialysis bag in water at 4-6 °C for 72-84 h, and changing the water every 4-8 h during this period.

[0022] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, in step C, the temperature of freeze-drying is -60--50 °C.

[0023] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, in step C, the time of freeze-drying is 24 to 48 h.

[0024] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, in step B, the yellow water polysaccharide is prepared by the following method:

[0025] a. After centrifuging the yellow water, collect the supernatant, add ethanol to the supernatant so that the final volume concentration of ethanol in the system is X 1 , after standing at low temperature and then centrifuging, precipitate 1 and supernatant 1 are obtained;

[0026] b. Concentrate supernatant 1 until the ethanol evaporates completely, then add ethanol so that the final volume concentration of ethanol in the system is X 2 , after standing at low temperature and then centrifuging, precipitate 2 and supernatant 2 are obtained;

[0027] c. Repeat the operation of step b, concentrate the supernatant obtained in the previous time until the ethanol evaporates completely, then add ethanol so that the final volume concentration of ethanol in the system is X 3 to X n , after standing at low temperature and then centrifuging, precipitate 3 to precipitate n and supernatant 3 to supernatant n are obtained respectively;

[0028] d. Respectively perform degreasing, deproteinization, dialysis and freeze-drying on precipitate 1 to precipitate n to obtain yellow water polysaccharide 1 to yellow water polysaccharide n;

[0029] Among them, X 1 to X n is selected from 40% to 80%, and gradually increases from X 1 to X n ;

[0030] The yellow water polysaccharide used in step B is selected from at least one of yellow water polysaccharide 1 to yellow water polysaccharide n.

[0031] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, the interval between X 1 and X n is not less than 5%.

[0032] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, when preparing the yellow water polysaccharide, the centrifugation is centrifugation at 6000 to 10000×g for 8 to 12 min.

[0033] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, when preparing the yellow water polysaccharide, the low-temperature placement is placement at 4 to 6°C for 24 to 36 h.

[0034] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, when preparing the yellow water polysaccharide, the water re-dissolution is: dissolving precipitate 1 to precipitate n with 2 to 3 times the volume of water.

[0035] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, when preparing the yellow water polysaccharide, the degreasing is as follows: successively washing with ethanol and acetone to remove lipids.

[0036] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, when preparing the yellow water polysaccharide, the deproteinization is as follows: using Sevag reagent to deproteinize until no white protein appears.

[0037] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, when preparing the yellow water polysaccharide, the dialysis is as follows: using a 3500 kd dialysis bag to dialyze in water at 4 - 6 °C for 72 - 84 h, and changing the water every 4 - 8 h during this period.

[0038] Among them, in the preparation method of the above-mentioned sulfated yellow water polysaccharide, when preparing the yellow water polysaccharide, the freeze-drying is as follows: freeze-drying at -60 to -50 °C for 24 - 48 h.

[0039] The present invention also provides the application of the above-mentioned sulfated yellow water polysaccharide or the sulfated yellow water polysaccharide prepared by the above method in the preparation of an antioxidant.

[0040] The sulfated modified yellow water polysaccharide of the present invention has better antioxidant properties and water solubility compared with the yellow water polysaccharide, and can be used to prepare a DPPH free radical scavenger.

[0041] In the present invention, water generally uses deionized water, and the reagent used for alcohol precipitation generally uses absolute ethanol.

[0042] The beneficial effects of the present invention:

[0043] In the present invention, sulfate groups are introduced into the yellow water polysaccharide. The introduction of sulfate groups can improve the ability of the polysaccharide to provide hydrogen ions. Hydrogen ions can combine with free radical ions to form a stable complex to terminate the free radical chain reaction, making the sulfated modified yellow water polysaccharide have better antioxidant properties; at the same time, the sulfated modification can also improve the water solubility of the yellow water polysaccharide and increase the utilization rate of the body.

[0044] In the present invention, the yellow water polysaccharide is more carefully extracted from a 40% ethanol solution to an 80% ethanol solution by fractional alcohol precipitation, reducing the purification steps, and a polysaccharide component with relatively higher purity can be obtained, which is beneficial for its subsequent application.

[0045] The process of the present invention is simple and the components are clear, providing a new idea for the high-value utilization of yellow water and yellow water polysaccharide. Description of the Drawings

[0046] Figure 1 It is the infrared spectrogram of the yellow water polysaccharide and the sulfated yellow water polysaccharide obtained in Example 1 of the present invention; among them, A is HSP40 and its sulfated derivative; B is HSP60 and its sulfated derivative; C is HSP80 and its sulfated derivative.

[0047] Figure 2 This is the monosaccharide composition diagram of the yellow water polysaccharide and sulfated yellow water polysaccharide obtained in Example 1 of the present invention; among them, A is HSP40; B is S 1:2 -HSP40; C is S 1:4 -HSP40.

