Method for synchronously extracting and separating polysaccharide, protein and grease in idesia oil meal

Abstract

The embodiment of the invention discloses a method for synchronously extracting and separating polysaccharide, protein and grease in idesia oil meal, which comprises the following steps: uniformly mixing the idesia oil meal with an inorganic salt solution, adding dimethyl carbonate, and uniformly mixing to obtain a mixture; performing ultrasonic treatment, shaking and centrifuging on the mixture to form an upper phase solution with grease, a middle phase solution with protein and a lower phase solution with polysaccharide; carrying out reduced pressure evaporation on the upper phase solution to obtain a grease extract, and recovering dimethyl carbonate for recycling; dissolving the middle-phase solution in a buffer solution, dialyzing and drying to obtain a protein extract; and dialyzing and drying the lower phase solution to obtain the polysaccharide extract. The method disclosed by the invention is simple, efficient, economical and green, can be used for industrially and synchronously extracting and separating the grease, the protein and the polysaccharide in the idesia oil meal, and is high in product yield and good in quality. In addition, the extracted polysaccharide extract is high in thermal stability and has remarkable inhibitory activity on alpha-glucosidase and non-enzymatic glycosylation.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for synchronously extracting and separating polysaccharides, proteins and oils in tung oil cake. Background Art

[0002] Idesia polycarpa Maxim, also known as oil grape, water winter melon, water winter tung, doushuanghong, fengshuanghong, mountain sycamore, chair tree, chair tung, etc., is a deciduous tree of the genus Idesia in the family Flacourtiaceae. It has strong adaptability, fast growth, easy planting and promotion, and is distributed in Zhejiang, Anhui, Fujian, Jiangxi, Shandong, Hunan, Sichuan, Guizhou, Yunnan and other places. The fruit of Idesia polycarpa Maxim has a high oil content and is rich in polyunsaturated fatty acids. It is a high-quality and high-yield woody edible oil tree species.

[0003] As the raw material for oil production, the large amount of oil cake produced after oil pressing by the tung oil nut fruit is still in the initial development stage, resulting in only a very small amount of tung oil cake used as compost, and a large amount of tung oil cake is still in the state of being developed and utilized, resulting in the waste of tung oil cake resources and affecting the development of the entire tung oil industry chain. Therefore, the lack of high-value utilization technology of tung oil cake and the lack of relevant high-value-added product development and creation capabilities have become bottlenecks restricting the upgrading and development of the tung oil industry.

[0004] In the traditional production process, before extracting protein and polysaccharide, it is necessary to defat first, otherwise it will affect the subsequent utilization of polysaccharide and protein, and then continue to separate protein and polysaccharide. The traditional process is complicated, requires a large amount of organic reagents, has low extract recovery rate and low efficiency. Therefore, it is necessary to develop a simple, efficient, green, economical and industrializable extraction and separation technology for tung oil meal, protein and polysaccharide. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art and provide a method for synchronously extracting and separating polysaccharides, proteins and oils in Castanopsis schrenkiana oil cake.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The method for simultaneously extracting and separating polysaccharides, proteins and oils from tung oil cake comprises the following steps:

[0008] Mix the tung oil cake with the inorganic salt solution, add dimethyl carbonate, and mix well to obtain a mixture;

[0009] The mixture is sonicated, shaken, and centrifuged to form a three-phase solution including an upper phase solution containing oil, a middle phase solution containing protein, and a lower phase solution containing polysaccharide;

[0010] The upper phase solution is evaporated under reduced pressure to obtain a fat extract, and dimethyl carbonate is recovered for recycling;

[0011] The middle phase solution is dissolved in a buffer solution, dialyzed, and dried to obtain a protein extract;

[0012] The lower phase solution was dialyzed and dried to obtain the polysaccharide extract.

[0013] The invention adopts ultrasound combined with three-phase extraction technology to simultaneously extract and separate oil, protein and polysaccharide in tung oil cake. Under the joint action of ultrasound, salting out, plasma precipitation, co-solvent precipitation and the like, water, solid salt and green environmentally friendly solvent dimethyl carbonate are added to the tung oil cake and fully mixed to form three phases. The upper phase is evaporated under reduced pressure to obtain oil, and the dimethyl carbonate can be recovered for recycling. The middle phase is dissolved in a buffer solution, dialyzed and dried to obtain refined protein, and the lower phase is dialyzed and dried to obtain refined pectin polysaccharide.

[0014] The ultrasound combined three-phase extraction technology of the present invention is used as a novel and environmentally friendly alternative to extract and utilize valuable food functional components in the tung oil meal. The technology not only improves the extraction efficiency, but also reduces environmental pollution, so that the useful components in the tung oil meal can be more comprehensively and effectively utilized.

[0015] In the present invention, dimethyl carbonate and inorganic salt form a three-phase water system, and the main factors affecting the extraction yield include the type of inorganic salt, solid-liquid ratio, inorganic salt concentration, pH, ultrasonic time, temperature and the volume of dimethyl carbonate.

[0016] Preferably, the concentration of the inorganic salt solution is 10-50%, w / v; the pH is 2-9; and the solid-liquid ratio of the Castanopsis schrenkiana oil cake to the inorganic salt solution is 1:15-40.

[0017] Preferably, the added amount of dimethyl carbonate is 5 to 25 mL.

[0018] Preferably, the mixture is ultrasonicated for 10 to 50 minutes, and then the mixture is placed in a constant temperature oscillator at a temperature of 20-45°C and shaken for 30 minutes. After shaking, the mixture is centrifuged at 4000 rpm for 15 minutes to form a three-phase liquid of upper phase solution, middle phase solution and lower phase solution.

[0019] Preferably, the inorganic salt solution is an ammonium citrate solution with a concentration of 30% (m / v) and a pH of 3; the solid-liquid ratio of the tung oil cake to the ammonium citrate solution is 1:26; the ultrasonic time is 31 min, the temperature is 30° C., and the volume of dimethyl carbonate is 15 mL.

[0020] The present invention utilizes single factor test (inorganic salt type, solid-liquid ratio, inorganic salt concentration, pH, ultrasonic time, temperature and volume of dimethyl carbonate) and response surface experiment to obtain the best extraction process conditions.

[0021] Preferably, the lower phase solution is adjusted to pH 6.5 with 1 mol / L sodium hydroxide solution, concentrated to 1 / 3 of the original volume, and then dialyzed for 48 hours with tap water in a dialysis bag with a molecular weight cutoff of 8000-14000Da; 4 times the volume of anhydrous ethanol is slowly added under stirring, and alcohol precipitation is carried out at 4°C overnight; centrifugation is performed to collect the precipitate; then the precipitate is washed twice with anhydrous ethanol and redissolved in 50-60°C distilled water, and insoluble impurities are removed by centrifugation again; and finally vacuum freeze-drying is performed to obtain a polysaccharide extract.

[0022] Preferably, the middle phase solution is dissolved in 0.05 mol / L Tris-HCl buffer at pH 8.0, placed at 4°C for more than 12 hours, dialyzed with tap water in a dialysis bag with a molecular weight cutoff of 8000 to 14000 Da, and the dialysate is freeze-dried to obtain a protein extract.

[0023] The present invention also provides a polysaccharide extract obtained by the method for simultaneously extracting and separating polysaccharides, proteins and oils in Castanopsis japonica oil cake as described above.

[0024] The present invention determines the chemical composition (total sugar, protein, uronic acid, degree of methylation), main structural characteristics (molecular weight, monosaccharide composition, triple helix structure, thermal stability, crystal structure, etc.), in vitro hypoglycemic and anti-glycosylation activities of the pectin polysaccharide of the tung oil meal prepared by ultrasound-three-phase extraction; determines the fatty acid composition of the oil in the tung oil meal prepared by ultrasound-three-phase extraction; determines the amino acid composition of the protein of the tung oil meal prepared by ultrasound-three-phase extraction. This study provides key data for the potential commercial application and high value-added utilization of the tung oil meal.

[0025] Chemical analysis of the oil extract of the present invention determines that linoleic acid is the main unsaturated fatty acid. The protein extract of the present invention is characterized by typical plant seed protein, in which glutamic acid, aspartic acid and serine are the most abundant amino acids. The polysaccharide extract is mainly composed of RG-I pectin with a molecular weight of 226.58kDa, has a triple helical structure, strong thermal stability, and has significant inhibitory activity against α-glucosidase and non-enzymatic glycosylation. Therefore, the present invention also provides an application of the polysaccharide extract as described above in the preparation of α-glucosidase inhibitors.

[0026] The present invention also provides a use of the polysaccharide extract as described above in the preparation of an anti-glycosylation agent.

[0027] The polysaccharide extract of the present invention inhibits α-glucosidase through a mixed mechanism, quenching the fluorescence of the enzyme mainly through static interaction and binding at a single site, highlighting the potential of the polysaccharide extract as a new type of α-glucosidase inhibitor and anti-glycosylation agent, suitable for functional foods and dietary supplements.

[0028] The beneficial effects of the present invention compared with the prior art are:

[0029] (1) The present invention is a simple, efficient, green, economical, and industrializable synchronous extraction and separation technology of fat, protein, and polysaccharide from ash tree oil cake. The method adopts ultrasound combined with three-phase extraction technology to simultaneously extract fat, protein, and polysaccharide from ash tree oil cake. Under the joint action of ultrasound, salting out, plasma precipitation, co-solvent precipitation, etc., water, solid salt, and green environmentally friendly solvent methyl carbonate are added to ash tree oil cake and fully mixed to form three phases. The upper phase is evaporated under reduced pressure to obtain fat, and dimethyl carbonate is recycled after recovery. The middle phase is dissolved in a buffer solution and dialyzed and dried to obtain refined protein, and the lower phase is dialyzed and dried to obtain refined polysaccharide. Finally, a simple, efficient, economical, green, and industrializable synchronous extraction and separation of fat, protein, and polysaccharide from ash tree oil cake is achieved, and the product yield is high and the quality is good.

[0030] (2) The polysaccharide extract obtained by the present invention has strong thermal stability and has significant inhibitory activity against α-glucosidase and non-enzymatic glycosylation. It can be used as a new type of α-glucosidase inhibitor and anti-glycosylation agent, and is suitable for functional foods and dietary supplements.

