Kiwi fruit young fruit pectin polysaccharide or modified pectin polysaccharide thereof as well as preparation method and application of kiwi fruit young fruit pectin polysaccharide or modified pectin polysaccharide

Abstract

The invention provides kiwi fruit young fruit pectin polysaccharide or modified pectin polysaccharide thereof, a preparation method of the kiwi fruit young fruit pectin polysaccharide and application of the kiwi fruit young fruit pectin polysaccharide or the modified pectin polysaccharide in preparation of drugs or health-care food with an immune enhancement function. The kiwi fruit young fruit pectin polysaccharide and the modified pectin polysaccharide thereof are rich in polygalacturonic acid and I-type polyrhamnogalacturonic acid type pectin structural domains, and have remarkable in-vivo and in-vitro immunoregulation activity. The preparation method mainly comprises the following steps: efficiently preparing high-purity kiwi fruit young fruit pectin polysaccharide by adopting a deep eutectic solvent assisted heating extraction technology and an ultrafiltration membrane separation technology, and carrying out structure modification on the kiwi fruit young fruit pectin polysaccharide by adopting a low-temperature alkaline deesterification technology, an ultrasonic Fenton degradation technology and a controllable partial acid hydrolysis technology, so as to obtain the kiwi fruit young fruit pectin polysaccharide. By comparing the in-vivo and in-vitro immunomodulatory activity of the kiwi fruit young fruit pectin polysaccharide before and after modification, it is found that the kiwi fruit young fruit modified pectin polysaccharide obtained through different modification technologies is better in immunomodulatory activity; and the low-esterification-degree kiwi fruit young fruit modified pectin polysaccharide prepared by the low-temperature alkaline deesterification technology shows the strongest in-vivo and in-vitro immunostimulation effect.

Description

Technical Field

[0001] The invention relates to kiwifruit young fruit pectin polysaccharide or modified pectin polysaccharide thereof, and a preparation method and application thereof, belonging to the field of health food or medicine. Background Art

[0002] Immunomodulation refers to the process of adjusting the function of an organism's immune system, aiming to maintain or restore immune balance and ensure that the immune system can effectively fight pathogens while avoiding damage to the body's own tissues. Through immunomodulation, the intensity and duration of the immune response can be effectively controlled, protecting the body from damage caused by immune imbalance. Natural polysaccharides are a class of high-molecular-weight compounds composed of multiple monosaccharide molecules linked by glycosidic bonds. Natural polysaccharides, which are widely present in plants, can regulate the immune system through various mechanisms, including activating immune cells, promoting phagocytosis, regulating lymphocyte proliferation and transformation, enhancing complement system activation, and inducing cytokine secretion.

[0003] Kiwifruit is a large deciduous woody vine of the genus Actinidia in the family Actinidia, also known as kiwi. Kiwifruit has high nutritional, medical and health value. Kiwifruit is an economic crop with global economic value. It is rich in polysaccharides, flavonoids, polyphenols and other nutrients. Among them, polysaccharides are one of the main active ingredients of kiwifruit. Kiwifruit polysaccharides have the effects of regulating immunity, anti-oxidation, lowering blood pressure, anti-tumor, and improving obesity. Kiwifruit young fruits (thinning fruits) are a by-product of the kiwifruit cultivation industry. They are generally produced during physiological fruit drop or artificial fruit thinning. Because these young fruits are of low maturity and cannot be eaten, most of them are currently discarded directly, resulting in a waste of resources. Application number 202410372497.3, invention name: Use of kiwifruit polysaccharide extract in the preparation of drugs for preventing or / and treating colitis. This invention patent uses microwave-assisted low eutectic solvent extraction to prepare polysaccharide extracts of kiwifruit young fruit and kiwifruit mature fruit, and uses kiwifruit young fruit polysaccharide extracts and kiwifruit mature fruit polysaccharide extracts to treat ulcerative colitis. The efficacy is clear, especially the efficacy of kiwifruit young fruit polysaccharide extract is significant, which increases the commercial value of kiwifruit by-products. Application number 202311186897.7, invention name: A kiwifruit young fruit polysaccharide extract and its preparation method and use. This invention patent provides a kiwifruit young fruit polysaccharide extract and its preparation method and use. This invention patent uses traditional hot water extraction and microwave-assisted low eutectic solvent to prepare crude polysaccharides from kiwifruit young fruit. The molecular weight distribution of crude polysaccharides from kiwifruit young fruit is a heterogeneous polysaccharide, and its polydispersity coefficient (M w / M n ) ranged from 2.312 to 2.947, and the molecular weight of crude polysaccharide ranged from 1.265×10 5 Up to 6.520×10 5Da. This invention patent optimized the extraction parameters of kiwifruit young fruit crude polysaccharides extracted by microwave-assisted low eutectic solvent through single-factor experiments and Box-Behnken central composite design-response surface methodology, achieving a higher extraction rate and a shorter extraction time. Kiwifruit young fruit crude polysaccharides have in vitro antioxidant activity and in vitro immunomodulatory activity. Studies have found that although kiwifruit young fruit crude polysaccharides have potential in vitro immunomodulatory activity, their molecular weight distribution is uneven, their molecular weight is large, their degree of esterification is high, and their water solubility is relatively poor, which affects their immunomodulatory activity. In addition, there are no relevant literature reports on the structural characteristics of kiwifruit young fruit pectin polysaccharides and their modified pectin polysaccharides, and there are no relevant literature reports on their in vivo immunomodulatory activity and mechanism of action. Summary of the Invention

[0004] The present invention provides a kiwifruit young fruit pectin polysaccharide or a modified pectin polysaccharide thereof, a preparation method thereof, and use thereof in preparing a medicine or health food having an immune-enhancing effect.

[0005] The invention provides a kiwifruit young fruit pectin polysaccharide, which is prepared from kiwifruit young fruit as a raw material and has high purity. Each 100 mg of the polysaccharide contains 90.05 mg ± 0.65 mg of total polysaccharide, 25.49 mg ± 1.38 mg of total uronic acid, 2.79 mg ± 0.09 mg of total protein, and 7.83 mg GAE ± 0.09 mg GAE (gallic acid equivalent) of total bound phenols. The polysaccharide has an esterification degree of 41.56% ± 0.16% and a molecular weight (M w ) is (10.47±0.12)×10 4 Da, polydispersity coefficient (M w / M n ) is 1.868. It contains the following monosaccharides in molar percentages: mannose (Man) 2.10 mol%, rhamnose (Rha) 5.38 mol%, glucuronic acid (GlcA) 1.42 mol%, galacturonic acid (GalA) 16.74 mol%, glucose (Glc) 4.68 mol%, galactose (Gal) 52.16 mol%, and arabinose (Ara) 17.52 mol%. The molar proportion of polygalacturonic acid (HG) is 11.36 mol%, and the molar proportion of type I polyrhamnogalacturonic acid (RG-I) is 80.44 mol%. The side chain length of RG-I is 12.95.

[0006] The present invention provides a method for preparing the pectin polysaccharide from young kiwifruit, which is prepared by a deep eutectic solvent-assisted heating extraction method. The preparation method is as follows:

[0007] a. Freeze-dry the young kiwifruit, grind into powder and sieve:

[0008] b. Removal of alcohol-soluble components: Extract the freeze-dried powder of young kiwifruit with 80% (v / v) ethanol, centrifuge and discard the supernatant to obtain a precipitate;

[0009] c. Deep eutectic solvent-assisted heating extraction: according to a solid-liquid ratio of 1:50 (w / v), the extraction temperature was 95°C, and the extraction time was 4 hours; wherein the deep eutectic solvent was composed of choline chloride, ethylene glycol, and ultrapure water, and the molar ratio of choline chloride to ethylene glycol was 1:3, and the water content of the deep eutectic solvent was 40%. After the extraction was completed, the supernatant was retained by centrifugation;

[0010] d. Alcohol precipitation: Use 75% (v / v) ethanol (final concentration) for graded precipitation, then centrifuge and collect the precipitate;

[0011] e. Redissolve: Redissolve the precipitate from step d with ultrapure water;

[0012] f. Starch removal: Use α-amylase and saccharifying enzyme to remove starch in the reconstituted solution, centrifuge, and reserve the supernatant;

[0013] g. Ultrafiltration: The supernatant is subjected to centrifugal ultrafiltration to collect purified fractions with molecular weights between 3 kDa and 100 kDa, and then freeze-dried to obtain high-purity kiwifruit young fruit pectin polysaccharide (YKDHP).