[0048] Figure 3 This is the molecular weight distribution diagram of the yellow water polysaccharide and sulfated yellow water polysaccharide obtained in Example 1 of the present invention; among them, A is HSP40; B is S 1:2 -HSP40; C is S 1:4 -HSP40.

[0049] Figure 4 This is the standard curve diagram for determining the degree of substitution by the barium chloride gelatin method.

[0050] Figure 5 This is the standard curve diagram for determining the protein content by the Coomassie brilliant blue method.

[0051] Figure 6 This is the standard curve diagram for determining the neutral sugar content by the phenol-sulfuric acid method.

[0052] Figure 7 This is the standard curve diagram for determining the uronic acid content by the carbazole-sulfuric acid method.

[0053] Figure 8 This is the effect diagram of the antioxidant effect of the yellow water polysaccharide and sulfated yellow water polysaccharide obtained in Example 1 of the present invention. Detailed implementation mode

[0054] The present invention first provides a sulfated yellow water polysaccharide, which is a sulfated derivative of yellow water polysaccharide and is obtained by sulfation reaction of yellow water polysaccharide.

[0055] There is currently no report on the sulfation modification of yellow water polysaccharide. The present invention can achieve the sulfation modification of yellow water polysaccharide to obtain sulfated yellow water polysaccharide. The yellow water polysaccharide subjected to sulfation modification in the present invention is not limited to the yellow water polysaccharide obtained by fractional alcohol precipitation in the present invention, and common yellow water polysaccharides in the art are applicable. After different yellow water polysaccharides are sulfated and modified, the molecular weight, degree of substitution, etc. are different; as shown in the examples of the present invention, when HSP40 is used for sulfation reaction, the degree of substitution of the sulfate group of the obtained sulfated yellow water polysaccharide is 0.451-0.625, and its molecular weight is 3251-64909 Da.

[0056] Specifically, a preparation method of a sulfated yellow water polysaccharide includes the following steps:

[0057] A. Prepare a sulfation reagent: Mix pyridine and chlorosulfonic acid to obtain a sulfation reagent;

[0058] B. Prepare a homogeneous solution of Huangshui polysaccharide: Dissolve Huangshui polysaccharide in an organic solvent to obtain a homogeneous solution of Huangshui polysaccharide;

[0059] C. Mix the sulfuric acid esterification reagent obtained in step A and the homogeneous solution of Huangshui polysaccharide obtained in step B, and heat for sulfuric acid esterification reaction. After the reaction is completed, neutralize the system to neutral, and then perform alcohol precipitation (that is, mix the neutralized material with absolute ethanol) to obtain a precipitate. The obtained precipitate is successively redissolved in water, dialyzed, and freeze-dried to obtain sulfated Huangshui polysaccharide.

[0060] In step A of the present invention, the volume ratio of pyridine to chlorosulfonic acid is 1-6:1. When preparing the sulfuric acid esterification reagent, anhydrous pyridine is generally used for pyridine and it is fully cooled in an ice bath. Since chlorosulfonic acid is a strong acid, to ensure safety, generally chlorosulfonic acid is added dropwise to pyridine. After the addition is completed, first stir in an ice bath for 20-50 min to accelerate cooling, and then stir at room temperature for 20-50 min for natural cooling. By controlling the volume ratio of chlorosulfonic acid to pyridine, sulfated Huangshui polysaccharides with different degrees of sulfation can be obtained.

[0061] In step B of the present invention, the concentration of the homogeneous solution of Huangshui polysaccharide is 5-20 mg / mL; in step B, at least one of formamide, N,N-dimethylformamide, dimethyl sulfoxide, or dimethylacetamide is used as the organic solvent to dissolve Huangshui polysaccharide.

[0062] In step C of the present invention, control the volume ratio of the sulfuric acid esterification reagent to the homogeneous solution of Huangshui polysaccharide to be 1:0.5-2; in step C, in order to ensure full contact and complete reaction, the sulfuric acid esterification reagent is added dropwise to the homogeneous solution of Huangshui polysaccharide.

[0063] In step C of the present invention, the temperature of the sulfuric acid esterification reaction is 60-80 °C; the time of the sulfuric acid esterification reaction is 2-4 h.

[0064] In step C of the present invention, a 2-5 M NaOH solution is used to neutralize the system to neutral; in step C, the alcohol precipitation is to mix the neutralized system with ethanol 4-6 times the volume of the neutralized system for precipitation; in step C, the time of the alcohol precipitation is 12-15 h; in step C, the redissolution in water is to dissolve the precipitate obtained by alcohol precipitation with water 2-4 times the volume of the precipitate; in step C, the operation of dialysis is: dialyze the redissolved system with a 3500 kd dialysis bag in water at 4-6 °C for 72-84 h, and change the water every 4-8 hours during this period; in step C, the temperature of the freeze-drying is -60--50 °C; in step C, the time of the freeze-drying is 24-48 h.