[0031] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a schematic diagram of the process of this embodiment;

[0033] Figure 2 The effects of extraction parameters on the yields of Alpinia trefolia pectin polysaccharide IPMPP and Alpinia trefolia oil meal protein IPMP (where A is the effect of inorganic salt types, B is the effect of ammonium citrate concentration, C is the effect of solid-liquid ratio, D is the effect of pH, E is the effect of ultrasonic time, F is the effect of temperature, and G is the effect of dimethyl carbonate volume);

[0034] Figure 3 It is the IPMPP response surface analysis (in the figure, A, B, C, and D are X1, X2, X3, and X4 respectively; A is the relationship between the actual extraction rate and the predicted extraction rate of IPMPP under the optimal extraction conditions; B is the normal probability plot of the residual; C is the relationship between the studentized residual and the predicted response; and D is the perturbation plot of the influence of each factor on the yield);

[0035] Figure 4 It is the response surface analysis of IPMP (in the figure, A, B, C, and D are X1, X2, X3, and X4 respectively; A is the relationship between the actual extraction rate and the predicted extraction rate of IPMP under the optimal extraction conditions; B is the normal probability plot of the residual; C is the relationship between the studentized residual and the predicted response; and D is the perturbation plot of the influence of each factor on the yield);

[0036] Figure 5 Figure 1 is the IPMPP response surface analysis (A is the contour map of the interaction between inorganic salt concentration (X1) and solid-liquid ratio (X2) on IPMPP yield; B is the contour map of the interaction between inorganic salt concentration (X1) and pH value (X3) on IPMPP yield; C is the contour map of the interaction between inorganic salt concentration (X1) and ultrasonic time (X4) on IPMPP yield; D is the contour map of the interaction between solid-liquid ratio (X2) and pH value (X3) on IPMPP yield; E is the contour map of the interaction between solid-liquid ratio (X2) and ultrasonic time (X4) on IPMPP yield; F is the contour map of the interaction between pH value (X3) and ultrasonic time (X4) on IPMPP yield). (G) is the response surface diagram of the interaction between inorganic salt concentration (X1) and solid-liquid ratio (X2) on IPMPP yield; H is the response surface diagram of the interaction between inorganic salt concentration (X1) and pH value (X3) on IPMPP yield; I is the response surface diagram of the interaction between inorganic salt concentration (X1) and ultrasonic time (X4) on IPMPP yield; J is the response surface diagram of the interaction between solid-liquid ratio (X2) and pH value (X3) on IPMPP yield; K is the response surface diagram of the interaction between solid-liquid ratio (X2) and ultrasonic time (X4) on IPMPP yield; L is the response surface diagram of the interaction between pH (X3) and ultrasonic time (X4) on IPMPP yield);

[0037] Figure 6Figure 1 is the response surface analysis of IPMP, (where A is the contour map of the interaction between inorganic salt concentration (X1) and solid-liquid ratio (X2) on IPMP yield; B is the contour map of the interaction between inorganic salt concentration (X1) and pH value (X3) on IPMP yield; C is the contour map of the interaction between inorganic salt concentration (X1) and ultrasonic time (X4) on IPMP yield; D is the contour map of the interaction between solid-liquid ratio (X2) and pH value (X3) on IPMP yield; E is the contour map of the interaction between solid-liquid ratio (X2) and ultrasonic time (X4) on IPMP yield; F is the contour map of the interaction between pH value (X3) and ultrasonic time (X4) on IPMP yield. (G) is the response surface diagram of the interaction between inorganic salt concentration (X1) and solid-liquid ratio (X2) on IPMPP yield; (H) is the response surface diagram of the interaction between inorganic salt concentration (X1) and pH value (X3) on IPMP yield; (I) is the response surface diagram of the interaction between inorganic salt concentration (X1) and ultrasonic time (X4) on IPMP yield; (J) is the response surface diagram of the interaction between solid-liquid ratio (X2) and pH value (X3) on IPMP yield; K is the response surface diagram of the interaction between solid-liquid ratio (X2) and ultrasonic time (X4) on IPMP yield; L is the response surface diagram of the interaction between pH (X3) and ultrasonic time (X4) on IPMP yield);

[0038] Figure 7 The gas chromatograms of fatty acid standards and IPMO of tung oil (Note: 1, 2, 3, 4, 5 in the figure are palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, erucic acid, and 10.25min is the solvent peak);

[0039] Figure 8 is the amino acid composition of IPMP;

[0040] Fig. 9 Ion chromatograms of standard monosaccharides and IPMPP;

[0041] Fig.10 is the molecular weight distribution diagram of IPMPP;

[0042] Fig.11 is the particle size distribution diagram of IPMPP;

[0043] Fig.12 is the infrared absorption spectrum of IPMPP;

[0044] Fig.13 It is the Congo red reaction result of IPMPP;

[0045] Fig.14 is the X-ray diffraction pattern of IPMPP;

[0046] Fig.15 is the DSC diagram of IPMPP;

[0047] Fig.16 is the scanning electron microscope image of IPMPP;

[0048] Fig.17 The inhibitory effect of IPMPP on α-glucosidase activity;

[0049] Fig.18 Lineweaver-Burk curve of IPMPP inhibition of α-glucosidase;

[0050] Fig.19 is the fluorescence emission spectrum after the action of IPMPP and α-glucosidase;

[0051] Fig. 20 for the inhibitory effect of MPP on fructosamine;

[0052] Fig.21 for the inhibitory effect of IPMPP on α-dicarbonyl compounds;

[0053] Fig. 22 The inhibitory effect of IPMPP on AGEs;

[0054] Fig.23 Figure 2 is the fluorescence emission spectra of AG and IPMPP with AGEs. DETAILED DESCRIPTION

[0055] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0056] This embodiment is a method for simultaneously extracting and separating polysaccharides, proteins and oils from Castanopsis altissima oil cake.

[0057] Material selection: Fresh Castanopsis schrenkiana fruits were collected on November 4, 2022 in Jiubao Qiang Township, Pingwu County, Mianyang City, Sichuan Province, with coordinates of 32°20′54.18″N, 104°23′47.50″E, and 1584 meters above sea level. The fruits were dried using a commercial microwave vacuum dryer through a segmented process at a vacuum level of 0.07MPa and different microwave powers: 2kW, 50min; 1kW, 20min; 0.5kW, 10min; 0.3kW, 10min. The oil was extracted using a screw press at 110°C, and the Castanopsis schrenkiana oil meal was stored at -20°C for subsequent analysis. The oil meal components included 5.52% protein, 8.85% oil, and 10.67% water-soluble polysaccharides.

[0058] The extraction and separation steps include:

[0059] First, a certain concentration of inorganic salt solution (10-50%, w / v, pH=2-9, pH adjusted with 1M NaOH solution or HCl solution) is prepared, a certain amount of tung oil meal is weighed, 20 ml of inorganic salt solution is added according to an appropriate material-liquid ratio (1:15-1:40), and mixed. Then dimethyl carbonate (5-25 mL) is added, mixed, and ultrasonicated (10-50 min). Subsequently, the mixture is placed in a constant temperature oscillator (temperature is 20-45°C) and shaken for 30 min. After the shaking is completed, it is centrifuged at 4000 rpm for 15 minutes to form a three-phase liquid. The upper phase composed of organic solvent (dimethyl carbonate and oil in tung oil meal) is evaporated under reduced pressure to obtain the oil IPMO in the oil meal, and the dimethyl carbonate is recovered for recycling. The middle layer is mainly composed of protein. The precipitate of the middle layer is dissolved in 0.05mol / L Tris-HCl buffer (pH 8.0), placed at 4℃ for more than 12h, dialyzed with tap water in a dialysis bag with a molecular weight cutoff of 8000-14000Da, and the dialyzate is freeze-dried to obtain the oil meal protein IPMP. The lower phase is mainly composed of inorganic salts, polysaccharides and tung oil meal. The pH of the lower phase solution is adjusted to 6.5 with 1mol / L sodium hydroxide solution, and then the pectin polysaccharide sample solution is concentrated to 1 / 3 of the original volume, and then dialyzed with tap water in a dialysis bag with a molecular weight cutoff of 8000-14000Da for 48h. Slowly add 4 times the volume of anhydrous ethanol under stirring, and precipitate overnight at 4℃. Centrifuge at 4000r / min for 10min, collect the precipitate; then wash it twice with anhydrous ethanol and redissolve it in 50-60℃ distilled water, and centrifuge it again (4000r / min, 10min) to remove insoluble impurities. Finally, vacuum freeze-drying was performed to obtain pectin polysaccharide IPMPP extracted by three-phase extraction.

[0060] Single-factor experimental design of IPMP and IPMPP

[0061] The main factors affecting the extraction yield of IPMP and IPMPP include the type of inorganic salt, solid-liquid ratio, inorganic salt concentration, pH, ultrasonic time, temperature and volume of dimethyl carbonate. The scope of their effects was determined through single-factor experiments to provide data and reference for orthogonal experiments.

[0062] (1) Effect of inorganic salt ratio on the extraction rate of IPMP and IPMPP

[0063] The same concentration and pH (20%, w / v, pH=6, pH adjusted with 1M NaOH solution or HCl solution), dimethyl carbonate volume (15mL), ultrasonic time 20min, temperature 30°C were used. Different inorganic salts were added to the solution. The volumes of the middle phase and the lower phase after removing the tung oil cake were collected with a pipette to further measure the yields of IPMP and IPMPP, and to study the effects of different inorganic salts on the yields of IPMP and IPMPP.

[0064] (2) Effect of inorganic salt concentration on the extraction rate of IPMP and IPMPP

[0065] Ammonium citrate solutions of different concentrations (10%-50%, w / v, pH=6) were added, and the subsequent steps were the same as the above steps.

[0066] (3) Effect of solid-liquid ratio on the extraction rate of IPMP and IPMPP

[0067] Ammonium citrate solution was added according to different material-liquid ratios (1:20, 1:25, 1:30, 1:35 and 1:40), and the subsequent steps were the same as the above steps.

[0068] (4) Effect of pH on the extraction rate of IPMP and IPMPP

[0069] Ammonium citrate solutions of different pH values ​​(pH=2, 3, 4, 5, 6, 7, 8, 9, pH adjusted with 1 M NaOH solution or HCl solution) were added, and the subsequent steps were the same as the above steps.

[0070] (5) Effect of ultrasonic time on the extraction rate of IPMP and IPMPP

[0071] The ultrasonic time was set to (10, 20, 30, 40 and 50 min), and the subsequent steps were the same as above.