[0014] Compared with Patent 202311186897.7, the main difference between the present invention and Patent 202311186897.7 is that the extraction method is low eutectic solvent-assisted heating extraction, and the extraction parameters are different; in addition, the present invention uses ultrafiltration membranes of different molecular weights to efficiently separate samples to prepare high-purity kiwifruit young fruit pectin polysaccharides, while the patent document uses a dialysis method to prepare crude kiwifruit young fruit polysaccharides.

[0015] The present invention also provides a modified pectin polysaccharide from young kiwifruit fruit. The pectin polysaccharide from young kiwifruit fruit is structurally modified by a low-temperature alkaline deesterification technology, an ultrasonic Fenton degradation technology, or a controlled partial acid hydrolysis technology. The modified pectin polysaccharide from young kiwifruit fruit has high purity, and contains 90.11mg±1.74mg-90.80mg±1.75mg of total polysaccharide, 23.23mg±1.37mg-58.52mg±1.24mg of total uronic acid, 2.34mg±0.08mg-2.68mg±0.04mg of total protein, and 3.21mg GAE±0.14mg GAE-6.78mg GAE±0.10mg GAE (gallic acid equivalent) of total bound phenols per 100mg. The degree of esterification is 5.59%±0.46%-47.48%±0.43%, and the molecular weight (M w ) is (0.609±0.021)×10 4 Da–(8.133±0.068)×10 4Da, polydispersity coefficient (M w / M n ) is 1.361–1.781. It contains the following monosaccharides in the following molar percentages: mannose (Man) 2.08mol%–5.66mol%, rhamnose (Rha) 4.99mol%–16.02mol%, glucuronic acid (GlcA) 1.49mol%–5.44mol%, galacturonic acid (GalA) 14.97mol%–57.62mol%, glucose (Glc) 1.72mol%–3.33mol%, galactose (Gal) 13.54mol%–52.55mol%, and arabinose (Ara) 0.00mol%–19.90mol%. The molar proportion of HG is 9.29mol%–41.60mol%, the molar proportion of RG-I is 45.58mol%–83.81mol%, and the side chain length of RG-I ranges from 0.84mol% to 12.76.

[0016] Further preferably, the modified pectin polysaccharide of kiwifruit young fruit has high purity, wherein each 100 mg contains 90.36 mg ± 1.81 mg of total polysaccharide, 23.23 mg ± 1.37 mg of total uronic acid, 2.68 mg ± 0.04 mg of total protein, and 6.78 mg GAE ± 0.10 mg GAE (gallic acid equivalent) of total bound phenols; its degree of esterification is 16.76% ± 0.09%, and its molecular weight (M w ) is: (8.133±0.068)×10 4 Da, polydispersity coefficient (M w / M n ) is 1.781; it contains the following monosaccharides in molar percentages: mannose (Man) 2.08 mol%, rhamnose (Rha) 5.68 mol%, glucuronic acid (GlcA) 1.49 mol%, galacturonic acid (GalA) 14.97 mol%, glucose (Glc) 3.33 mol%, galactose (Gal) 52.55 mol%, arabinose (Ara) 19.90 mol%; the molar proportion of HG is 9.29 mol%, the molar proportion of RG-I is 83.81 mol%, and the side chain length of RG-I is 12.76.

[0017] The present invention also provides a method for preparing the modified pectin polysaccharide of young kiwifruit, which comprises modifying the pectin polysaccharide YKDHP of young kiwifruit by low-temperature alkaline deesterification technology, ultrasonic Fenton degradation technology or controlled partial acid hydrolysis technology;

[0018] Wherein, the preparation method of the low-temperature alkaline deesterification technology is:

[0019] a. Take young kiwifruit pectin polysaccharide YKDHP and add it to NaOH solution with a pH value of 11.0, and stir at 4°C;

[0020] b. Add hydrochloric acid to the solution of step a until the pH value is neutral 7.0, and ultrafiltration is performed through an ultrafiltration centrifuge tube with a molecular weight limit of 3 kDa to retain the upper retained component, and then freeze-dry to obtain a kiwifruit young fruit modified pectin polysaccharide (YKDHP-D) with a reduced degree of esterification;

[0021] The preparation method of the ultrasonic Fenton degradation technology is:

[0022] a. Take young kiwifruit pectin polysaccharide YKDHP, add it to Vc solution and H2O2 solution, and ultrasonicate it at 520W and 24kHZ for 2h.

[0023] b. The solution of step a was centrifuged and ultrafiltered through an ultrafiltration centrifuge tube with a molecular weight limit of 3 kDa, the upper retained component was retained, and then freeze-dried to obtain a kiwifruit young fruit modified pectin polysaccharide (YKDHP-U) with reduced molecular weight;

[0024] The preparation method of the controllable partial acid hydrolysis technology is:

[0025] a. Take kiwifruit pectin polysaccharide YKDHP and add it to trifluoroacetic acid solution to make the final concentration of trifluoroacetic acid in the system 0.25M, then place it at 95℃ for 3 hours;

[0026] b. NaOH was added to the solution in step a until the pH value was neutral at 7.0, and the solution was ultrafiltered through an ultrafiltration centrifuge tube with a molecular weight limit of 3 kDa, followed by freeze-drying to obtain a modified pectin polysaccharide (YKDHP-A) of kiwifruit young fruit with reduced side chain length.

[0027] The present invention also provides use of the modified pectin polysaccharide from young kiwifruit in preparing health-care food or medicine that helps to enhance immunity.

[0028] The present invention uses a deep eutectic solvent-assisted heating extraction technology, supplemented by ultrafiltration membrane separation technology, to efficiently prepare high-purity kiwifruit pectin polysaccharides from young fruits. The kiwifruit pectin polysaccharides are then structurally modified by low-temperature alkaline deesterification technology, ultrasonic Fenton degradation technology, and controlled partial acid hydrolysis technology. By comparing the in vivo and in vitro immunomodulatory activities of kiwifruit pectin polysaccharides before and after modification, it was found that the kiwifruit pectin polysaccharides obtained by different modification technologies had better immunomodulatory activities, and the low-esterification kiwifruit pectin polysaccharide prepared by low-temperature alkaline deesterification technology exhibited the strongest in vivo and in vitro immunostimulatory effects. This invention not only helps to clarify the structure-function relationship and mechanism of action of the immunomodulatory activity of kiwifruit pectin polysaccharides, but also improves the utilization rate of kiwifruit by-products, reduces resource waste, and provides a theoretical basis for the development of high-value-added kiwifruit pectin polysaccharide products. It is of great significance to promote the added value of the kiwifruit industry and increase the income of growers. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 High-performance gel exclusion chromatography of pectin polysaccharides from young kiwifruit fruits and their modified pectin polysaccharides (Note 1) YKDHP, YKDHP-D, YKDHP-U and YKDHP-A are respectively high-purity pectin polysaccharides from young kiwifruit fruits prepared by deep eutectic solvent-assisted heating extraction, modified pectin polysaccharides prepared by low-temperature controlled alkaline deesterification technology, modified pectin polysaccharides prepared by ultrasonic Fenton degradation technology, and modified pectin polysaccharides prepared by controlled partial acid hydrolysis technology);