[0065] The Huangshui polysaccharide used in the present invention can be prepared by the following method:

[0066] a. After centrifuging the yellow water, collect the supernatant, add ethanol to the supernatant to make the final volume concentration of ethanol in the system X 1 , after standing at low temperature, centrifuge again to obtain precipitate 1 and supernatant 1;

[0067] b. Concentrate supernatant 1 until the ethanol has completely evaporated, then add ethanol to make the final volume concentration of ethanol in the system X 2 , after standing at low temperature, centrifuge again to obtain precipitate 2 and supernatant 2;

[0068] c. Repeat the operation of step b, concentrate the supernatant obtained in the previous time until the ethanol has completely evaporated, then add ethanol to make the final volume concentration of ethanol in the system X 3 ~X n , after standing at low temperature, centrifuge again to obtain precipitate 3~precipitate n and supernatant 3~supernatant n respectively;

[0069] d. Dissolve precipitate 1~precipitate n in water successively, degrease, deproteinize, dialyze and freeze-dry to obtain yellow water polysaccharide 1~yellow water polysaccharide n;

[0070] Among them, X 1 ~X n is selected from 40% to 80%, and gradually increases from X 1 to X n ;

[0071] The yellow water polysaccharide used in step B is selected from at least one of yellow water polysaccharide 1~yellow water polysaccharide n.

[0072] In the present invention, an appropriate preparation method of yellow water polysaccharide can be selected according to the yellow water polysaccharide to be sulfated as needed. If only yellow water polysaccharide 1 is sulfated, only yellow water polysaccharide 1 needs to be prepared, and the subsequent steps do not need to be carried out; if yellow water polysaccharide 10 (including yellow water polysaccharide 10 alone or a mixed yellow water polysaccharide of yellow water polysaccharide 1~10 including yellow water polysaccharide 10) is sulfated, yellow water polysaccharide 1~10 need to be prepared successively, and the subsequent steps do not need to be carried out.

[0073] In the present invention, the principle of fractional alcohol precipitation is to use ethanol aqueous solutions with different polarities to extract polysaccharides with different polarities. The polarity of the ethanol solution gradually decreases from 40% to 80%, so the molecular weight of the polysaccharides that can be extracted becomes smaller and smaller. When preparing yellow water polysaccharide 2~n in the present invention, there is no need to take fresh yellow water (if fresh yellow water is taken, it will cause a decrease in component purity) for alcohol precipitation, but to carry out more detailed alcohol precipitation on the supernatant obtained in the previous time to obtain yellow water polysaccharide, which can reduce the purification steps, obtain polysaccharide components with relatively higher purity, and also reduce the raw material usage. At the same time, in order to obtain yellow water polysaccharide with obvious excellent components, the present invention can control that the interval between X 1 and X n is not less than 5% for each pair.

[0074] In the present invention, each time when preparing the yellow water polysaccharide, ethanol remains in the previous operations, and the subsequent operations have requirements for the ethanol concentration. Therefore, it is necessary to first volatilize all the remaining ethanol in the previous steps (generally, remove ethanol by rotary evaporation under reduced pressure at -0.1 MPa and 55 - 65 °C), and then add new ethanol to a fixed concentration. This operation is an important requirement in the fractional alcohol precipitation of the present invention.

[0075] When preparing the yellow water polysaccharide in the present invention, the centrifugation is carried out at 6000 - 10000×g for 8 - 12 min. Two centrifugations are required for preparing precipitate 1, and one centrifugation is required respectively when preparing precipitate 2 - precipitate n. Each time during centrifugation, the parameters can be the same or different. When preparing the yellow water polysaccharide in the present invention, the low-temperature placement is carried out at 4 - 6 °C for 24 - 36 h. One low-temperature placement is required respectively when preparing precipitate 1 - precipitate n. Each time during low-temperature placement, the parameters can be the same or different.

[0076] When preparing the yellow water polysaccharide in the present invention, the water re-dissolution is as follows: dissolve precipitate 1 - precipitate n with 2 - 3 times the volume of water; the degreasing is as follows: wash successively with ethanol and acetone to remove lipids. When preparing the yellow water polysaccharide, the deproteinization is as follows: use Sevag reagent (CHCl 3 / nBuOH = 4:1, v / v) to deproteinize until no white protein appears. When preparing the yellow water polysaccharide, the dialysis is as follows: use a 3500 kd dialysis bag to dialyze in water at 4 - 6 °C for 72 - 84 h, and change the water every 4 - 8 h during this period. When preparing the yellow water polysaccharide, the freeze-drying is as follows: carry out freeze-drying at -60 - -50 °C for 24 - 48 h.