[0072] (6) Effect of temperature on the extraction rate of IPMP and IPMPP

[0073] The temperature was set to 20, 25, 30, 35, 40, and 45 °C, and the subsequent steps were the same as above.

[0074] (7) Effect of dimethyl carbonate volume on the extraction rate of IPMP and IPMPP

[0075] The volume of dimethyl carbonate was 5, 10, 15, 20, and 25 mL, and the subsequent steps were the same as above.

[0076] Response Surface Methodology (RSM) Design

[0077] The response surface methodology was used to optimize the extraction conditions of IPMP and IPMPP using a 4-factor 3-level Box-Behnken design (BBD) to obtain the maximum response value. According to the single-factor experimental results of the above-mentioned polysaccharide from Castanopsis schrenkiana oil cake, the four factors with the greatest influence were selected, and the ammonium citrate concentration (X1), material-liquid ratio (X2), pH (X3) and ultrasonic time (X4) were used as independent variables, and the response values ​​of IPMP and IPMP extraction rate were used as functions. 29 4-factor 3-level design experiments were conducted. The experimental levels and coding of the 4 factors are shown in Table 1. The range of values ​​of each extraction parameter was independently determined according to the single-factor experiment to determine the extraction conditions.

[0078] Table 1 Factor levels and codes of the Box-Behnken (BBD) experimental design for IPMPP and IPMP yields

[0079]

[0080] Analytical methods

[0081] Determination of the extraction rate of polysaccharide IPMPP, protein IPMP and oil IPMO from Castanopsis altissima

[0082] IPMPP extraction rate determination

[0083] The lower phase after removing the tung oil cake was collected and collected with a pipette to further measure the polysaccharide content in the lower phase to calculate the polysaccharide yield according to formula (2.1):

[0084]

[0085] Where M is the weight of the tung oil cake weighed (g), C L and V L are the concentration and volume (mL) of the polysaccharide solution in the lower phase at equilibrium, respectively. The polysaccharide concentration was calculated using the phenol-sulfuric acid method.

[0086] IPMP extraction rate determination

[0087] Collect the middle phase and use a pipette to further measure the protein content in the middle phase and calculate the protein yield according to formula (2.2):

[0088]

[0089] Where M is the weight of the tung oil cake weighed (g), C L is the concentration of protein solution in the lower phase at equilibrium, V L is the volume of the polysaccharide solution in the lower phase at equilibrium (mL), and the protein concentration was determined by the Coomassie Brilliant Blue method.

[0090] Determination of IPMO extraction rate

[0091] Collect the upper organic phase (mainly dimethyl carbonate and oil in the tung oil cake) and remove the organic phase by vacuum evaporation. Dry at 105℃ for 2h and calculate the oil extraction rate according to formula (2.3):

[0092]

[0093] Wherein, M1 is the mass (g) of oil after vacuum evaporation of the organic phase, and M is the mass (g) of the weighed Castanopsis schrenkiana oil cake.

[0094] Determination of uronic acid content of IPMPP

[0095] The total uronic acid content in polysaccharides was determined by aminobenzenesulfonic acid-m-hydroxybiphenyl method. Prepare 0.1mg / mL galacturonic acid standard, take 0, 50, 100, 200, 300, 400μL, add distilled water to 400μL, then add 40μL of 4mol / L potassium aminosulfonate and 2.5mL concentrated sulfuric acid, shake well, and boil in boiling water for 20min. After cooling, add 40μL of 3% (w / v) m-hydroxybiphenyl and shake well. Let stand at room temperature for 15min, measure the absorbance at 525nm, and draw a standard curve. Weigh the sample and prepare it into a 0.1mg / mL solution, take 400μL of the sample solution, measure it according to the above method, and calculate the total uronic acid content in the sample.

[0096] Determination of fatty acid composition of IPMO

[0097] The fatty acid composition was determined by methyl esterification combined with gas chromatography. Weigh 30 mg of IPMO and add 1 mL of n-hexane, then add 200 μL of 2 mol / L methanol-sodium hydroxide to prepare the fatty acid methyl esterification product. The mixture was heated in a 50 °C bath for 15 min, and then 200 μL of 2 mol / L HCl was added. Then, 1 μL of the supernatant was injected into a gas chromatograph (Agilent 8890A) equipped with a flame ionization detector (FID) and a SH-Rt2560 capillary column (60 m × 0.25 mm × 0.20 μm). The heating program was: the initial temperature was 130 °C for 5 min, then the temperature was increased to 240 °C at a rate of 4 °C / min and maintained for 20 min.

[0098] Determination of the amino acid composition of IPMP

[0099] Place an appropriate amount of sample in a digestion tube, add 3 drops of phenol, and then add 5mL of 6mol / L hydrochloric acid. Then inject nitrogen into the tube, seal it, and hydrolyze it at 100℃ for 48h. After appropriate dilution, derivatize it using the phenyl isothiocyanate (PITC) method. Add 50μL of triethylamine-acetonitrile solution (1mol / L) and 50μL of PITC acetonitrile solution (0.1mol / L) to 100μL of sample solution, mix, and react at room temperature for 2h. After the reaction, add 200μL of n-hexane, mix thoroughly, and let stand for 10min. Then collect the lower layer solution and analyze it using a high-resolution mass spectrometer (Thermo Fisher Q-Exactive) system. Shimazu InsertSustain C18 chromatographic column (4.6×250mm). The elution program was as follows: initial conditions were 10% A to 10% B, washing for 0-1 min; 10% A to 25% B, 1-1.1 min; 25% A to 30% B from 1.1-7 min; maintained at 30% A to 30% B in 7-10 min; gradually increased from 30% A to 3% B from 10-15 min; sharply increased from 33% A to 90% B from 15-15.1 min; maintained at 90% A to 90% B for 15.1-20 min; recovered to 90% A to 10% B from 20-20.1 min; and finally from 10% A to 10% B from 20.1-25 min. The mass spectrometry conditions included an ionization transfer tube temperature of 400 °C, an evaporation temperature of 500 n °C, a sheath gas pressure of 75 arb, an auxiliary gas pressure of 20 arb, a spray voltage of 3.5 kV, and monitoring of target ions.

[0100] IPMPP monosaccharide composition determination

[0101] The monosaccharide composition was determined by ion chromatography. 5 mg of polysaccharide sample was weighed into a 10 mL Abe bottle, 4 mL of trifluoroacetic acid (3M) was added, the bottle was sealed with an alcohol blowtorch, and hydrolyzed at 120°C for 3 h. The residual trifluoroacetic acid was removed by vacuum rotary evaporation, a small amount of methanol was added, and the trifluoroacetic acid was removed by vacuum rotary evaporation again. The volume was fixed with a 50 mL volumetric flask and then filtered through a 0.22 μm nylon filter for chromatographic analysis. The ion chromatography detection conditions are: Thermo Fisher ICS6000 ion chromatograph, chromatographic column: Carbopac PA20 (3×150 mm); detector: electrochemical detector; column temperature: 30°C; flow rate: 0.5 mL / min; injection volume: 20 μL; mobile phase gradient elution program: 0-16.05 min, 10% NaOH (20 mM) and 90% ultrapure water; 16.05-30.05 min, 10% NaOH (20 mM), 20% CH3COONa (500 mM) and 70% ultrapure water. Various monosaccharide standards with a concentration of 1-10 ppm were prepared respectively, and chromatographic analysis was performed according to the above ion chromatography method. The monosaccharide composition and molar percentage in the polysaccharide sample can be determined based on the retention time of the standard and the standard curve.

[0102] Molecular weight determination of IPMPP

[0103] High performance liquid gel permeation chromatography (HPGPC) was used for determination. The test conditions were: Agilent 1260 GPC high performance liquid chromatograph; refractive index detector (1260RID); gel chromatography column: TSK-Gel GMPWXL column (7.8×300mm), mobile phase: 0.02M potassium dihydrogen phosphate; flow rate: 0.5mL / min; column box temperature: 35°C; injection volume: 25μL; instrument test time: 32min.

[0104] Drawing of standard curve: A series of pullulan standards with different molecular weights (6.3, 9.8, 22, 49.7, 107, 216, 334, 739 kDa) were dissolved in 0.02 M potassium dihydrogen phosphate to prepare a standard solution with a concentration of 4 mg / mL. After filtering through a 0.22 μm aqueous filter membrane, the solution was tested on the machine according to the HPGPC test conditions and its retention time was recorded. The standard curve was drawn with the retention time (min) as the horizontal axis and the logarithm of the molecular weight of the dextran standard (LogMW) as the vertical axis. The regression equation was fitted to calculate the molecular weight of different components of IPMPP. The linear fitting equation obtained was y = -0.4501x + 11.86, R 2 =0.9927.

[0105] Determination of molecular weight: 4 mg of IPMPP sample was dissolved in 2 mL of 0.02 M potassium dihydrogen phosphate to prepare a polysaccharide solution with a concentration of 2 mg / mL after it was fully dissolved. After filtering through a 0.22 μm aqueous filter membrane, it was analyzed on a machine according to the HPGPC test conditions. The chromatogram of the purified IPMPP component was recorded, and the purity of the polysaccharide sample was evaluated based on the number of peaks and peak symmetry on the chromatogram. The molecular weight of the pectin polysaccharide sample was calculated based on the retention time of the pectin polysaccharide sample on the chromatogram and compared with the molecular weight standard curve of the dextran standard.

[0106] Particle size and Zeta potential determination of IPMPP

[0107] Ultrapure water was used to prepare an IPMPP solution with a concentration of 1 mg / mL, and the Zeta potential value and particle size of the IPMPP solution were measured using a nanoparticle size potential analyzer at a test temperature of 25°C. The particle size of the sample was expressed as the Z-average diameter.

[0108] Infrared Spectrum and Determination of Esterification Degree of IPMPP

[0109] The infrared absorption spectrum of the IPMPP sample was determined by the KBr tablet method: 1-2 mg of IPMPP dry sample and 200 mg of pre-dried spectrally pure KBr powder were weighed, placed in a dry agate mortar and ground evenly, pressed into a tablet by a tablet press, and scanned at 450-4000 cm by a Fourier transform infrared spectrometer. -1 Infrared spectra of polysaccharide samples from Castanopsis alpiniae oil meal.