[0030] Figure 2 Monosaccharide composition of pectin polysaccharides from young kiwifruit fruits and their modified pectin polysaccharides (Note 1) YKDHP, YKDHP-D, YKDHP-U and YKDHP-A are respectively high-purity pectin polysaccharides from young kiwifruit fruits prepared by deep eutectic solvent-assisted heating extraction, modified pectin polysaccharides prepared by low-temperature controlled alkaline deesterification technology, modified pectin polysaccharides prepared by ultrasonic Fenton degradation technology, and modified pectin polysaccharides prepared by controlled partial acid hydrolysis technology);

[0031] Figure 3 Fourier transform infrared spectral characteristics of pectin polysaccharides from young kiwifruit fruits and their modified pectin polysaccharides (Note 1) YKDHP, YKDHP-D, YKDHP-U and YKDHP-A are high-purity pectin polysaccharides from young kiwifruit fruits prepared by deep eutectic solvent-assisted heating extraction, modified pectin polysaccharides prepared by low-temperature controlled alkaline deesterification technology, modified pectin polysaccharides prepared by ultrasonic Fenton degradation technology, and modified pectin polysaccharides prepared by controlled partial acid hydrolysis technology, respectively);

[0032] Figure 4 Pectin polysaccharides and modified pectin polysaccharides from young kiwifruit 1H NMR and 13 C NMR spectra (Note 1) YKDHP, YKDHP-D, YKDHP-U, and YKDHP-A are respectively high-purity pectin polysaccharides from young kiwifruit prepared by deep eutectic solvent-assisted heating extraction, modified pectin polysaccharides prepared by low-temperature controlled alkaline deesterification technology, modified pectin polysaccharides prepared by ultrasonic Fenton degradation technology, and modified pectin polysaccharides prepared by controlled partial acid hydrolysis technology);

[0033] Figure 5 Effects of pectin polysaccharides from young kiwifruit and their modified pectin polysaccharides on the cytotoxicity (A), nitric oxide (NO) (B), and TNF-α (C) and IL-6 cytokine (D) contents of RAW 264.7 immune cells (Note 1) YKDHP, YKDHP-D, YKDHP-U, and YKDHP-A are high-purity pectin polysaccharides from young kiwifruit prepared by deep eutectic solvent-assisted heating extraction, modified pectin polysaccharides prepared by low-temperature controlled alkaline deesterification technology, modified pectin polysaccharides prepared by ultrasonic Fenton degradation technology, and modified pectin polysaccharides prepared by controlled partial acid hydrolysis technology, respectively; Note 2) Error bars are standard deviations; significant differences (p < 0.05) between different samples are indicated by different letters (ad); significant differences between the tested samples and the blank control are indicated by *p < 0.05 and **p < 0.01).

[0034] Figure 6 Effects of C29 or TAK-242 on the production of NO (A), TNF-α (B), and IL-6 (C) in RAW 264.7 macrophages, and the effect of modified pectin polysaccharides in young kiwifruit on protein expression (D) (Note 1) YKDHP-D is a modified pectin polysaccharide prepared by low-temperature controlled alkaline deesterification technology; Note 2) Error bars are standard deviations; significant differences between the tested samples and the blank control are indicated by *p < 0.05 and **p < 0.01; significant differences between the tested samples and the TAK-242-treated group are indicated by # p < 0.05 and ## p<0.01 indicates a significant difference between the tested sample and the C29-treated group. ※ p < 0.05 and ※※ p < 0.01);

[0035] Figure 7Effects of kiwifruit pectin polysaccharides and modified pectin polysaccharides on body weight (A) and organ indexes (B and C) of immunosuppressed mice (Note 1) NC, MC, PC, YKDHP, YKDHP-D, YKDHP-U, and YKDHP-A represent blank control group, model group, positive group, gavage kiwifruit pectin polysaccharide group, gavage kiwifruit modified pectin polysaccharide group with reduced esterification degree, gavage kiwifruit modified pectin polysaccharide group with reduced molecular weight, and gavage kiwifruit modified pectin polysaccharide group with reduced RG-I side chain length, respectively; error bars represent standard deviations; significant differences (p < 0.05) between different samples are represented by different letters; significant differences are represented by *p < 0.05 and **p < 0.01).

[0036] Figure 8 Effects of kiwifruit pectin polysaccharides and modified pectin polysaccharides on the spleen tissue morphology of immunosuppressed mice (pathological observation of mouse spleen) (Note 1) NC, MC, PC, YKDHP, YKDHP-D, YKDHP-U, and YKDHP-A represent blank control group, model group, positive group, gavage kiwifruit pectin polysaccharide group, gavage kiwifruit modified pectin polysaccharide group with reduced esterification degree, gavage kiwifruit modified pectin polysaccharide group with reduced molecular weight, and gavage kiwifruit modified pectin polysaccharide group with reduced RG-I side chain length, respectively);

[0037] Figure 9 Effects of kiwifruit pectin polysaccharides and modified pectin polysaccharides on the levels of immune cytokines and immunoglobulins in the serum of immunosuppressive mice (Note 1) NC, MC, PC, YKDHP, YKDHP-D, YKDHP-U, and YKDHP-A represent blank control group, model group, positive group, gavage kiwifruit pectin polysaccharide group, gavage kiwifruit modified pectin polysaccharide group with reduced esterification degree, gavage kiwifruit modified pectin polysaccharide group with reduced molecular weight, and gavage kiwifruit modified pectin polysaccharide group with reduced RG-I side chain length, respectively; error bars represent standard deviations; significant differences (p < 0.05) among different samples are represented by different letters). DETAILED DESCRIPTION

[0038] Example 1 Preparation of Kiwifruit Young Fruit Pectin Polysaccharides and Modified Pectin Polysaccharides

[0039] 1. Preparation of high-purity and uniform kiwifruit pectin polysaccharides by deep eutectic solvent-assisted heating extraction method

[0040] After freeze-drying, the young kiwifruit fruit was ground into powder and sieved (80 mesh) to prepare kiwifruit freeze-dried powder. Subsequently, 10 g of the freeze-dried powder was mixed with 80% ethanol at a material-to-liquid ratio of 1:10 (w / v), and then sonicated at 480 W for 30 minutes to remove alcohol-soluble components. After the ultrasound is completed, the mixture is centrifuged at 5000×g for 10 minutes, and the supernatant is discarded to obtain a precipitate; subsequently, a low eutectic solvent is used to assist heating to extract pectin polysaccharides from young kiwifruit fruits, according to a material-liquid ratio of 1:50 (w / v), an extraction temperature of 95°C, and an extraction time of 4 hours, wherein the low eutectic solvent is composed of choline chloride, ethylene glycol and ultrapure water, and the molar ratio of choline chloride to ethylene glycol is 1:3, and the water content of the low eutectic solvent is 40%. After the extraction is completed, the mixture is centrifuged at 5000×g for 10 minutes, and the supernatant is retained; subsequently, 75% v / v ethanol (final concentration) is used for graded precipitation, and after the alcohol precipitation is allowed to stand overnight, the precipitate is collected after centrifugation at 5000×g for 10 minutes; the precipitate after alcohol precipitation is further The product was reconstituted with ultrapure water; subsequently, α-amylase (5 U / mL, 85°C, 10 h) and saccharifying enzyme (5 U / mL, 59°C, 10 h) were used to remove starch from crude polysaccharides of young kiwifruit fruits; the product was inactivated at 95°C for 30 min, centrifuged at 5000×g for 15 min, and the supernatant was retained. The supernatant was used for subsequent ultrafiltration membrane centrifugation and ultrafiltration purification, that is, the supernatant was placed in ultrafiltration centrifuge tubes with molecular weight limits of 100 kDa and 3 kDa, respectively, and ultrafiltration centrifuged at 4000×g for 15 min (this step was repeated 3-5 times). Subsequently, purified fractions with molecular weights between 3 kDa and 100 kDa were collected, and finally freeze-dried to obtain high-purity kiwifruit young fruit pectin polysaccharide (YKDHP).