[0077] The sulfated modified yellow water polysaccharide of the present invention is not limited to the yellow water polysaccharide obtained by fractional alcohol precipitation as described above, and common yellow water polysaccharides in the art are all applicable; however, the yellow water polysaccharide obtained by the above fractional alcohol precipitation is a polysaccharide component with a relatively single composition and high purity. If it is to be applied subsequently, generally no complex purification steps are required. For example, in the present invention, it can be directly used for sulfation modification without purification; or different polar polysaccharides can be obtained only by anion exchange chromatography separation without complex purification steps.

[0078] The present invention will be further described in detail below through examples, but the protection scope of the present invention is not limited to the scope of the examples. The raw materials and equipment used in the examples of the present invention are all known products and are obtained by purchasing commercially available products.

[0079] In the present invention, unless otherwise specified, the raw materials to be prepared are all commercially available products well-known to those skilled in the art.

[0080] Example 1

[0081] Preparation of Huangshui polysaccharide:

[0082] After centrifuging the Huangshui sample at 8000×g for 10 min, collect the supernatant, add absolute ethanol to make the final volume concentration of ethanol 40% (v / v), place it at 4 °C for 24 h, then centrifuge at 8000×g for 10 min to obtain the precipitate, labeled as 40%;

[0083] The supernatant after taking the 40% precipitate above is rotary evaporated under reduced pressure (-0.1 MPa, 55 °C) until the ethanol is completely volatilized, add absolute ethanol to make the final volume concentration of ethanol 60% (calculated according to the actual remaining volume after rotary evaporation), perform ethanol precipitation at 4 °C for 24 h, then take the precipitate, labeled as 60%;

[0084] The supernatant after taking the 60% precipitate above is rotary evaporated under reduced pressure (-0.1 MPa, 55 °C) until the ethanol is completely volatilized, add absolute ethanol to make the final volume concentration of ethanol 80% (calculated according to the actual remaining volume after rotary evaporation), perform ethanol precipitation at 4 °C for 24 h, then take the precipitate, labeled as 80%;

[0085] Take the precipitates collected three times and dissolve them in water respectively, then wash them successively with ethanol and acetone to remove lipids, and then use Sevag reagent (CHCl 3 / BuOH = 4:1, v / v) to deproteinize three times. Finally, dialyze the mixed solution with a 3500 kd dialysis bag in deionized water at 4 °C for 72 h, change the water every 4 h during this period, and freeze-dry to obtain three kinds of Wuliangye Huangshui crude polysaccharides, labeled as HSP40, HSP60 and HSP80.

[0086] Preparation of sulfated Huangshui polysaccharide: Sulfate the above HSP40.

[0087] Dropwise add chlorosulfonic acid to pyridine (the volume ratio of chlorosulfonic acid to pyridine is 1:2), stir for 30 min each in an ice bath and at room temperature to obtain a sulfating reagent; dropwise add the sulfating reagent (10 mL) to a 10 mL anhydrous formamide solution (10 mg / mL) of Huangshui polysaccharide HSP40, magnetically stir at 70 °C for 2.5 h for sulfation modification, neutralize the obtained product with 4M NaOH solution to neutrality, perform ethanol precipitation with absolute ethanol for 12 h, after redissolving in water, dialyze and freeze-dry the obtained product to obtain the sulfated modified Huangshui polysaccharide, denoted as S 1:2 -HSP40;

[0088] To obtain sulfated Huangshui polysaccharides with other degrees of substitution, dropwise add chlorosulfonic acid to pyridine (the volume ratio of chlorosulfonic acid to pyridine is 1:4 respectively), and the subsequent operations are the same as above to obtain another degree of substitution of sulfated Huangshui polysaccharide, denoted as S 1:4 -HSP40.

[0089] According to the above method, replace HSP40 with HSP60 or HSP80 to obtain S 1:2 - HSP60 and S 1:4 - HSP60, S 1:2 - HSP80 and S 1:4 - HSP80.

[0090] Characterization and testing

[0091] 1. Infrared spectroscopy (FTIR) characterization

[0092] Select 2 mg of the dried sample and analyze it using the ATR mode. The wavenumber range is 400 - 4000 cm -1 , the spectrometer resolution is 4 cm -1 , the signal-to-noise ratio is 50000:1, and scan 64 times.

[0093] Figure 1 For HSP40, S prepared in Example 1 1:2 - HSP40, S 1:4 - HSP40, HSP60, S 1:2 - HSP60, S 1:4 - HSP60 and HSP80, S 1:2 - HSP80, S 1:4 - FT-IR spectra of HSP80. From Figure 1 It can be seen that the spectra of HSP40, HSP60, HSP80, and S 1:2 - HSP40, S 1:4 - HSP40, S 1:2 - HSP60, S 1:4 - HSP60, S 1:2 - HSP80, S 1:4 - The spectral basic profiles of HSP80 are similar, and the typical peaks of polysaccharides near 3410 cm -1 , 2930 cm -1 and 11420 cm -1 are clearly visible. They are due to intermolecular hydrogen bonds, C-H stretching of CH 2 groups, asymmetric stretching and symmetric stretching vibrations of C=O in carboxylic acid (-COO-) groups respectively. In the sulfated modified Huangshui polysaccharides, characteristic absorption peaks of sulfate groups were detected, which are due to S=O stretching vibration and C=O=S symmetric stretching vibration at 1250 cm -1 and 820 cm -1 respectively. It indicates that the six sulfated polysaccharides are successfully modified.