[0110] The methyl ester and free carboxyl groups are at 1735 cm -1 and 1628cm -1 The peak area was used to calculate the degree of esterification (DM) of IPMPP.

[0111]

[0112] Determination of triple helix in IPMPP

[0113] Deionized water was used to prepare an IPMPP solution with a concentration of 2 mg / mL, 2 mL of the polysaccharide solution was mixed with 2 mL of 80 μmol / L Congo red solution, and different volumes of 1 mol / L NaOH solution were slowly added to increase the NaOH concentration in the mixed solution to 0, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45 and 0.5 mol / L, respectively. The mixed solution was allowed to stand at room temperature for 10 min, and then the absorption spectrum in the wavelength range of 300-550 nm was scanned using a UV-visible spectrophotometer at room temperature, and the maximum absorption wavelength was recorded.

[0114] X-ray diffraction analysis of IPMPP

[0115] Place the dried IPMPP sample in a sample cell and then into an X-ray diffractometer to measure its crystal properties. Test conditions: copper target Tube voltage 40 kV; scanning range (2θ) 5~80°, scanning speed 10° / min.

[0116] Thermal Stability Analysis of IPMPP

[0117] The thermal stability of IPMPP was determined using a differential scanning calorimeter (DSC). The sample (about 4 mg) was placed in a crucible and the polysaccharide sample was subjected to DSC analysis in a dynamic nitrogen atmosphere with a detection temperature range of 45 to 445°C and a heating rate of 10°C / min.

[0118] Scanning electron microscopy (SEM) analysis

[0119] First, fix the double-sided tape on the stage, take a small amount of dry IPMPP and disperse it on the double-sided tape, so that the polysaccharide sample forms a uniform thin layer on the double-sided tape. Then place the stage in a vacuum coating machine for gold spraying, and finally place the gold-sprayed stage in the scanning electron microscope sample chamber to observe the microscopic morphological characteristics of the solid molecules of the polysaccharide purified components and collect image information at the same time.

[0120] Study on the hypoglycemic activity of IPMPP in vitro

[0121] IPMPP inhibition of α-glucosidase activity assay

[0122] Phosphate buffer solution (100 mM, pH 6.9) was used to prepare different concentrations of IPMPP (0.5-6.0 mg / mL), α-glucosidase (0.5 U / mL) and p-nitrophenyl-α-D-galactopyranoside (pNPG, 5 mmol / L). 100 μL α-glucosidase was added to 100 μL IPMPP, incubated at 37 °C for 10 min, 100 μL pNPG was added, incubated at 37 °C for 20 min, and 1 mL 1 mol / L sodium carbonate was added to terminate the reaction. Control 1 was a mixture of pPNG solution and IPMPP without enzyme; Control 2 was a mixture of pNPG and enzyme without IPMPP. Acarbose was used as a positive control. The absorbance at 405 nm was measured using an ELISA reader.

[0123]

[0124] Inhibition kinetics of IPMPP on α-glucosidase

[0125] 100 μL of pNPG (1, 2, 3, 4 mM) of different concentrations was taken as substrate, and an equal volume of α-glucosidase (0.5 U / mL) and an equal volume of IPMPP solution of different concentrations (0, 1, 3, 6 mg / mL) were added. The reaction solution was mixed and reacted at 37°C for 20 minutes. After 1 mL of 1 mol / L sodium carbonate solution was added to the reaction solution to stop the reaction, the absorbance value was measured at a wavelength of 405 nm. The Lineweaver-Burk double reciprocal plotting method was used, with the reciprocal of the IPMPP substrate as the horizontal axis and the reciprocal of the reaction rate as the vertical axis to draw a curve, determine the inhibition type, and analyze the inhibition kinetic parameters of the IPMPP inhibitor on α-glucosidase. The Michaelis-Menten constant (Km) and the maximum reaction rate (Vmax) were calculated according to the Michaelis-Menten equation (Formula 2.6).

[0126]

[0127] Fluorescence quenching effect of IPMPP on α-glucosidase

[0128] All sample solutions were prepared with pH = 6.9 phosphate buffer. The endogenous fluorescence intensity of α-glucosidase was measured using an F-7000 fluorescence spectrometer and a constant temperature circulating water bath for temperature control. 1.5 mL of α-glucosidase solution (0.75 U / mL) was transferred and different concentrations of IPMPP solution (1-7 mg / mL) were added respectively, and incubated at 30°C for 10 min. The excitation and emission grating slits were set to 5 nm, the excitation wavelength was set to 280 nm, the scanning wavelength range was 300-500 nm, the scanning rate was 1200 nm / min, and the resolution was 1.0 nm. The fluorescence emission spectrum intensity after the action of α-glucosidase and IPMPP solution was measured. The fluorescence quenching constant was calculated according to the Stem-Volmer formula (Formula 2.7). The binding constant and number of binding sites of α-glucosidase and IPMPP can be calculated by the following formula (Formula 2.8):

[0129] In formulas (2.7) and (2.8), F0 is the total fluorescence emission of the α-glucosidase solution; F is the total fluorescence emission after the α-glucosidase reacts with IPMPP; [Q] is the IPMPP concentration; KSV is the Stem-Volmer quenching constant; Kq is the dynamic quenching rate constant. Generally, the maximum dynamic quenching rate constant of various fluorescence quenchers for biomacromolecules is 2.0×1010L·moL -1 ·S -1 ; τ0 is the endogenous fluorescence lifetime of biomacromolecules, τ0 = 10 -8s. In formula (2.8), Ka is the Stern-Volmer binding constant, [Q] is the concentration of IPMPP solution, and n is the number of binding sites between α-glucosidase and IPMPP.

[0130] IPMPP in vitro anti-glycosylation assay

[0131] Establishment of the Glycosylation System of Bovine Serum Albumin (BSA)

[0132] The BSA-fructose model was used to study the effect of polysaccharides on BSA glycosylation. 2 mL of bovine serum albumin (20 mg / mL), 2 mL of fructose (90.1 mg / mL), and 1 mL of polysaccharides of different concentrations or aminoguanidine sulfate (AG) were mixed and saccharified in PBS solution (0.2 mol / L, pH 7.4, 0.02% NaN3) and incubated at 50°C for 24 h. The resulting solution was used in the following experiments.

[0133] Determination of fructosamine content

[0134] Fructosamine is an Amdaori rearrangement product of protein glycosylation and a target compound for monitoring the accumulation of early glycosylation products. The nitro blue-tetrazolium (NBT) method was used to determine the concentration of fructosamine. The BSA-fructose-water and BSA-fructose-polysaccharide solution reaction systems were incubated at 50°C for 24h, respectively. After incubation, 400μL of the reaction mixture, 320μL of ultrapure water and 1.6mL of NBT (0.3mmol / L) were dissolved in 100mM sodium carbonate buffer, pH 10.35) and mixed, and incubated at 25°C for 15 minutes. Its absorbance was measured at 530nm. Fructosamine can reduce NBT to the tetrazolium radical NBT + , and then a highly colored formaldehyde dye is generated in proportion to it, which has a strong absorbance at 530nm. Aminoguanidine hydrochloride (AG) was used as a positive control. The inhibition rate (%) of fructosamine was calculated by the following formula:

[0135]

[0136] Where A0 and A represent the absorbance of the glycosylation system in the absence of polysaccharide and in the presence of polysaccharide, respectively.

[0137] Determination of α-dicarbonyl compound content

[0138] The inhibitory effect of polysaccharides on mid-stage glycosylation products was evaluated by quantification of α-dicarbonyl compounds. 500 μL of the above culture solution, 40 μL of ultrapure water, 50 μL of 500 mmol / L Girard-T reagent (dissolved in ultrapure water) and 400 μL of sodium formate (500 mmol / L, pH 2.9) were mixed and incubated at 25°C for 1 hour. The absorbance of the mixture was then measured at 290 nm. With AG as the positive control, the inhibition rate (%) of α-dicarbonyl compounds was calculated by the following formula:

[0139]

[0140] Where A0 and A represent the absorbance of the glycosylation system in the absence of polysaccharides and in the presence of polysaccharides, respectively.

[0141] Fluorescence AGEs (Advanced Glycation End Products) Assay

[0142] The saccharification solution (60 μL) was mixed with 2 mL PBS (0.2 mmol / L, pH 7.4), and the fluorescence intensity of the mixture was measured at an excitation wavelength of 370 nm and an emission wavelength of 440 nm using a fluorescence spectrophotometer (model F-7000). AG was used as a positive control, and the fluorescence intensity reflected the number of fluorescent AGEs. The inhibition rate of fluorescent AGEs was calculated as follows:

[0143]

[0144] Where F0 and F represent the fluorescence intensity of the saccharification solution without IPMPP and with IPMPP, respectively.

[0145] Data processing and analysis

[0146] All experimental data were expressed as mean ± standard deviation from three replicates. The response surface experimental design was statistically analyzed using Design Expert 12 software (Stat-Ease Inc., Minneapolis, MN, USA). SPSS statistical software version 23.0 was used to analyze the data. P < 0.05 was considered statistically significant, and statistical differences were represented by different letters in the figure. OriginPro 2021 (OriginLab Inc, USA) was used for drawing.

[0147] Results and Analysis

[0148] Single factor experimental results of IPMPP

[0149] Effect of inorganic salt types

[0150] In the three-phase extraction system, (NH4)2SO4 is the first choice due to its compatibility with proteins and high solubility. Citric acid is also often used due to its cost-effectiveness in extracting pectin polysaccharides. Adding salt to the sample can increase the cell permeability due to osmotic shock. Adding salt will cause salting-in, thereby increasing the solubility of proteins in the mixture. Figure 2 A shows that when ammonium citrate is used, the yields of IPMP (3.90±0.10%) and IPMPP (6.73±0.20%) are significantly higher than those of ammonium sulfate and sodium citrate (p<0.05). Therefore, ammonium citrate is selected as the inorganic salt in the subsequent three-phase extraction system.