[0041] 2. Preparation of modified pectin polysaccharides from kiwifruit using low-temperature alkaline deesterification technology

[0042] The high-purity kiwifruit young fruit pectin polysaccharide YKDHP prepared in step 1 was added to a NaOH solution with a pH value of 11.0 (the final concentration of YKDHP was 5 mg / mL), and the deesterification reaction was stirred at 4°C for 30 minutes; subsequently, hydrochloric acid (1M) was added to the mixed solution until the mixed solution was neutral (pH value was 7.0), and then centrifuged and ultrafiltered through an ultrafiltration centrifuge tube with a molecular weight limit of 3 kDa (4000×g, 15 minutes, and this step was repeated 3-5 times), and finally freeze-dried to obtain a modified kiwifruit young fruit pectin polysaccharide (YKDHP-D) with a reduced degree of esterification.

[0043] 3. Preparation of modified pectin polysaccharides from kiwifruit using ultrasonic Fenton degradation technology

[0044] The highly purified kiwifruit pectin polysaccharide YKDHP prepared in step 1 was added to a 20 mM vitamin C solution and a 40 mM H₂O₂ solution (final YKDHP concentration of 6 mg / mL) and subjected to ultrasonic degradation under ultrasonic conditions of 520 W and 24 kHz for 2 h. Subsequently, the supernatant was ultrafiltered through an ultrafiltration tube with a molecular weight cutoff of 3 kDa (4000 × g, 15 min, repeated 3-5 times), and finally freeze-dried to obtain a modified kiwifruit pectin polysaccharide with a reduced molecular weight (YKDHP-U).

[0045] 4. Modified pectin polysaccharides from young kiwifruit prepared by controlled acid hydrolysis technology:

[0046] The high-purity kiwifruit young fruit pectin polysaccharide YKDHP prepared in step 1 was added to a 0.5M trifluoroacetic acid solution (the final concentration of YKDHP was 5 mg / mL), and the final concentration of trifluoroacetic acid in the mixed solution was 0.25M. The mixed solution was placed at 95°C for reaction for 3 hours. Subsequently, NaOH (1M) was added to the mixed solution until the mixed solution was neutral (pH value was 7.0), and then centrifuged and ultrafiltered in an ultrafiltration centrifuge tube with a molecular weight limit of 3 kDa (4000×g, 15 min, repeating this step 3-5 times), and finally freeze-dried to obtain a kiwifruit young fruit modified pectin polysaccharide (YKDHP-A) with reduced side chain length.

[0047] Example 2 Characterization of the physicochemical properties of the pectin polysaccharides of young kiwifruit and their modified pectin polysaccharides of the present invention

[0048] 1. Experimental methods:

[0049] 1. Chemical composition analysis

[0050] The total polysaccharide content of pectin polysaccharides and modified pectin polysaccharides in young kiwifruit was determined using the phenol-sulfuric acid method. 4 mg of the sample to be tested was accurately weighed and dissolved in 10 mL of ultrapure water to prepare a 0.4 mg / mL solution. 100 μL of the sample solution was accurately measured and placed in a 2 mL EP tube. 50 μL of 6% phenol solution was added and mixed thoroughly. After mixing, 250 μL of concentrated sulfuric acid was added in the dark, and the mixture was incubated at 90°C for 10 min. After cooling the reaction solution to room temperature, the OD value was measured at 490 nm. Three replicates were performed, and the average of the experimental results was calculated. Ultrapure water was used as a blank control. 10 mg each of galactose and galacturonic acid standards were accurately weighed and placed in a 10 mL volumetric flask. 10 mL of ultrapure water was added to prepare a 2 mg / mL stock solution. The stock solution was then diluted to 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively. The determination was performed according to the above method, and a standard curve was drawn with the standard concentration X (mg / mL) as the abscissa and the absorbance value Y as the ordinate.

[0051] The total uronic acid content of pectin polysaccharides and modified pectin polysaccharides in young kiwifruit was determined using the m-hydroxybiphenyl method. Accurately weigh 5 mg of the sample to be tested and dissolve it in 10 mL of ultrapure water to prepare a 0.5 mg / mL sample solution. Pipette 50 μL of the sample solution to be tested, add 500 μL of 12.5 mM sodium tetraborate-sulfuric acid solution, mix well, and incubate at 95°C in the dark for 10 minutes. Rapidly cool the reaction solution to room temperature, add 10 μL of 0.15% 3-phenylphenol solution, and react at room temperature in the dark for 15 minutes before measuring its OD value at 520 nm. Three parallel experiments were performed, and the experimental results were averaged. Ultrapure water was used as a blank control. Accurately weigh 10 mg of galacturonic acid standard into a 10 mL volumetric flask. Add 10 mL of ultrapure water to prepare a 1 mg / mL standard solution. Then dilute the standard solution to 0.1, 0.15, 0.2, 0.25, and 0.3 mg / mL. Determine the concentration of galacturonic acid (X) as the abscissa and the absorbance (Y) as the ordinate to create a standard curve.

[0052] The total protein content of kiwifruit pectin polysaccharides and modified pectin polysaccharides was determined using the Coomassie Brilliant Blue method. The sample to be tested was configured as an 8 mg / mL stock solution. 50 μL of sample solution was drawn, and 250 μL of Coomassie Brilliant Blue solution was added. After mixing, the mixture was protected from light and reacted for 15 minutes, and the OD value was measured at 595 nm. Three parallel groups were set up, and the experimental results were averaged. Ultrapure water was used as a blank control. 10 mg of bovine serum albumin (BSA) was accurately weighed in a 2 mL volumetric flask, and 2 mL of ultrapure water was added to prepare a 5 mg / mL BSA stock solution. The BSA stock solution was then diluted to 0.1, 0.15, 0.2, 0.25, and 0.3 mg / mL. The determination was performed according to the above method, and a standard curve was drawn with BSA concentration (X) (mg / mL) as the horizontal coordinate and absorbance value (Y) as the vertical coordinate.

[0053] The total bound phenol content of pectin polysaccharides and modified pectin polysaccharides in young kiwifruit was determined using the Folin-phenol method. The sample to be tested was prepared as a 1 mg / mL stock solution. 50 μL of the sample solution was aspirated and 250 μL of Folin-phenol solution was added. The mixture was mixed and reacted for 3 minutes. 20% NaCO₃ was then added to the solution in the dark for 30 minutes. The OD value was then measured at 760 nm. Three replicates were performed, and the results were averaged. Ultrapure water was used as a blank control. 10 mg of gallic acid was accurately weighed into a 2 mL volumetric flask. 2 mL of ultrapure water was added to prepare a 5 mg / mL stock solution of gallic acid. The stock solution was then diluted to 0.02, 0.04, 0.06, 0.08, and 0.10 mg / mL. The assay was performed according to the above method, and a standard curve was plotted with the gallic acid concentration (X) (mg / mL) as the abscissa and the absorbance (Y) as the ordinate.

[0054] 2. Molecular Weight Determination

[0055] The molecular weight and distribution of pectin polysaccharides and modified pectin polysaccharides from young kiwifruit were analyzed by high performance gel exclusion chromatography coupled with multi-angle laser light scattering and differential detection (SEC-MALLS-RID). The chromatographic column was a Shodex OHpakSB-804HQ column (8.0×300 mm), the mobile phase was a 0.9% NaCl aqueous solution, the flow rate was 0.5 mL / min, the sample concentration was approximately 2.5 mg / mL, and the injection volume was 100 μL.