[0094] 2. Determination of monosaccharide composition

[0095] The acetylated derivatives of polysaccharides were prepared by hydrolysis, reduction and acetylation methods for GC-MS analysis. GC-MS conditions: RXI-5SIL MS chromatographic column 30m×0.25mm×0.25mm; The programmed temperature conditions were as follows: the initial temperature was 120°C, and it was heated to 250°C at a rate of 3°C / min; it was held for 5 min; the inlet temperature was 250°C, the detector temperature was 250°C / min, the carrier gas was helium, and the flow rate was 1 mL / min.

[0096] Figure 2 For the HSP40 prepared in Example 1 ( Figure 2 A) and S 1:2 -HSP40 ( Figure 2 B) and S 1:4 -HSP40 ( Figure 2 C) monosaccharide composition diagrams, Table 1 shows the monosaccharide composition. As shown in the figure, there are no other monosaccharide peaks in the liquid chromatogram except for the 10 monosaccharides, indicating that all the monosaccharides in the yellow water polysaccharide sample have been detected. The chromatogram baseline is stable, and the peak shape is symmetrical and sharp, indicating that this method can be used to detect the monosaccharide composition of yellow water polysaccharides. As shown in the figure, the monosaccharide compositions of the three polysaccharides are basically the same, and all of them contain mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, xylose, galactose and arabinose. Among them, the higher contents of HSP40 are glucose and mannose, and S 1:2 -HSP40 and S 1:4 -HSP40 contain more glucose and arabinose.

[0097] Table 1 Monosaccharide composition of three polysaccharides

[0098]

[0099] 3. Determination of molecular weight

[0100] High performance gel permeation chromatography (HPGPC, Shimadzu Corporation, Kyoto, Japan) equipped with an LC high performance liquid chromatography pump, a RID-20 refractive index detector and a 7725i manual syringe (Rheodyne L.P. Corporation, USA) was used to identify the average molecular weight. A TSK gel GMPWXL column (Tosoh Bioscience, Japan) was used for sample separation. Polysaccharide concentration: 2.0 mg / mL; Mobile phase: containing 0.1% NaNO 3 and 0.06% NaN 3Distilled water; flow rate: 0.8 mL / min; temperature: 40 °C; injection volume: 20 μL. Calibration curves were generated using T-series dextran standards with molecular weights of 5.20 kDa, 11.60 kDa, 23.80 kDa, 48.60 kDa, 148.00 kDa, 273.00 kDa, 410.00 kDa, and 668.00 kDa.

[0101] Figure 3 For the HSP40 prepared in Example 1 ( Figure 3 A) and S 1:2 -HSP40 ( Figure 3 B) and S 1:4 -HSP40 ( Figure 3 C), the molecular weight distribution diagrams show that the polysaccharide HSP40 in yellow water polysaccharide contains three types of polysaccharides with molecular weights including 11441459 Da, 303844 Da, and 8566 Da; polysaccharide S 1:2 -HSP40 contains two types of polysaccharides with molecular weights including 14142 Da and 3521 Da; polysaccharide S 1:4 -HSP40 contains two types of polysaccharides with molecular weights including 64909 Da and 4012 Da. After sulfation modification, the molecular weight decreases, which may be because under acidic conditions, the polysaccharide chain undergoes hydrolysis and the molecular weight decreases.

[0102] Table 2 Molecular weight conditions of three polysaccharides

[0103]

[0104]

[0105] 4. Degree of substitution

[0106] Preparation of solutions: 0.5% gelatin solution: 0.5 g of gelatin was made up to 100 mL with ultrapure water; 0.5% barium chloride-gelatin solution: 0.5 g of barium chloride was dissolved in 100 mL of gelatin solution.

[0107] Preparation of standard solutions: 0.1088 g of potassium sulfate dried to constant weight was accurately weighed and made up to 100 mL with ultrapure water. 1, 2, 3, 4, 5, and 6 mL of the above standard solution were respectively taken and made up to 10 mL in a volumetric flask to obtain a series of potassium sulfate standard curves with concentration gradients of 60, 120, 180, 240, 300, and 360 μg / mL.