[0151] Effect of ammonium citrate concentration

[0152] like Figure 2 As shown in Figure B, as the amount of ammonium citrate added increased from 10g / 100mL to 50g / 100mL, the yields of IPMPP and IPMP both showed a trend of increasing first and then decreasing. This is because as the salt concentration in the system increases, the salting-out effect becomes stronger. The optimal concentration of ammonium citrate will cause the protein to flocculate and separate, maximizing the release of proteins bound to lipids. When the concentration of ammonium citrate is 30%, the highest extraction rates of IPMPP and IPMP are 7.63% and 4.10%, respectively. As the amount of ammonium citrate added increases, the extraction rate of IPMPP also gradually decreases, and IPMP also shows a similar trend of change. Because as the concentration of salt ions increases, salt ions bind more water molecules, resulting in a weakening of the hydrogen bond network formed between polysaccharides and water molecules, thereby affecting the extraction of polysaccharides. When the concentration of ammonium citrate is 30%, the yields of IPMPP and IPMP are the highest, so 30% ammonium citrate is selected as the center point of the subsequent response surface experiment.

[0153] Effect of material-liquid ratio

[0154] In the initial step of three-phase extraction, a certain concentration of inorganic salt solution is added to the oil meal, the cells absorb water, and the mixture begins to increase its viscosity. If a small volume of inorganic salt solution is added, the solution will become more viscous, and the increase in viscosity will affect the extraction rate. However, adding more inorganic salt solution will increase the process cost because it increases the salt demand and the total volume of the extraction mixture. Therefore, the optimization of the material-liquid ratio is an important parameter, such as Figure 2As shown in Figure C, 1:25 is the most suitable ratio, and the extraction rates of IPMP and IPMPP are the highest, which are 7.51% and 3.91%, respectively. When the solid-liquid ratio increases from 1:15 to 1:25, the IPMPP yield increases from 5.98% to 7.51%. When the ratio increases from 1:30 to 1:45, the yield decreases from 7.03% to 5.79%, and IPMP also shows a similar trend of change. This may be because the higher solid-liquid ratio reduces its viscosity, which may hinder the extraction of IPMPP and IPMP. On this basis, the solid-liquid ratio of 1:25 was selected as the center point of the subsequent response surface experiment.

[0155] Effect of pH

[0156] The separation efficiency of three-phase extraction is highly correlated with changes in pH. It affects the ionization of amino acids in proteins, which in turn affects other properties of macromolecules. Therefore, the distribution of proteins and polysaccharides in three-phase extraction systems changes with changes in pH. Figure 2 As shown in D, within the pH range of 2.0-3.0, the extraction rate of IPMPP gradually increased, while when the pH was higher than pH 3.0, the extraction rate of IPMPP gradually decreased. At pH 3.0 and pH 4.0, the extraction rates of IPMP were the highest, which were 3.73% and 3.71%, respectively. There was no significant difference in the effects on IPMP (p>0.05)( Figure 2 D). Adjusting the pH of the system may affect the surface charge of ionized proteins, thereby affecting the extraction of polysaccharides and proteins. In this experiment, the three-phase extraction system was more effective in extracting and separating IPMPP under acidic conditions (pH 3.0), which may be due to the separation of some acidic polysaccharides from the oil meal. The highest yield of IPMPP was 8.33% at pH 3.0. The results of some studies show that polysaccharides have the lowest solubility under acidic conditions, the net charge of polysaccharides is zero, and the amount of protein precipitation is the largest, which is beneficial to the separation of oil and protein in oil meal, and then promotes the dissolution of oil in dimethyl carbonate. As the pH continues to increase, it will affect the separation of glycoproteins bound to polysaccharides, thereby affecting the yield of IPMPP. Therefore, considering the effect of pH on the yield of IPMP and IPMPP, pH 3 was selected as the center point of the subsequent response surface experiment.

[0157] Effect of ultrasound time

[0158] Ultrasonic time is one of the important factors affecting ultrasonic combined three-phase extraction, because it has a significant impact on the yield and production cost. Figure 2As shown in Figure E, when the extraction time was extended from 10 min to 30 min, the extraction rate of IPMPP increased from 6.54% to 8.81%, and IPMP also showed a similar trend, increasing from 3.77% to 4.89%. Longer ultrasonic time (>30 min) significantly reduced the extraction rate of IPMPP and IPMP, because ultrasound can achieve effective extraction of substances in a shorter extraction time. Ultrasound combined with three-phase extraction may help to increase the yield of pectin polysaccharides. The reason may be that ultrasound decomposes or disperses pectin polysaccharides in the aqueous phase, thereby increasing mass transfer during the three-phase extraction separation process. Therefore, this method is conducive to the full extraction of IPMPP and IPMP. Therefore, the experiment selected an ultrasonic time of 30 min as the center point of the subsequent response surface experiment.

[0159] Effect of temperature

[0160] Figure 2 F is the effect of temperature on the extraction rate of IPMPP and IPMP in the range of 15-40℃. In the temperature range of 20℃~30℃, the extraction rate of IPMPP and IPMP increased significantly with the increase of temperature (p<0.05). In the temperature range of 30℃~45℃, the yield decreased with the increase of temperature. In the ultrasound-three-phase extraction system, the increase of temperature is conducive to the enrichment of IPMPP in the low phase. This may be because the increase of temperature is conducive to the expansion of macromolecules with a large number of exposed hydroxyl groups, so that they form more hydrogen bonds or links, further making IPMPP more hydrophilic, thus concentrating in the low phase. However, too high extraction temperature will affect the solubility of protein, which may cause the precipitation phase to dissolve again in the aqueous phase, thereby reducing the protein yield. In addition, higher temperature will have a negative impact on the cavitation effect in the ultrasound-three-phase extraction process, thereby reducing the extraction rate. Based on these results, 30℃ was determined to be the temperature for subsequent experiments.

[0161] Effect of volume of dimethyl carbonate

[0162] The advantage of using DMC is that it does not cause salting out due to its excessive use, causing the protein to transfer from the aqueous phase to the interphase. In addition, DMC is partially soluble in water (solubility up to 13%), which helps dissolve the protein in the interphase. As DMC is a green solvent, the process is green and environmentally friendly. The increase in the volume of DMC promotes the floating of the precipitated protein above the lower aqueous phase. This promotes the separation of protein from lipids and pectin. Figure 2G It can be seen that when the volume of DMC is 15mL, the extraction rates of IPMP (4.16%) and IPMPP (7.31%) are the highest. When the volume of DMC continues to increase to 25mL, the yields of IPMP and IPMPP are significantly reduced. This may be due to protein denaturation caused by excessive organic solvents, which hinders the ability of proteins to enter the intermediate phase. Based on these results, the experiment determined the volume of 15mL of DMC as the volume for subsequent experiments.

[0163] Response surface optimization experiment

[0164] Model fitting and statistical analysis

[0165] According to the results of the single factor experiment, four factors with the greatest influence on the yield of IPMPP and IPMP were selected for response surface optimization experiments. The extraction conditions of ultrasound combined with three-phase extraction were optimized, and the experimental design of 4 factors and 3 levels and the experimental results are listed in Table 2. Using Design Expert software, regression analysis was performed on the experimental data in the table, and the quadratic polynomial regression equations (2.12) and (2.13) of the yield of IPMPP (Y) and the yield of IPMP (Y) on ammonium citrate concentration (X1), solid-liquid ratio (X2), pH (X3) and ultrasonic time (X4) were obtained respectively.

[0166] Y IPMP =5.31-0.1099X1+0.4096X2-0.0551X3+0.0149X4+0.0614X1X2-0.4436X1X3-0.3074X1X4+0.2211X2X3+0.1852X2X4-0.1725X3X4-0.5935X1 2 -0.026X2 2 -0.6999X3 2 -0.4250X4 2 (2.12)

[0168] Y IPMPP =9.82-0.1508X1+0.5950X2-0.1442X3+0.0217X4+0.2975X1X2-0.5950X1X3-0.0150X1X4+0.3650X2X3+0.1725X2X4-0.0875X3X4-1.03X1 2 -1.18X2 2 -1.14X3 2 -0.5996X4 2 (2.13)

[0170] Table 2 Box-Behnken experimental design and experimental results

[0171]

[0172] The variance analysis was performed on the multiple regression models (2.12, 2.13) of IPMPP and IPMP yield, and the results are shown in Table 3. From the data analysis of the table, it can be seen that:

[0173] (1) The F values ​​of IPMPP and IPMP were 36.88 and 10.08 respectively, and the P value was less than 0.0001, indicating that the established regression model was significant;

[0174] (2) Correlation coefficient R between IPMPP and IPMP 2 The values ​​of are 0.9736 and 0.9472, respectively, which are close to 1, indicating that the predicted values ​​are in good agreement with the experimental values, that is, the regression model can predict the yield of IPMPP and IPMP with reasonable accuracy; further indicating that the regression model has a good fit;

[0175] (3) The F values ​​of the lack of fit of IPMPP and IPMP were 3.61 and 0.9297, respectively, and the P values ​​were 0.1137>0.05 and 0.4422>0.05, indicating that the lack of fit of the regression model was not significant, the regression was significant, and the correlation between it and the pure error was not significant;

[0176] (4) The coefficient of variation (CV) values ​​of IPMPP and IPMP were 2.69% and 6.55%, respectively, which were relatively low, indicating that the regression model had high accuracy and confidence.

[0177] (5) The significance of the correlation between the regression coefficients of IPMPP and IPMP was determined by the F test and P value. The smaller the P value, the more significant the contribution of the variable. According to the P value, the linear coefficients (X1, X2, X3, X1X2, X1X3, X2X3) and the quadratic coefficients (X1 2 , X2 2 、X3 2 、X4 2 ) all had significant differences (P<0.05), indicating that they had significant effects on the extraction rate of IPMPP; the linear coefficients (X2, X1X3, X1X4) and quadratic coefficients (X1 2 , X2 2 、X3 2 、X4 2 ) have significant differences (P<0.05), indicating that they have significant effects on the IPMP extraction rate. According to the F value of the linear coefficient, the influence of the four factors on IPMPP and IPMP yield is in the following order: solid-liquid ratio>inorganic salt concentration>pH>ultrasonic time.

[0178] Table 3 Analysis of variance table of response surface regression model of IPMPP and IPMP yield

[0179]

[0180]

[0181] The accuracy of the regression model was further tested by Figure 3 A and 4A can intuitively show that it is feasible to use the response surface method to predict and analyze IPMPP and IPMP yield, and the predicted values ​​are highly consistent with the experimental results. Figure 3 B and 4B), the residuals of IPMPP and IPMP yield are normally distributed along the straight line, which further verifies the accuracy of the model. Figure 3 C and 4C), it can be found that all studentized residuals are randomly distributed between -2 and 2, indicating that the difference terms follow a normal distribution. In addition, from the perturbation diagram of the impact of various factors on IPMPP and IPMP ( Figure 3 D and Figure 4 D) It can be seen intuitively that the curve corresponding to the solid-liquid ratio is the steepest (with the largest slope), and it has the greatest impact on the yield of IPMPP and IPMP, which is consistent with the results of variance analysis. The above results further prove that the regression model established by the response surface methodology for the yield of IPMPP and IPMP extracted from UTPP in this experiment has high accuracy and confidence.