[0056] 3. Sugar Composition Determination

[0057] Accurately weigh 6 mg of each sample to be tested and dissolve it in 1 mL of ultrapure water. Once dissolved, add 1 mL of 4 mM trifluoroacetic acid (TFA) and hydrolyze at 95°C for 12 h. After cooling the hydrolyzate to room temperature, add 1 mL of methanol solution, mix, and spin dry on a vacuum rotary evaporator at 60°C. Repeat this process four times to completely remove residual TFA. Finally, dissolve the dried hydrolyzed sample in 1 mL of ultrapure water for subsequent derivatization. For derivatization, accurately pipette 50 μL of the hydrolyzate into a 5 mL EP tube, add 50 μL of 0.6 mM sodium hydroxide solution and 100 μL of 0.5 mM 1-phenyl-3-methyl-5-pyrazolone (PMP) solution, mix thoroughly, and incubate at 70°C in the dark for 100 min. The reaction solution was cooled to room temperature, and 30 μL of 0.3 mM HCl solution was added. Extraction was then performed with 1 mL of chloroform solution, and the chloroform layer was removed. This process was repeated six times to remove any residual PMPs. The neutral derivatization solution was then adjusted to a total volume of 1 mL with ultrapure water. Finally, the derivatized solution was filtered through a 0.22 μm organic phase filter and used for HPLC analysis. The mixed standard solution for this experiment consisted of rhamnose, mannose, galacturonic acid, glucuronic acid, galactose, glucose, xylose, and arabinose at a concentration of 0.5 mg / mL. The standard solution was derivatized using the above steps. The derivatized products were detected by HPLC. The HPLC detection conditions were as follows: the chromatographic column was ZORBAX Eclipse XDB-C18 (particle size was 5 μm), the injection volume was 20 μL, the column temperature was controlled at 35°C, the mobile phase was: phase A was 0.1 mol / L phosphate buffer (pH = 6.7), phase B was acetonitrile, the ratio of phase A to phase B was 82.5:17.5 (v / v), the flow rate was 1.0 mL / min, and the DAD detection wavelength was 245 nm.

[0058] 4. Infrared spectroscopy

[0059] Weigh 1.5 mg of the sample to be tested and add it to dry potassium bromide powder. Then, mix and grind them evenly and compress them into tablets. The compressed tablets are placed in a Fourier transform infrared spectrometer at 4000-500 cm -1 The infrared spectrum is drawn by scanning the range.

[0060] 5. Nuclear Magnetic Resonance Spectroscopy

[0061] 40 mg of the sample to be tested was accurately weighed and then dissolved in 1.0 mL of D2O. The one-dimensional hydrogen spectrum of the sample to be tested was measured using a Bruker Ascend 600 MHz spectrometer with a z-gradient probe ( 1 H) and one-dimensional carbon spectra ( 13 C). The acquisition temperature of the hydrogen and carbon spectra was 25°C, the carbon frequency was 150.90 MHz, and the proton frequency was 600.13 MHz.

[0062] 2. Experimental results:

[0063] 1. Chemical composition of pectin polysaccharides and modified pectin polysaccharides from young kiwifruit

[0064] The chemical compositions of kiwifruit pectin polysaccharide (YKDHP) and its modified pectin polysaccharides (YKDHP-D, YKDHP-U, and YKDHP-A) are summarized in Table 1. The results showed that the total polysaccharide contents of kiwifruit pectin polysaccharide (YKDHP) and its modified pectin polysaccharides (YKDHP-D, YKDHP-U, and YKDHP-A) were relatively stable, at 90.05±0.65 mg / 100 mg, 90.36±1.81 mg / 100 mg, 90.11±1.74 mg / 100 mg, and 90.80±1.75 mg / 100 mg, respectively, indicating that the kiwifruit pectin polysaccharide and its modified pectin polysaccharide had extremely high polysaccharide purity. Compared with the original kiwifruit young fruit pectin polysaccharide (YKDHP), the total uronic acid content of the modified pectin polysaccharide of kiwifruit young fruit with reduced esterification degree (YKDHP-D) prepared by low-temperature alkaline deesterification technology slightly decreased to 23.23±1.37 mg / 100 mg, the total uronic acid content of the low molecular weight modified pectin polysaccharide of kiwifruit young fruit (YKDHP-DU) prepared by ultrasonic Fenton degradation technology slightly increased to 28.76±1.57 mg / 100 mg, and the total uronic acid content of the modified pectin polysaccharide of kiwifruit young fruit with reduced side chain length (YKDHP-A) prepared by controlled acid hydrolysis technology significantly increased to 58.52±1.24 mg / 100 mg. In addition, compared with the original kiwifruit pectin polysaccharides, the total protein content of the modified kiwifruit pectin polysaccharides modified by low-temperature alkaline deesterification technology, ultrasonic Fenton degradation technology, and controlled partial acid hydrolysis technology was slightly reduced.

[0065] Table 1 Chemical composition, molecular weight, dispersion coefficient and monosaccharide composition of pectin polysaccharides and modified pectin polysaccharides from young kiwifruit

[0066]

[0067] Note 1) YKDHP, YKDHP-D, YKDHP-U and YKDHP-A are respectively high-purity kiwifruit pectin polysaccharides prepared by deep eutectic solvent-assisted heating extraction, modified pectin polysaccharides prepared by low-temperature controlled alkaline deesterification technology, modified pectin polysaccharides prepared by ultrasonic Fenton degradation technology, and modified pectin polysaccharides prepared by controlled partial acid hydrolysis technology; 2) mg GAE / 100 mg, gallic acid equivalent per 100 mg of polysaccharide; 3) Superscript (ad) indicates significant differences among different groups (p < 0.05); 4) Calculation formula: HG (mol%) = GalA (mol%) - Rha (mol%); RG-I (mol%) = GalA (mol%) - HG (mol%) + Rha (mol%) + Gal (mol%) + Ara (mol%); RG-I side chain length = (Ara (mol%) + Gal (mol%)) / Rha (mol%)

[0068] 2. Structural characteristics of pectin polysaccharides and modified pectin polysaccharides from young kiwifruit

[0069] (1) Molecular weight distribution

[0070] High performance gel exclusion chromatography (HPSEC) of pectin polysaccharides and modified pectin polysaccharides from young kiwifruit fruits Figure 1 The results showed that both kiwifruit pectin polysaccharide (YKDHP) and modified pectin polysaccharide with reduced esterification degree (YKDHP-D) showed symmetrical elution curves, and the elution curves mostly overlapped and the retention times were close, indicating that the molecular weight of modified pectin polysaccharide with reduced esterification degree prepared by low-temperature alkaline deesterification technology was slightly reduced. As shown in Table 1, the molecular weight (M w ) were (10.47±0.12)×10 4 Da sum (8.133±0.068)×10 4 Da, their polydispersity coefficient (M w / M n ) were 1.868 and 1.781 respectively; however, the high performance gel exclusion chromatograms of the modified pectin polysaccharides (YKDHP-U and YKDHP-A) of kiwifruit young fruits modified by ultrasonic Fenton degradation technology and controlled partial acid hydrolysis technology were obviously shifted to the right and narrowed, indicating that their molecular weight and dispersion coefficient were significantly reduced. As shown in Table 1, the molecular weight (Mw ) were reduced to (1.176±0.026)×10 4 Da sum (0.609±0.021)×10 4 Da, polydispersity coefficient (M w / M n ) decreased to 1.514 and 1.361.