[0108] Determination of standard curves:

[0109] Using the barium chloride gelatin method, 1.0 mL of each concentration of K 2 SO 4The standard solution was placed in a stoppered test tube. 1.0 mL of 1 moL / L HCl solution and 0.5 mL of 0.5% barium chloride - gelatin solution were respectively added thereto. After the above three solutions were shaken and mixed evenly, they were subjected to a constant temperature reaction in a water bath at 25 °C for 20 min. After the reaction ended, the absorbance value A1 of each tube was quickly measured at 320 nm. At the same time, an equal volume of gelatin solution was used to replace the barium chloride - gelatin solution, and the same amount of K 2 SO 4 The standard solution and the HCl solution were mixed and reacted in a water bath, and the absorbance value A0 at 320 nm was measured. With the absorbance value A1 - A0 as the ordinate and the concentration of sulfate ion as the abscissa, a standard curve was made.

[0110] Sample determination:

[0111] Precisely weigh 5 mg of yellow water polysaccharide or sulfated derivative of yellow water polysaccharide, and hydrolyze it in a sealed tube with dilute hydrochloric acid at 100 °C for 12 h. After the hydrolysis was completed, wait for the hydrolysis solution to return to room temperature, accurately transfer 0.1 mL of the above solution, and refer to the measurement method of the standard curve to obtain the absorbance value of the sample solution at 320 nm. According to the standard curve, calculate the content of sulfate groups in the sample and convert it into the degree of substitution according to the following formula.

[0112] DS = (1.62 × S%) / (32 - 1.02 × S%)

[0113] Among them, DS is the degree of substitution, and S% is the mass fraction of S.

[0114] Figure 4 It is the standard curve for the determination of the degree of substitution by the barium chloride - gelatin method. The degree of substitution of Example 1 was calculated through this standard curve. Sulfated yellow water polysaccharide S 1:2 -HSP40 has a degree of substitution of sulfate groups reaching 0.625, and sulfated yellow water polysaccharide S 1:4 -HSP40 has a degree of substitution of sulfate groups reaching 0.56, indicating that the sulfation modification was successful.

[0115] 5. Determination of protein content

[0116] The neutral sugar content of yellow water polysaccharide and its sulfated derivative was determined by the method of using a BCA protein concentration assay kit.

[0117] Figure 5 It is the standard curve graph for the determination of protein content by the Coomassie brilliant blue method. The protein content in Example 1 was calculated through this standard curve. The protein content of HSP40 is 2.81%. After sulfation modification, the protein content of S 1:2 -HSP40 is 2.32%, and the protein content of S 1:4 -HSP40 is 2.20%.

[0118] 6. Determination of neutral sugar content

[0119] The neutral sugar content of the yellow water polysaccharide and its sulfated derivatives was determined by the phenol-sulfuric acid method.

[0120] Preparation of phenol solution: Weigh 5 g of phenol precisely, dissolve it in an appropriate amount of ultrapure water, and make up the volume to 100 mL with a volumetric flask to obtain a 5% phenol solution.

[0121] Preparation of glucose standard curve: Weigh 10.2 mg of glucose precisely and make up the volume to 100 mL. Respectively pipette 2, 4, 6, 8 mL of the standard solution into 10-mL volumetric flasks, dilute to the mark, and shake well to prepare a series of glucose standard solutions with concentrations of 20.4, 40.8, 61.2, 81.6, 102 μg / mL. Respectively pipette 2 mL of the above standard solutions into 10-mL stoppered test tubes, accurately add 1 mL of 5% phenol solution, shake well, slowly add 5.0 mL of concentrated sulfuric acid, shake well, heat in a water bath at 90 °C for 20 min. After cooling to room temperature, measure the absorbance value at 495 nm. Taking the glucose concentration (X) as the abscissa and the absorbance value (Y) as the ordinate, draw the standard curve.

[0122] Sample determination: Precisely prepare a 1 mg / mL sample solution of the yellow water polysaccharide and its sulfated yellow water polysaccharide. Pipette 2 mL of the test solution and place it in a 10-mL stoppered test tube. Referring to the determination method of the standard curve, add phenol and concentrated sulfuric acid successively. After heating in a water bath, measure the absorbance value A1 at 495 nm. Calculate the neutral sugar content of the sample according to the standard curve.

[0123] Figure 6 It is the standard curve graph for the determination of neutral sugar content by the phenol-sulfuric acid method. Calculate the neutral sugar content in Example 1 through this standard curve. The results of the phenol-sulfuric acid method show that the neutral sugar content of HSP40 is 93.61%. After sulfated modification, the neutral sugar content of S 1:2 -HSP40 is 70.62%, and the sugar content of S 1:4 -HSP40 is 80.80%.

[0124] 7. Determination of uronic acid content

[0125] The uronic acid content of the yellow water polysaccharide and its sulfated derivatives was determined by the carbazole-sulfuric acid method.