[0182] Response surface analysis

[0183] Through the 3D response surface plot and 2D contour plot, we can intuitively judge the interaction between the variables in the model and the change in the relationship between the variables and the response value. The closer the shape of the 2D contour plot is to an ellipse, the stronger the interaction between the variables. Conversely, the closer the contour line is to a circle, the weaker the interaction between the independent variables. In the 3D response surface plot, the steeper the response surface, the more significant the effect of the variable on the response value. Figure 5 and Figure 6It can be seen that when the other factor is fixed at the 0 level, the interaction between salt concentration (X1) and liquid-to-solid ratio (X2) and salt concentration (X1) and pH value (X3) has a quadratic effect on the yield of pectin and protein. Therefore, these interactions are significant and consistent with the results in Table 3. When the inorganic salt concentration is 20-30 mg / mL, the liquid-to-solid ratio is 1:20-1:25, and the other parameters are all 0, the yield of IPMPP and IPMP increases. When the other parameter is 0, as the inorganic salt concentration and pH increase from 20 mg / mL to 30 mg / mL and the pH increases from 2 to 3, the yield of IPMPP and IPMP also increases. These results confirm the accuracy and reliability of the established regression model.

[0184] Model Validation

[0185] The process conditions of IPMP and IPMPP were optimized by response surface analysis. The optimized extraction process conditions of IPMPP were as follows: ammonium citrate concentration of 29.84%, solid-liquid ratio of 1:26.24, pH 2.98, and ultrasonic time of 30.55 min; the optimized extraction process conditions of IPMP were as follows: no ammonium citrate concentration of 29.43%, solid-liquid ratio of 1:26.45, pH 3.08, and ultrasonic time of 30.85 min. Considering the feasibility of actual operation, the optimized extraction process conditions were adjusted to ammonium citrate concentration of 30%, solid-liquid ratio of 1:26, pH 3, and ultrasonic time of 31 min. Three verification experiments were carried out under the adjusted optimal process conditions, and the yields of IPMPP and IPMP were 10.03±0.31% and 5.03±0.11%, respectively, which were basically consistent with the model prediction results (IPMP was 4.91% and IPMPP was 9.90±0.31%). Therefore, the regression model obtained in the experiment is feasible for predicting and analyzing the yield of IPMPP and IPMP, and its accuracy and feasibility are good.

[0186] Yield Analysis of IPMPP, IPMP and IPMO

[0187] As shown in Table 4, when the extraction conditions optimized by response surface analysis were used, the extraction rate of lipids was 8.10%, the extraction rate of proteins was 5.03%, and the extraction rate of polysaccharides was 10.03%. The recovery rates of oil, protein, and polysaccharides were 91.62%, 83.08%, and 93.95%, respectively. These results show that ultrasound combined with three-phase extraction technology can effectively recover most of the oil, protein, and polysaccharide components in the oil cake of Castanopsis schrenkiana, highlighting the practicality and reliability of this extraction method. The experimental results show that ultrasound combined with three-phase extraction system can achieve the simultaneous extraction of oil, protein, and polysaccharides.

[0188] Table 4 Extraction rates of IPMPP, IPMP and IPMO

[0189]

[0190] Fatty acid composition analysis of IPMO

[0191] The gas chromatogram of the fatty acid composition of IPMO extracted by UTPP is shown in Figure 7 As shown in Table 5, it can be concluded that linoleic acid (84.57%) is the main unsaturated fatty acid. The high content of linoleic acid in the ash oil can reduce the cholesterol level in the blood, which is essential for preventing atherosclerosis. It can also serve as a precursor of important signaling molecules and plays a vital role in skin health, inflammation, etc. In addition, palmitic acid (5.69%) and palmitoleic acid (4.37%) are the main saturated fatty acids. In addition, oleic acid, stearic acid and erucic acid are 3.53%, 1.37% and 0.47% respectively. Similar to previous research results, the researchers collected ash fruits from different regions to study their fatty acid composition. The results showed that linoleic acid was the main fatty acid in the fruit oil, and the linoleic acid content in each region was above 60%. In general, the results show that the effect of extraction technology on the fatty acid composition of ash oil is negligible or has no effect.

[0192] Table 5 Fatty acid composition of tung oil meal

[0193]

[0194] Amino acid composition analysis of IPMP

[0195] The quality of a protein is usually determined by its essential amino acid content. The amino acid composition of IPMP is as follows: Figure 8As shown in the figure, IPMP contains a total of 17 amino acids. IPMP has a high nutritional quality because it meets the types of essential amino acids for the human body except tryptophan, including 7 essential amino acids for the human body, such as threonine (Thr), valine (Val), methionine (Met), leucine (Leu), isoleucine (Ile), phenylalanine (Phe) and lysine (Lys). According to the polarity of the side chain, amino acids can be divided into hydrophilic amino acids and hydrophobic amino acids, among which hydrophobic amino acids mainly include 8 amino acids such as glycine (Gly), proline (Pro), alanine (Ala), valine (Val), methionine (Met), leucine (Leu), isoleucine (Ile) and phenylalanine (Phe). Hydrophilic amino acids mainly include 9 amino acids such as aspartic acid (Asp), glutamic acid (Glu), histidine (His), serine (Ser), arginine (Arg), threonine (Thr), tyrosine (Tyr), cysteine ​​(Cys) and lysine (Lys). The main amino acids are Glu (24.9%), Asp (12.2%) and Ser (9.9%), which are typical amino acid profiles of plant seed proteins. This finding is similar to the results of simultaneous extraction of oil, protein and polysaccharide from oil-tea camellia seed cake using three-phase extraction. The protein in oil-tea camellia seed cake is rich in glutamic acid (Glu) and aspartic acid (Asp)

[35] . It can be seen that IPMP extracted by UTPP can be used as an excellent supplementary choice for grains and grain-based feeds.

[0196] The main chemical composition of IPMPP

[0197] The main chemical components of IPMPP are shown in Table 6, and the neutral sugar content is 56.17%. IPMPP contains 35.56% uronic acid, which is a typical acidic heteropolysaccharide. Uronic acid is an important ingredient in hypoglycemic drugs and moisturizing cosmetics. In addition, the protein content of IPMPP is 4.48%, indicating the presence of polysaccharide-protein complexes in the extract. This residual protein may be due to its coupling with IPMPP. The degree of esterification is an important indicator for evaluating the functional properties and applications of pectin, and a value below 50% can be identified as low methoxyl pectin. The esterification value of pectin polysaccharide extracted from UTPP is 36.64%, indicating that it is a low methoxylated pectin, and therefore can be used as a potential food raw material for low-sugar or sugar-free products.

[0198] Table 6 Main chemical composition of IPMPP

[0199]

[0200] Analysis of monosaccharide composition of IPMPP

[0201] Fig. 9The ion chromatogram peaks of the samples showed that 9 monosaccharides, including fucose (Fuc), arabinose (Ara), rhamnose (Rha), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), galacturonic acid (GalA), and glucuronic acid (GlcA), were detected from IPMPP, with molar percentages of 4.02%, 14.01%, 7.50%, 12.97%, 13.91%, 6.59%, 4.37%, 30.04%, and 6.60%, respectively (Table 7). The presence of a large amount of GalA, Ara, and Gal indicated that IPMPP was a pectin polysaccharide. The "smooth" region of pectin contained a high galacturonic acid (HG) domain, which was composed of partially methylated and acetylated galacturonic acid. The "hair" region contains more complex structures such as rhamnogalacturonan-I (RG-I), rhamnogalacturonan-II (RG-II) and xyluronic acid (XGA), which are characterized by branched chains. As shown in Table 7, the RG-I content of IPMPP exceeds 41.97%, indicating that UTPP is an effective method for isolating RG-I-rich pectin from Castanopsis japonica oil cake. The Rha / GalA ratio of RG-I pectin represents the distribution range between HG and RG-I domains, usually ranging from 0.05 to 1. IPMPP is within this range with a ratio of 0.25. The (Ara+Gal) / Rha ratio reflects the level of branching in the RG-I domain, and its higher value indicates that the side chain connected by rhamnose is longer. In general, the (Ara+Gal) / Rha ratio ranges from 1.89 to 29.83. The ratio of IPMPP was 3.60, indicating a relatively high degree of branching within its RG-I domain, highlighting its potential as a functional dietary supplement.

[0202] Table 7 Monosaccharide composition of IPMPP

[0203]

[0204] Note: HG%=(GalA-Rha)mol%, RG-I%=(2Rha+Gal+Ara)mol%.

[0205] Molecular weight distribution of IPMPP

[0206] Fig.10This is the molecular weight distribution diagram of HPGPC of tung oil meal polysaccharide. The weight average molecular weight (Mw), number average molecular weight (Mn) and dispersion index (PDI, Mw / Mn ratio) were calculated. PDI can be used to represent the dispersion index of the polymer. The larger the PDI, the wider the molecular weight distribution. The Mw of IPMPP is 226.58±8.03kDa, Mn is 89.79±2.33kDa, and Mw / Mn is 2.52±0.02, indicating that its molecular weight distribution is relatively narrow. These results confirm that IPMPP is composed of highly uniform polysaccharides with a concentrated molecular weight distribution.

[0207] Analysis of Particle Size and Zeta Potential of IPMPP

[0208] like Fig.11 As shown, the particle size distribution of IPMPP shows a single peak, indicating a relatively uniform particle size distribution. The particle dispersion index (PDI) is 0.34, reflecting a high degree of uniformity in the particle size distribution. The average particle size of IPMPP is 1331.67nm. IPMPP is negatively charged with a zeta potential value of -35.13, exceeding |±30|, a property that helps prevent large-scale aggregation of particles.