[0071] (2) Monosaccharide composition type

[0072] like Figure 2As shown in Table 1, the monosaccharide compositions of kiwifruit young fruit pectin polysaccharides (YKDHP) and kiwifruit young fruit modified pectin polysaccharides (YKDHP-D, YKDHP-U and YKDHP-A) are similar, mainly including rhamnose (Rha), mannose (Man), glucuronic acid (GlcA), galacturonic acid (Gal), glucose (Glc), galactose (Gal) and arabinose (Ara). As shown in Table 1, the molar percentages of the constituent sugars of YKDHP, YKDHP-D, YKDHP-U and YKDHP-A are as follows: mannose (Man) 2.08 mol%–5.66 mol%, rhamnose (Rha) 4.99 mol%–16.02 mol%, glucuronic acid (GlcA) 1.42 mol%–5.44 mol%, galacturonic acid (GalA) 14.97 mol%–57.62 mol%, The molar proportions of glucose (Glc) ranged from 1.72 mol% to 4.68 mol%, galactose (Gal) from 13.54 mol% to 52.55 mol%, and arabinose (Ara) from 0.00 mol% to 19.90 mol%. The molar proportions of HG ranged from 9.29 mol% to 41.60 mol%, and the molar proportions of RG-I ranged from 45.58 mol% to 83.81 mol%. The side chain lengths of RG-I ranged from 0.84 to 12.95. Compared with YKDHP, the molar proportions of the constituent sugars in YKDHP-D, YKDHP-U, and YKDHP-A prepared by low-temperature alkaline deesterification, ultrasonic Fenton degradation, and controlled partial acid hydrolysis all changed to varying degrees. The molar percentages of the constituent sugars in YKDHP and YKDHP-D were very similar, indicating that the low-temperature alkaline deesterification modification technique did not alter the chemical structure of the pectin polysaccharides in young kiwifruit. However, compared with YKDHP, the galacturonic acid content of YKDHP-U increased from 16.74 mol% to 22.48 mol%. Compared with YKDHP, the arabinose content of YKDHP-A was removed, and the galactose content was significantly reduced from 52.16 mol% to 13.54 mol%, while the galacturonic acid content was significantly increased from 16.74 mol% to 57.62 mol%. In addition, compared with YKDHP, the HG molar ratio of YKDHP-A increased significantly from 11.36 mol% to 41.6 mol%, the RG-I molar ratio decreased significantly from 80.44 mol% to 45.58%, and the RG-I side chain length decreased significantly from 12.95 to 0.84.

[0073] (3) Infrared spectral characteristics

[0074] like Figure 3As shown in the figure, compared with the pectin polysaccharide (YKDHP) of young kiwifruit fruit, the main infrared spectral characteristics of the modified pectin polysaccharides (YKDHP, YKDHP-D, YKDHP-U, and YKDHP-A) of young kiwifruit fruit did not change significantly after being modified by different techniques, including the 3430 cm -1 (OH vibration), 2920cm -1 (CH vibration), 1744cm -1 (COO-R vibration), 1614cm -1 (COO-H vibration), 1441cm -1 (C-OH vibration), 1256cm -1 (COC vibration), 1102cm -1 (CO vibration) and 1020cm -1 (CC vibration) and other characteristic infrared spectrum absorption peaks. Compared with YKDHP, YKDHP-D and YKDHP-A have the peaks at 1744cm -1 The peak intensity at around 1614 cm -1 Compared with YKDHP, YKDHP-U has a peak intensity of 1744 cm -1 The peak intensity at around 1614 cm -1 The peak intensity around 1744cm -1 The peak area of 1744 cm -1 and 1614cm -1 The degree of esterification of pectin polysaccharides was estimated by the average ratio of the sum of the peak areas of YKDHP and YKDHP. As shown in Table 1, after the low-temperature alkaline deesterification technology was used to modify YKDHP (esterification degree of 41.56%), its esterification degree was significantly reduced to 16.76% (YKDHP-D); after the ultrasonic Fenton degradation technology was used to modify it, its esterification degree slightly increased to 47.48% (YKDHP-U); after the controlled partial acid hydrolysis technology was used to modify it, its esterification degree was significantly reduced to 5.59% (YKDHP-A).

[0075] (4) Nuclear magnetic resonance spectroscopy

[0076] The chemical structure characteristics of kiwifruit pectin polysaccharides and modified pectin polysaccharides were further analyzed by one-dimensional nuclear magnetic resonance spectroscopy. Figure 4As shown in the figure, there are different degrees of differences in the 1D NMR spectra of kiwifruit pectin polysaccharide (YKDHP) and modified pectin polysaccharides (YKDHP-D, YKDHP-U, YKDHP-A). In YKDHP, the signals at 1.25ppm, 5.25ppm, and 16.40ppm are attributed to 1,2,4-α-L-Rhap; 1.30ppm and 5.32ppm are attributed to 1,2-α-L-Rhap; the signals at 4.98ppm, 100.39ppm, and 170.62ppm are attributed to 1,4-α-D-GalAMep; the signals at 5.03ppm, 99.51ppm, and 173.31ppm are attributed to 1,4-α-D-GalAp; and the signals at 5.10ppm and 107.4ppm are attributed to 1,4-α-D-GalAp. The signal at 9 ppm is assigned to 1,5-α-L-Araf; the signals at 5.14 ppm and 109.18 ppm are related to H-1 and C-1 of T-α-L-Araf / 1,3,5-α-L-Araf, respectively; the signals at 5.18 ppm and 107.27 ppm are assigned to 1,3-α-L-Araf; and the signals at 3.81 ppm and 52.82 ppm are from GalA-OCH3. The signals at 2.08 ppm, 2.18 ppm, and 20.07 ppm represent the O-acetyl group of GalAp. The signals at 4.47 ppm and 103.24 ppm are assigned to 1,3,6-β-D-Galp. The signals at 4.53 ppm and 103.24 ppm are assigned to 1,3-β-D-Galp. These signals indicate that YKDHP is primarily composed of HG and RG-I type pectin domains. Compared to YKDHP, the signal intensities at 3.81 ppm, 52.82 ppm, 20.07 ppm, 4.98 ppm, and 170.62 ppm in YKDHP-D were weakened, indicating that the present invention can reduce or remove the methylated and acetylated chemical groups of GalAp residues in pectin polysaccharides in young kiwifruit fruit through low-temperature alkaline deesterification. The NMR spectra of YKDHP-U and YKDHP are similar, indicating that ultrasonic Fenton degradation has no significant effect on the primary chemical structure of pectin polysaccharides in young kiwifruit fruit. Compared with YKDHP, in YKDHP-A, the signal intensities of 2.08ppm, 2.18ppm, 3.81ppm, 4.47ppm, 4.53ppm, 5.10ppm, 5.14ppm, 5.18ppm, 20.07ppm, 52.82ppm, 103.24ppm, 107.27ppm, 107.49 and 109.18ppm were weakened or even disappeared, indicating that the present invention can successfully reduce or remove Araf and Galp residues of kiwifruit pectin polysaccharides through controllable partial acid hydrolysis technology, and can significantly reduce the methylation and acetylation chemical groups of GalAp residues, and significantly increase the proportion of RG-I main chain (the signal at 16.40ppm is significantly enhanced).

[0077] The beneficial effects of the kiwifruit young fruit pectin polysaccharide and its modified pectin polysaccharide of the present invention are demonstrated by efficacy tests below.

[0078] Example 1 In vitro immunological activity analysis and mechanism evaluation of pectin polysaccharides and modified pectin polysaccharides of young kiwifruit of the present invention

[0079] 1. Experimental Methods

[0080] 1. Cytotoxicity assay:

[0081] RAW 264.7 macrophages were cultured at a volume of 5 × 10 3 Cells were plated at a concentration of 100 μL / well in a 96-well microplate and cultured overnight in an incubator. The supernatant was aspirated, and 100 μL of YKDHP, YKDHP-D, YKDHP-U, or YKDHP-A (25, 50, 100, 200, or 400 μg / mL) was added to each well and incubated for 24 hours. The blank culture medium served as the blank control group, and LPS (1 μg / mL) served as the positive control group. The supernatant was aspirated, and 100 μL of MTT solution (1 mg / mL) was added to each well and cultured for another 4 hours. The supernatant was aspirated, and 100 μL of DMSO was added to each well. The absorbance was measured at 570 nm. The cell proliferation rate was calculated using the following formula:

[0082] Cell proliferation rate (%) = 1-(A1-A2) / A1×100%

[0083] Among them, A1 is the absorbance value of the blank group, and A2 is the absorbance value of the sample group.