[0126] Preparation of carbazole-ethanol solution: Weigh 50 mg of carbazole precisely and dissolve it in 50 mL of 95% ethanol to obtain a 0.1% carbazole-ethanol solution;

[0127] Preparation of standard curve: Accurately weigh 14.8 mg of galacturonic acid and make up the volume to 100 mL, place it in a 100 mL volumetric flask, add distilled water to make up the volume, and prepare a standard stock solution of 148 μg / mL. Respectively pipette 2, 4, 6, 8 mL into 10 mL volumetric flasks, and add ultrapure water to make up the volume to obtain a series of standard solutions of galacturonic acid with concentrations of 29.6, 59.2, 88.8, 118.4, 148 μg / mL. Accurately pipette 1 mL of the above standard solution into a 10 mL stoppered test tube. Under the condition of ice-water bath, slowly add 6 mL of concentrated sulfuric acid, shake well and mix evenly. Then, react all the test tubes in a water bath at 85 °C for 20 min. After cooling to room temperature, accurately pipette 0.2 mL of carbazole ethanol solution into the test tube. React at room temperature for 2 h, and measure the absorbance value A520 at 520 nm. Take the galacturonic acid concentration (X) as the abscissa and the absorbance value (Y) as the ordinate to draw the standard curve.

[0128] Figure 7 It is the standard curve diagram for the determination of uronic acid content by the carbazole-sulfuric acid method. Through this standard curve, the neutral sugar content in Example 1 can be calculated. The uronic acid content of HSP40 determined by the carbazole-sulfuric acid method is 0.90%. After sulfation modification, 1:2 the uronic acid content of S-HSP40 is 0.52%, 1:4 the uronic acid content of S-HSP40 is 0.61%. The difference before and after sulfation modification is not significant, indicating that sulfation modification does not drastically change the basic components of the polysaccharide.

[0129] The main component of yellow water polysaccharide is neutral sugar, and the contents of protein and uronic acid are relatively low. The main components are shown in Table 3.

[0130] Table 3 Main components of three polysaccharides

[0131] Polysaccharide sample Protein Neutral sugar Uronic acid HSP40 2.80％ 93.61％ 0.90％ <![CDATA[S 1:2 -HSP40]]> 2.30％ 70.62％ 0.52％ <![CDATA[S 1:4 -HSP40]]> 2.20％ 80.80％ 0.61％

[0132] 8. Determination of DPPH free radical scavenging rate

[0133] Prepare polysaccharide sample solutions with mass concentrations of 0.5, 1, 1.5, 2 mg / mL. Take 2 mL of each concentration sample solution and add 2 mL of DPPH solution with a mass concentration of 0.04 mg / mL, shake well and place in the dark for 30 min, and measure the absorbance value at 517 nm.

[0134]

[0135] Figure 8 For HSP40 and sulfated yellow water polysaccharide (S 1:4 -HSP40 and S 1:2-HSP40) DPPH free radical scavenging rate curve. Free radical scavenging activity is an important antioxidant capacity of natural active polysaccharides. The DPPH method is a classic experimental method for evaluating the antioxidant activity of polysaccharides in vitro. DPPH is purple in ethanol solution and can exist stably, but it needs to be stored at low temperature in the dark. It has a single electron, so it can accept an electron or a hydrogen ion and has the maximum absorption at a wavelength of 517 nm. When there is a free radical scavenger, the single electron of DPPH is captured and its color becomes lighter. Therefore, the level of polysaccharide free radical scavenging activity can be evaluated by the degree of decrease in its absorbance value. This antioxidant capacity is expressed by the inhibition rate. The greater the inhibition rate, the stronger the antioxidant property.

[0136] In this experiment, the yellow water polysaccharide HSP40 and its sulfated derivatives (S 1:4 -HSP40 and S 1:2 -HSP40) both have significant scavenging activities against DPPH free radicals. The DPPH free radical scavenging ability was found to be dose-dependent in all samples. Moreover, the antioxidant activity of the sulfated yellow water polysaccharide derivatives is higher than that of the yellow water polysaccharide. In particular, the antioxidant activity of S 1:2 -HSP40 is significantly improved. These results indicate that the addition of sulfate groups promotes the scavenging ability against DPPH free radicals.

[0137] In summary, sulfated modification of yellow water polysaccharide enhances the antioxidant property of yellow water polysaccharide, improves the utilization value of yellow water, and provides a new idea for the high-value utilization of yellow water.