[0209] Infrared Spectrum Analysis of IPMPP

[0210] The functional groups of IPMPP are Fig.12 The infrared spectrum of IPMPP shows the characteristics of pectin, with a peak at 3400 cm -1 A strong and broad absorption peak caused by -OH stretching vibration was observed nearby, which belongs to the stretching vibration of hydroxyl groups of polysaccharides and water involved in hydrogen bonding, indicating that the pectin chain has strong intermolecular forces. -1 The absorption peak nearby is the CH extension peak of the CH2 group, 1735cm -1 and 1625cm - The absorption peaks at 1419 cm-1 are esterified carboxyl (-COOR) and free carboxyl (-COO-), which are closely related to the esterification degree of pectin. -1 The weaker absorption peaks appearing nearby are attributed to the bending vibration of OH or CH2. -1 、1155cm -、 1015cm -1 and 780cm -1 The peaks of further confirmed the existence of β-type or α-(1→6) glucosidic bonds in IPMPP

[45] . Overall, IPMPP is a typical pectin polysaccharide.

[0211] Analysis of the triple helix structure of IPMPP

[0212] Congo red experiments are often used to study the sugar chain conformation of polysaccharides in aqueous solutions, because it can form complexes with polysaccharides with triple helical structures, which are closely related to the biological activity of polysaccharides. When polysaccharides are complexed with Congo red, the maximum absorption wavelength of the complex will move toward the long wavelength direction in the visible light range compared with Congo red, that is, a red shift will occur. However, within a certain concentration range of NaOH, the maximum absorption wavelength of the complex will gradually decrease, indicating that the polysaccharide conformation has changed. Fig.13 As shown, as the concentration of NaOH solution increases, the λmax of the complex formed by IPMPP and Congo red shifts redward. When the NaOH concentration is 0.05 mol / L, its λmax reaches a maximum of 496.33. The above results show that IPMPP maintains a triple helical structure at lower alkaline concentrations, indicating its potential biologically active conformation. This structural integrity at a specific pH value highlights the relevance of IPMPP in biological environments, indicating its practicality in applications where molecular conformation affects functional efficacy.

[0213] X-ray diffraction analysis of IPMPP

[0214] XRD is a convenient analytical technique for distinguishing between crystalline and amorphous materials. Fig.14 As shown, the XRD pattern of IPMPP shows a broad dispersion peak at a diffraction angle of 22.55°, and no obvious sharp peak in the diffraction range of 5° to 90°, indicating that IPMPP lacks a crystal structure and exists mainly in an amorphous form.

[0215] Thermal Stability Analysis of IPMPP

[0216] Differential scanning calorimetry (DSC) was used to evaluate the thermodynamic properties of pectin samples. Generally, the DSC thermogram of pectin samples showed obvious endothermic and exothermic peaks, among which the higher melting temperature (Tm) and melting enthalpy (ΔHm) indicated that pectin had a strong water retention capacity. Fig.15 As shown in the endothermic stage, the melting temperature (Tm) and melting enthalpy (ΔHm) of IPMPP are 107.06°C and 122.01 J / g, respectively. In the exothermic stage, IPMPP shows a single exothermic peak, the onset temperature (To) of the exothermic phase is 330.46°C, the glass transition temperature (Tg) is 357.72°C, and the glass transition enthalpy (ΔHg) is -12.71 J / g. The above results show that IPMPP has high thermal stability.

[0217] Scanning electron microscopy (SEM) analysis of IPMPP

[0218] like Fig.16As shown, the surface microstructure of IPMPP was analyzed using SEM at magnifications ranging from 100× to 10,000×. At 100× magnification, IPMPP exhibited a lamellar structure characterized by a curled surface and some debris, indicating its complex physical composition. At 5,000× magnification, the fragments were small, consistent with the size characteristics associated with pectin; in addition, the surface exhibited varying degrees of protrusions, indicating structural diversity. At 10,000× magnification, it was observed that the surface was uneven and some particles exhibited a rod-like structure.

[0219] IPMPP in vitro glucose-lowering test

[0220] Analysis of the inhibitory effect of IPMPP on α-glucosidase

[0221] α-Glucosidase inhibitors play a key role in diabetes management by slowing down the conversion of carbohydrates into glucose in the small intestine, thereby reducing postprandial hyperglycemia, enhancing insulin sensitivity, and preventing diabetes and its related complications. Fig.17 As shown in the figure, the inhibitory effect of IPMPP on α-glucosidase activity increased with the increase of concentration, reaching an inhibition rate of 68.82% at a concentration of 6.0 mg / mL, indicating that its inhibitory effect was concentration-dependent. The half-maximal inhibitory concentration (IC 50 ) is 1.40 mg / mL, which is significantly higher than 0.11 mg / mL of acarbose. Although its inhibitory effect is not as good as acarbose, IPMPP still has the potential as an anti-diabetic drug with the advantage of fewer side effects. The efficacy of polysaccharides (such as IPMPP) as α-glucosidase inhibitors is affected by many factors, including molecular weight, uronic acid content, and monosaccharide composition. It is worth noting that polysaccharides rich in arabinose and galacturonic acid have been shown to have significant α-glucosidase inhibitory activity. Therefore, the significant α-glucosidase inhibitory activity of IPMPP may be attributed to its high levels of arabinose and galacturonic acid, showing its potential therapeutic value in diabetes management.

[0222] Inhibition kinetics analysis of IPMPP on α-glucosidase

[0223] The Lineweaver-Burk double reciprocal plot was used to study the inhibition type of IPMPP on α-glucosidase. When the concentration of substrate pPNG was between 1 and 4 mmol / L, 1, 3, and 6 mg / mL of IPMPP were used to measure the change in the enzymatic reaction rate. The results are shown in Figure 2. Fig.18As shown, as the concentration of IPMPP increases, the slope of the curve increases. Double reciprocal mapping shows a good linear relationship, and both fitting curves intersect the third quadrant of the coordinate system, indicating that the inhibition of IPMPP to α-glucosidase meets the mixed inhibition type. As shown in Table 8, according to the Lineweaver-Burk kinetic equation, when the concentration of IPMPP is 0, the Km value of α-glucosidase is 16.629mM, and the Vmax value is 0.481 (ΔA405 / min). In the reaction system, when 1, 3 and 6mg / mL of IPMPP are added respectively, the Km value of α-glucosidase drops to 5.156mM, and the Vmax value drops to 0.058 (ΔA405 / min) respectively. After adding IPMPP, the Ki value is 3.595mg / mL, and the Kis value is 0.792mg / mL. Generally, the lower the Ki and Kis values ​​of the tested inhibitor, the tighter the binding of the inhibitor-enzyme complex and the inhibitor-enzyme-substrate complex, and the stronger the inhibitor's inhibitory effect on α-glucosidase. These results indicate that IPMPP inhibits α-glucosidase activity by forming a polysaccharide-enzyme-substrate complex, thereby significantly affecting the enzymatic reaction process. This mechanism shows the potential of IPMPP as an effective natural inhibitor in diabetes management by regulating enzyme activity.

[0224] Table 8 Inhibition kinetic parameters of IPMPP on α-glucosidase

[0225]

[0226]

[0227] Analysis of fluorescence quenching effect of IPMPP on α-glucosidase

[0228] Proteins containing aromatic amino acids such as tyrosine, phenylalanine, and tryptophan, such as α-glucosidase, show strong fluorescence. Tryptophan is particularly important as a major contributor to fluorescence emission. When α-glucosidase interacts with inhibitors or its microenvironment changes, its fluorescence intensity usually decreases. Fig.19As shown, the fluorescence intensity of α-glucosidase decreased with increasing IPMPP concentration, indicating that IPMPP effectively reduced the fluorescence intensity of α-glucosidase. The higher the IPMPP concentration, the more obvious the quenching effect. In addition, after IPMPP treatment, the maximum fluorescence emission wavelength of α-glucosidase changed from 334nm to 335.4nm under 300nm excitation. This red shift indicates that there is an interaction between IPMPP and α-glucosidase, which changes the polarity around tyrosine and tryptophan residues, transforming the microenvironment from hydrophilic to hydrophobic. This change can lead to the unfolding of fluorescent amino acid residues and the subsequent quenching of the fluorescent group, indicating that the structure of the enzyme has changed significantly.

[0229] The fluorescence quenching mechanism of enzyme inhibitors and enzymes is usually classified as static quenching, dynamic quenching, or a combination of the two. To determine the binding mechanism between IPMPP and α-glucosidase, the Stern-Volmer equation was applied to the fluorescence quenching data. The resulting Stem-Volmer equation is shown in Fig.19 As shown in C1, there is a significant linear relationship between α-glucosidase and IPMPP (R 2 =0.9828). The molecular weight of IPMPP is 226.58 kDa, so the Kq value of IPMPP is 1.034×1013 M-1s-1, and the KSV value is 1.034×105 M-1. Kq is significantly greater than the maximum value of the dynamic fluorescence quenching rate constant (2.0×1010 M-1s-1), indicating that the quenching process of IPMPP on α-glucosidase is mainly due to the static quenching caused by the interaction between molecules to form a complex, which is caused by the formation of a non-fluorescent complex that cannot emit light or has low luminescence efficiency, and is the result of the combined action of the fluorescent group and the quencher. Fig.19 As shown in C2, the Ka value of IPMPP inhibition of α-glucosidase was calculated to be 8.1227×104 L·mol by fitting the curve of lg[(F0-F) / F] versus lg[Q]. -1 (Table 8). The Ka value reflects the binding affinity between the inhibitor and the enzyme, and the larger the Ka value, the stronger the affinity. When the molecule binds to the site on the macromolecule, Ka and n can be used to evaluate the equilibrium state between the free molecule and the bound molecule. The n value is 0.9859 (Table 8), which is approximately equal to 1, indicating that α-glucosidase has 1 binding site for IPMPP, indicating that there is an interaction between IPMPP and α-glucosidase. The above results show that IPMPP has the potential to be used as a new type of α-glucosidase inhibitor in functional foods and dietary supplements, demonstrating its ability to effectively regulate postprandial blood glucose levels.