[0084] 2. Determination of nitric oxide and cytokine content:

[0085] RAW 264.7 cells were cultured at 1 × 10 5 Cells were cultured overnight in a 24-well plate at a concentration of 100 μg / well. The supernatant was aspirated, and 1 mL of YKDHP, YKDHP-D, YKDHP-U, or YKDHP-A (50, 100, or 200 μg / mL) was added to each well and incubated for 48 hours. The culture medium served as the blank control, and LPS (1 μg / mL) served as the positive control. Subsequently, the supernatant was collected and the levels of nitric oxide (NO) and cytokines (TNF-α and IL-6) were measured according to the Griess and ELISA kit instructions, respectively.

[0086] 3. Cellular immune mechanism of pectin polysaccharides in young kiwifruit

[0087] (1) Inhibit TLR2 / 4 signaling

[0088] RAW 264.7 cells were plated at 1 × 10 5Cells were cultured overnight at 1 mL / well in a 24-well plate. After removing the culture medium, 1 mL of culture medium containing or not TAK-242 (1 μM) or C29 (30 μM) was added to the wells and cultured for another 4 hours. Subsequently, the cells were treated with YKDHP-D (200 μg / mL) or LPS (1 μg / mL) for 48 hours. Total NO content was measured by the Griess method, and TNF-α and IL-6 levels were measured using ELISA kits.

[0089] (2) Western blot analysis

[0090] RAW 264.7 cells were plated at 4 × 10 5 Cells were cultured overnight in a 6-well plate. After removing the culture medium, 2 mL of YKDHP-D (200 μg / mL) was added to the wells and incubated for 48 hours. The culture medium was used as the blank control group, and LPS (1 μg / mL) was used as the positive control group. The supernatant was removed and the well plate was rinsed twice with PBS. 1 mL of PBS was added to each well, the cells were gently blown off and transferred to a 1.5 mL centrifuge tube, and the cells were collected after centrifugation. RIPA cell lysis buffer (containing 0.1% PMSF and protein phosphatase inhibitors) was added and lysed on ice for 30 minutes. Capillary protein immunoblot analysis was performed on the Protein Simple Wes system using a 12-230 kDa separation module (SM-W004) and an anti-rabbit detection module (DM-001) according to the manufacturer's instructions. Data were analyzed using Compass software, and the appropriate exposure time was set to ensure maximum signal.

[0091] 2. Experimental Results

[0092] 1. In vitro immunological activity of pectin polysaccharides and modified pectin polysaccharides from young kiwifruit

[0093] Figure 5 A shows the cytotoxic effects of kiwifruit pectin polysaccharide (YKDHP) and its modified pectin polysaccharides (YKDHP-D, YKDHP-U, and YKDHP-A) on RAW 264.7 cells. The results showed that at the experimental concentrations (25-400 μg / mL), YKDHP, YKDHP-D, YKDHP-U, and YKDHP-A had no toxic effects on the cell viability of RAW 264.7 macrophages. In addition, Figure 5As shown in Figure B-5D, different concentrations (50-200 μg / mL) of YKDHP, YKDHP-D, YKDHP-U, and YKDHP-A can significantly increase the levels of NO, IL-6, and TNF-α produced by RAW 264.7 macrophages, indicating that kiwifruit pectin polysaccharides and their modified pectin polysaccharides have significant in vitro immunostimulatory effects, and the in vitro immunostimulatory effects of modified kiwifruit pectin polysaccharides modified by different modification techniques are significantly enhanced. Specifically, compared with YKDHP, the immunostimulatory effect of YKDHP-D is significantly enhanced, indicating that the low-temperature alkaline deesterification technology enhances the immunostimulatory effect of kiwifruit pectin polysaccharides by reducing the esterification degree of kiwifruit pectin polysaccharides, indicating that the in vitro immunostimulatory effect of kiwifruit pectin polysaccharides is negatively correlated with their esterification degree. Compared with YKDHP, the molecular weight of YKDHP-U is significantly reduced, and its in vitro immunostimulatory effect is also enhanced, which indicates that ultrasonic Fenton degradation technology can improve the in vitro immunostimulatory effect of kiwifruit pectin polysaccharides by reducing the molecular weight. Compared with YKDHP, the in vitro immunostimulatory effect of YKDHP-A is also enhanced. However, the immunostimulatory effect of YKDHP-A is weaker than that of YKDHP-D and YKDHP-U. By comparing and analyzing their structural characteristics, it was found that the molecular weight of YKDHP-A is significantly reduced, the degree of esterification is significantly reduced, but its arabinose side chains are removed and the galactose side chains are also reduced. This indicates that controlled partial acid hydrolysis technology may improve the in vitro immunostimulatory effect of kiwifruit pectin polysaccharides by reducing the molecular weight and degree of esterification, but reducing the length of its RG I side chain may lead to a decrease in its immunostimulatory effect. In summary, the results of the present invention show that the degree of esterification and molecular weight of kiwifruit pectin polysaccharides may be negatively correlated with their immunostimulatory effect, while the length of the RG-I side chain may be positively correlated with its immunostimulatory effect. In particular, low-temperature controllable alkaline deesterification technology can significantly enhance the immunostimulatory effect of pectin polysaccharides in young kiwifruit fruits by selectively reducing the esterification degree of pectin polysaccharides in young kiwifruit fruits and maintaining their chemical structure characteristics.

[0094] 2. Potential cellular immune mechanisms of modified pectin polysaccharides from young kiwifruit

[0095] To further reveal the potential mechanism of macrophage activation mediated by modified kiwifruit pectin polysaccharide (YKDHP-D) obtained by low-temperature controlled alkaline deesterification technology, we analyzed the effects of C29 (a selective TLR2 signaling inhibitor) and TAK-242 (a selective TLR4 signaling inhibitor) inhibitors on the release of NO, TNF-α, and IL-6 from RAW 264.7 macrophages. Figure 6As shown in Figures A-6C, both C29 and TAK-242 inhibitors significantly inhibited the secretion of NO, TNF-α, and IL-6 by RAW 264.7 macrophages, and the inhibition rate of TAK-242 was significantly higher than that of C29. These results confirm that YKDHP-D can activate RAW 264.7 macrophages to exert immune stimulation by interacting with TLR2 or TLR4 receptors on the surface of macrophages, and that TLR4 receptors have a greater impact on the immune effects of modified pectin polysaccharides from young kiwifruit.

[0096] Polysaccharides can bind to pattern recognition receptors (PRRs) on the surface of immune cells, activating downstream signaling pathways and thereby regulating immune cell activation and cytokine secretion, thereby modulating immune function. As mentioned above, modified pectin polysaccharide from young kiwifruit (YKDHP-D) can activate RAW 264.7 macrophages through TLR2 or TLR4 receptors. Nuclear factor-κB (NF-κB) is a major immune regulatory signaling pathway in RAW264.7 cells. NF-κB protein is typically a dimer formed by the p65 / p50 subunits. Normally, NF-κB protein is associated with the inhibitory protein IκB, maintaining an inactive, "inactive" state. When cells are stimulated by external stimuli, the intracellular IKK kinase is awakened and activated, adding phosphate groups to the IκB protein (phosphorylation). The phosphorylated IκB protein then dissociates from the NF-κB-IκB trimer. At this time, the p65 subunit in NF-κB can quickly enter the cell nucleus, start gene transcription, and ultimately activate the NF-κB signaling pathway in the cell. In order to explore the regulatory effect of modified pectin polysaccharides from young kiwifruit on the NF-κB signaling pathway, the expression levels of key proteins p65 and phosphorylated p65 (pp 65) involved in the NF-κB signaling pathway were detected by Western blot. Figure 6 As shown in Figure D, compared with the control group, the positive group treated with 1 μg / mL LPS and the sample group treated with 200 μg / mL YKDHP-D significantly upregulated p-p65 expression. These results suggest that YKDHP-D may enhance immune function by regulating the NF-κB signaling pathway. In summary, the immunomodulatory mechanism of modified pectin polysaccharides from young kiwifruit fruit is closely related to the NF-κB signaling pathway mediated by TLR2 and TLR4 receptors.