Claims

1. Preparation method of sulfated yellow water polysaccharide, Characterized in that: The sulfated yellow water polysaccharide is a sulfated derivative of yellow water polysaccharide, obtained by sulfation reaction of yellow water polysaccharide; Comprising the following steps: A. Prepare a sulfation reagent: Mix pyridine and chlorosulfonic acid to obtain a sulfation reagent; B. Prepare a homogeneous solution of yellow water polysaccharide: Dissolve yellow water polysaccharide in an organic solvent to obtain a homogeneous solution of yellow water polysaccharide; C. Mix the sulfation reagent obtained in step A and the homogeneous solution of yellow water polysaccharide obtained in step B, heat for sulfation reaction, after the reaction is completed, neutralize the system to neutrality, then carry out alcohol precipitation to obtain a precipitate, and the obtained precipitate is successively redissolved in water, dialyzed and freeze-dried to obtain sulfated yellow water polysaccharide; In step A, the volume ratio of pyridine to chlorosulfonic acid is 1-2:1; In step C, control the volume ratio of the sulfation reagent to the homogeneous solution of yellow water polysaccharide to be 1:0.5-2; In step B, the yellow water polysaccharide is prepared by the following method: a. After centrifuging the yellow water, collect the supernatant, add ethanol to the supernatant to make the final volume concentration of ethanol in the system X 1 , after standing at low temperature, centrifuge again to obtain precipitate 1 and supernatant 1; b. Concentrate the supernatant 1 until the ethanol is completely volatilized, then add ethanol to make the final volume concentration of ethanol in the system X 2 , after standing at low temperature, centrifuge again to obtain precipitate 2 and supernatant 2; c. Repeat the operation in step b, concentrate the supernatant obtained in the previous time until the ethanol is completely volatilized, then add ethanol to make the final volume concentration of ethanol in the system X 3 ~X n , after standing at low temperature, centrifuge again to obtain precipitate 3 to precipitate n and supernatant 3 to supernatant n respectively; d. Redissolve precipitate 1 to precipitate n in water successively, degrease, deproteinize, dialyze and freeze-dry to obtain yellow water polysaccharide 1 to yellow water polysaccharide n; Among them, X 1 ~X n is selected from 40% to 80%, and gradually increases from X 1 to X n ; The yellow water polysaccharide used in step B is selected from at least one of yellow water polysaccharide 1 to yellow water polysaccharide n.

2. The preparation method of sulfated yellow water polysaccharide according to claim 1, Characterized in that: At least one of the following is satisfied: In step B, the concentration of the homogeneous solution of yellow water polysaccharide is 5-20 mg / mL; In step B, the organic solvent is at least one of formamide, N,N-dimethylformamide, dimethyl sulfoxide or dimethylacetamide.

3. The preparation method of sulfated yellow water polysaccharide according to claim 1, Characterized in that: At least one of the following is satisfied: In step C, add the sulfation reagent dropwise to the homogeneous solution of yellow water polysaccharide; In step C, the temperature of the sulfation reaction is 60-80 °C; In step C, the time of the sulfation reaction is 2-4 h.

4. The preparation method of sulfated yellow water polysaccharide according to claim 1, Characterized in that: At least one of the following is satisfied: In step C, use 2-5 M NaOH solution to neutralize the system to neutrality; In step C, the operation of alcohol precipitation is: Mix the neutralized system with ethanol 4-6 times the volume of the neutralized system for precipitation; In step C, the time of alcohol precipitation is 12-15 h; In step C, the operation of redissolving in water is: Dissolve the precipitate obtained by alcohol precipitation with water 2-4 times the volume of the precipitate; In step C, the operation of dialysis is: Dialyze the redissolved system with a 3500 kd dialysis bag in water at 4-6 °C for 72-84 h, and change the water every 4-8 hours during this period; In step C, the temperature of freeze-drying is -60 to -50 °C; In step C, the time of freeze-drying is 24-48 h.

5. The preparation method of sulfated yellow water polysaccharide according to claim 1, Characterized in that: X 1 to X n spaced apart from each other by not less than 5%.

6. The preparation method of sulfated yellow water polysaccharide according to claim 1, Characterized in that: At least one of the following is satisfied: When preparing the yellow water polysaccharide, the centrifugation is carried out at 6000-10000×g for 8-12 min; When preparing the yellow water polysaccharide, the low-temperature placement is carried out at 4-6°C for 24-36 h; When preparing the yellow water polysaccharide, the water re-dissolution is as follows: dissolving precipitate 1 to precipitate n with 2-3 times the volume of water; When preparing the yellow water polysaccharide, the defatting is as follows: sequentially washing with ethanol and acetone to remove lipids; When preparing the yellow water polysaccharide, the deproteinization is as follows: deproteinizing with Sevag reagent until no white protein appears; When preparing the yellow water polysaccharide, the dialysis is as follows: dialyzing with a 3500 kd dialysis bag in water at 4-6°C for 72-84 h, and changing the water every 4-8 h during this period; When preparing the yellow water polysaccharide, the freeze-drying is as follows: freeze-drying at -60 to -50°C for 24-48 h.

7. Use of the sulfated yellow water polysaccharide prepared according to any one of claims 1 to 6 in the preparation of an antioxidant.

8. According to the use described in claim 7, it is characterized in that: the antioxidant is a DPPH free radical scavenger.

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