[0230] In vitro anti-glycosylation assay of IPMPP

[0231] Chronic diabetes often leads to the development of nonenzymatic glycation end products (AGEs), which are associated with various diabetic complications such as microangiopathy, nephropathy, and atherosclerosis. The glycation process begins with the formation of Schiff bases, which are reversible intermediates that occur when amino groups in proteins react with carbonyl groups generated by reducing sugars. Schiff bases then undergo Amadori rearrangement to produce more stable products that participate in the Maillard reaction. This series of reactions ultimately forms harmful AGEs through processes such as dehydration, oxidation, and further molecular rearrangements. AGEs contribute to the worsening of diabetic complications by interacting with specific receptors called RAGEs. This interaction promotes the generation of reactive oxygen species (ROS) and triggers an inflammatory response, which amplifies oxidative stress and activates proinflammatory mediators, exacerbating the course of the disease. Therefore, targeted inhibition of AGE formation could be a strategy to mitigate diabetes-related complications. In this study, the effect of IPMPP on inhibiting nonenzymatic protein glycation was evaluated using a bovine serum albumin-fructose model with aminoguanidine (AG) as a positive control. The glycation process was analyzed through its early, intermediate, and late stages.

[0232] Fructosamine content analysis

[0233] In the early stages of glycosylation, Schiff base and Amadori products are formed, which react with nitro blue tetrazolium (NBT) to produce colored products that can be detected at a wavelength of 530 nm. Fructosamine is a marker of early glycosylation products and is used to measure the extent of this reaction. Fig. 20 As shown, IPMPP exhibited a significant inhibitory effect on the formation of fructosamine. As the concentration of IPMPP increased, the inhibition rate of fructosamine gradually increased, reaching 63.77% at a concentration of 6 mg / mL. The IC50 values ​​of AG and IPMPP were 2.45±0.10 mg / mL and 4.26±0.06 mg / mL, respectively. Although IPMPP was less potent than AG, its substantial inhibitory effect indicated its potential as an effective agent for managing the early glycation process. This suggests that IPMPP can be used as an alternative or complementary treatment in diabetes care, especially for alleviating the formation of early glycation products.

[0234] Analysis of α-dicarbonyl compound content

[0235] Studies have shown that dicarbonyl compounds contribute significantly to the rapid cross-linking of proteins, leading to the formation of stable AGEs. In fact, nearly 50% of AGEs are derived from these compounds. In the intermediate stages of glycation, fructosamine is broken down into α-dicarbonyl compounds such as methylglyoxal, glyoxal, and 3-deoxyglucose ketone, which serve as markers of intermediate glycation products. Fig.21The inhibitory effect of IPMPP on the formation of α-dicarbonyl compounds increased with the increase of its concentration, and when the concentration was 6 mg / mL, the inhibition rate of IPMPP was 41.21±1.37%. The IC50 of AG and IPMPP were 3.81±0.06g / mL and 7.29±0.54g / mL, respectively. Although IPMPP is less effective than AG, its ability to inhibit the formation of harmful intermediates shows its potential utility in managing the glycation process. This suggests that IPMPP can be used as a beneficial agent in interventions aimed at slowing the progression of diseases associated with AGEs.

[0236] Fluorescence AGEs (Advanced Glycation End Products) Content Analysis

[0237] The final stage of glycation is the formation of AGEs. AGEs are oxidative compounds formed in vivo and in vitro, produced by the reaction between proteins and reducing sugars. The fluorescence intensity of AGEs can be used as a quantitative reference for late glycation, reflecting the extent of the non-enzymatic glycation system and the inhibitory activity of the sample. Studies have shown that the molar concentration of monosaccharides, especially the high content of galacturonic acid units in pectin polysaccharides, can effectively inhibit the formation of AGEs. Different concentrations of IPMPP were introduced into the bovine serum albumin-glycosylation system to evaluate its concentration-dependent effect on AGE inhibition. Fig. 22 As shown, IPMPP significantly inhibited the formation of AGEs, and the inhibition rate increased with the increase of IPMPP concentration. When the concentration of IPMPP was 6 mg / mL, the inhibition rate reached a peak of 81.13±0.11%. The IC50 values ​​of AG and IPMPP were 0.98±0.01 mg / mL and 2.44±0.02 mg / mL, respectively, indicating that IPMPP can effectively remove AGEs and block the glycation reaction. Fig.23 As shown, when IPMPP and AG were used, the fluorescence intensity decreased in a dose-dependent manner, with AG showing a stronger quenching effect. The obvious blue shift of the maximum absorption peak indicated that the polarity around the fluorescent group increased due to the interaction with IPMPP, indicating the existence of a protective mechanism to prevent the formation of AGEs. The antioxidant properties of pectin polysaccharides, especially those rich in GalA, effectively inhibited the formation of AGEs by reducing the release of reactive oxygen species. The inhibitory ability of IPMPP in the late stage was significantly stronger than that in the early and middle stages, mainly inhibiting protein cross-linking and preventing the formation of late AGEs. This inhibitory effect may be related to the high content of galacturonic acid in IPMPP. The above results demonstrate the potential of IPMPP as a promising alternative therapeutic agent for the treatment of diseases related to glycation.

[0238] The use of n-butanol in conventional three-phase extraction (TPP) can cause environmental problems. Therefore, a sustainable ultrasound-coupled three-phase extraction (UTPP) system using dimethyl carbonate was developed to simultaneously extract and separate oil, protein, and polysaccharides from tung oil cake, while recycling the low-toxic dimethyl carbonate solvent. The optimization of various parameters such as inorganic salt type, solid-liquid ratio, salt concentration, pH value, ultrasound duration, temperature, and dimethyl carbonate volume was achieved through single factor experiments and response surface methodology. The most favorable conditions were determined to be 30% ammonium citrate concentration, 1:26 solid-liquid ratio, pH 3, ultrasound time 31 min, temperature 30 °C, and dimethyl carbonate volume 15 mL. These conditions resulted in the best extraction yields of 10.03 ± 0.31% for IPMPP, 5.03 ± 0.10% for IPMP, and 8.10 ± 0.75% for IPMO, with high recovery rates (91.62% for oil, 83.08% for protein, and 93.95% for polysaccharides). Chemical analysis of IPMO identified linoleic acid as the major unsaturated fatty acid. IPMPs are characterized by typical plant seed proteins, with glutamic acid, aspartic acid, and serine as the most abundant amino acids. IPMPP, which is mainly composed of RG-I pectin with a molecular weight of 226.58 kDa, has a triple helical structure, strong thermal stability, and has significant inhibitory activity against α-glucosidase and non-enzymatic glycosylation. In addition, IPMPP was found to inhibit α-glucosidase through a mixed mechanism, quenching the fluorescence of the enzyme mainly through static interactions and binding at a single site. These findings highlight the potential of IPMPP as a novel α-glucosidase inhibitor and anti-glycosylation agent suitable for functional foods and dietary supplements. Overall, this study highlights the effectiveness of ultrasound combined with three-phase extraction technology as a new and environmentally friendly alternative method for the extraction and utilization of valuable compounds from Castanopsis syringae oil meal, improving the traditional three-phase extraction method.

[0239] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A method for simultaneously extracting and separating polysaccharides, proteins and oils from tung oil cake, characterized in that: The following steps are involved: Mix the tung oil cake with the inorganic salt solution, add dimethyl carbonate, and mix well to obtain a mixture; The mixture is sonicated, shaken, and centrifuged to form a three-phase solution including an upper phase solution containing oil, a middle phase solution containing protein, and a lower phase solution containing polysaccharide; The upper phase solution is evaporated under reduced pressure to obtain a fat extract, and dimethyl carbonate is recovered for recycling; The middle phase solution is dissolved in a buffer solution, dialyzed, and dried to obtain a protein extract; The lower phase solution was dialyzed and dried to obtain the polysaccharide extract.

2. The method for synchronously extracting and separating polysaccharides, proteins and fats from Castanopsis japonica oil cake according to claim 1, characterized in that: The concentration of the inorganic salt solution is 10-50%, w / v; the pH is 2-9; and the material-liquid ratio of the tung oil cake to the ammonium citrate solution is 1:15-40.

3. The method for synchronously extracting and separating polysaccharides, proteins and fats from Castanopsis japonica oil cake according to claim 2, characterized in that: The added amount of dimethyl carbonate is 5 to 25 mL.

4. The method for synchronously extracting and separating polysaccharides, proteins and oils from Castanopsis japonica oil cake according to claim 1, characterized in that: The mixture was ultrasonicated for 10-50 minutes, and then the mixture was placed in a constant temperature oscillator at a temperature of 20-45° C. and shaken for 30 minutes. After shaking, the mixture was centrifuged at 4000 rpm for 15 minutes to form a three-phase liquid of an upper phase solution, a middle phase solution and a lower phase solution.

5. The method for synchronously extracting and separating polysaccharides, proteins and fats from Castanopsis altissima oil cake according to claim 1, characterized in that: The inorganic salt solution is an ammonium citrate solution with a concentration of 30% and a pH of 3; the solid-liquid ratio of the tung oil cake to the ammonium citrate solution is 1:26; the ultrasonic time is 31 minutes, the temperature is 30°C, and the volume of dimethyl carbonate is 15 mL.

6. The method for simultaneously extracting and separating polysaccharides, proteins and fats from Castanopsis japonica oil cake according to any one of claims 1 to 5, characterized in that: The lower phase solution was adjusted to pH 6.5 with 1 mol / L sodium hydroxide solution, concentrated to 1 / 3 of the original volume, and dialyzed with tap water in a dialysis bag with a molecular weight cutoff of 8000-14000Da for 48 hours; 4 volumes of anhydrous ethanol were slowly added under stirring, and the mixture was precipitated overnight at 4°C; the precipitate was collected by centrifugation; the precipitate was then washed twice with anhydrous ethanol and redissolved in 50-60°C distilled water, and the insoluble impurities were removed by centrifugation again; and the polysaccharide extract was finally obtained by vacuum freeze-drying.

7. The method for simultaneously extracting and separating polysaccharides, proteins and fats from Castanopsis japonica oil cake according to any one of claims 1 to 5, characterized in that: The middle phase solution was dissolved in 0.05 mol / L, pH 8.0 Tris-HCl buffer, placed at 4°C for more than 12 hours, dialyzed with tap water in a dialysis bag with a molecular weight cutoff of 8000-14000 Da, and the dialysate was freeze-dried to obtain a protein extract.

8. A polysaccharide extract obtained by the method for simultaneously extracting and separating polysaccharides, proteins and oils from Castanopsis japonica oil cake as described in any one of claims 1 to 6.

9. Use of the polysaccharide extract according to claim 8 in the preparation of α-glucosidase inhibitors.

10. Use of the polysaccharide extract according to claim 8 in the preparation of an anti-glycosylation agent.

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