[0097] Example 2 Evaluation of the in vivo immune activity of pectin polysaccharides and modified pectin polysaccharides of young kiwifruit of the present invention

[0098] 1. Experimental Methods

[0099] 1. Animal Experiment Design

[0100] Forty-two SPF male BALA / c mice were randomly divided into seven groups (n=6 per group): blank control (NC), model group (MC), YKDHP group, YKDHP-D group, YKDHP-U group, YKDHP-A group, and positive control group (PC). The dosing regimen for each group is shown in Table 2. Mouse body weights were recorded daily during the experiment. Mice were sacrificed at the end of the experiment, and blood and organs were collected.

[0101] Table 2 Dosage regimen for each group of mice

[0102]

[0103] 2. Body weight and organ index measurement

[0104] Weigh the mice daily. After the experiment, sacrifice the mice and remove the thymus and spleen to weigh them. Calculate the immune organ index using the following formula:

[0105]

[0106] 3. Histopathological Observation

[0107] The same part of the mouse spleen tissue was fixed in 4% paraformaldehyde and sent to Sewell Biotechnology Co., Ltd. for paraffin embedding and sectioning. The tissue was then stained with hematoxylin and eosin (H&E) and finally photographed and analyzed under a microscope.

[0108] 4. Determination of cytokine and immunoglobulin levels in serum

[0109] Blood was collected from the mouse orbital cavity and allowed to stand at room temperature for two hours before centrifugation to obtain the upper serum layer. Serum cytokine (TNF-α, IL-6, IFN-γ) and immunoglobulin (IgA and IgG) levels were measured using ELISA kits.

[0110] 2. Experimental Results

[0111] 1. Effects of kiwifruit pectin polysaccharides and modified pectin polysaccharides on body weight and organ indices in immunosuppressed mice

[0112] In the present invention, except for the blank control group (NC group), the mice in the other groups were intraperitoneally injected with cyclophosphamide for 3 consecutive days. Figure 7A. Compared with the NC group, the weight of mice in the other groups decreased significantly after modeling, preliminarily proving that the modeling was successful. Fourteen days after oral administration of the sample, the weight of mice in all groups increased, and compared with the model group (MC group), the weight of mice in the other groups increased significantly. The results showed that kiwifruit pectin polysaccharides and modified pectin polysaccharides had a good effect on restoring the weight of immunosuppressed mice. Compared with the original YKDHP, the kiwifruit pectin polysaccharides prepared by low-temperature controlled alkaline deesterification technology and controlled partial acid hydrolysis technology had a better effect on restoring the weight of immunosuppressed mice, especially YKDHP-D.

[0113] The thymus and spleen are vital immune organs of the body. They play a key role in central immunity and peripheral immunity respectively, and together maintain the balance and defense function of the immune system. Figure 7 As shown in Figures B and 7C, the thymus index and spleen index of each group of mice were measured. Compared with the NC group, the organ index of mice in the MC group was significantly decreased, indicating that the immunosuppressive model was successfully established. Compared with the MC group, the organ index of mice in the PC group and the polysaccharide groups (YKDHP, YKDHP-D, YKDHP-U, and YKDHP-A) was significantly increased, indicating that kiwifruit pectin polysaccharides and modified pectin polysaccharides can promote the regeneration and development of damaged cells in immunosuppressed mice, thereby improving the immunity of immunosuppressed mice to a certain extent. Compared with YKDHP, modified kiwifruit pectin polysaccharides prepared by low-temperature controlled alkaline deesterification and controlled partial acid hydrolysis were more effective in restoring the thymus index and spleen swelling index of immunosuppressed mice.

[0114] 2. Effects of kiwifruit pectin polysaccharides and modified pectin polysaccharides on spleen tissue morphology in immunosuppressed mice

[0115] H&E staining was used to observe the histomorphology of the spleen of each group of mice to further understand the effects of kiwifruit pectin polysaccharides and modified pectin polysaccharides on spleen swelling in immunosuppressed mice. Figure 8 As shown, the red and white pulp of the spleens of mice in the NC group had a clear demarcation line and were arranged in an orderly manner. In the MC group, the demarcation line between the white and red pulp of the spleens of mice was blurred, the white pulp area was reduced, and the cells were loosely arranged and disordered, with a disordered structure. Compared with the MC group, the red and white pulp of the spleens of mice in the PC group and the polysaccharide groups (YKDHP, YKDHP-D, YKDHP-U, and YKDHP-A) were more evenly distributed, with a relatively clear demarcation line, a larger white pulp area, and more orderly cell arrangement. These results indicate that both positive drugs and kiwifruit pectin polysaccharides and their modified products significantly enhance the recovery of the spleen in mice with CTX-induced immunosuppression.

[0116] 3. Effects of kiwifruit pectin polysaccharides and modified pectin polysaccharides on cytokines and immunoglobulins in the serum of immunosuppressed mice

[0117] like Figure 9 As shown, compared with the NC group, the serum levels of cytokines (TNF-α, IL-6, IFN-γ) and immunoglobulins (IgA and IgG) in immunosuppressed mice in the MC group were significantly lower, indicating that CTX injection resulted in a decrease in the mice's immunity. Administration of the positive drug and kiwifruit pectin polysaccharides (YKDHP, YKDHP-D, YKDHP-U, and YKDHP-A) significantly increased the serum cytokine and immunoglobulin levels. Compared with the original kiwifruit pectin polysaccharide (YKDHP), YKDHP-D, YKDHP-U, and YKDHP-A were more effective in restoring the immune cytokines and immunoglobulins in the serum of immunosuppressed mice. The modified kiwifruit pectin polysaccharide (YKDHP-D), prepared by low-temperature controlled deesterification, was most effective.

Claims

1. A kiwifruit young fruit pectin polysaccharide, characterized by: The invention is prepared from young kiwifruit fruits as raw materials, and contains 90.05mg±0.65mg of total polysaccharides, 25.49mg±1.38mg of total uronic acid, 2.79mg±0.09mg of total protein, and 7.83mg GAE±0.09mg GAE (gallic acid equivalent) of total bound phenols per 100mg; the esterification degree of pectin polysaccharides of young kiwifruit fruits is 41.56%±0.16%, and the molecular weight (M w ) is (10.47±0.12)×10 4 Da, polydispersity coefficient (M w / M n ) is 1.

868.

2. The kiwifruit young fruit pectin polysaccharide according to claim 1, characterized in that: It contains the following monosaccharides in the following molar percentages: Mannose (Man) 2.10 mol%, rhamnose (Rha) 5.38 mol%, glucuronic acid (GlcA) 1.42 mol%, galacturonic acid (GalA) 16.74 mol%, glucose (Glc) 4.68 mol%, galactose (Gal) 52.16 mol%, arabinose (Ara) 17.52 mol%; the molar proportion of polygalacturonic acid (HG) is 11.36 mol%, the molar proportion of type I polyrhamnogalacturonic acid (RG-I) is 80.44 mol%, and the side chain length of RG-I is 12.

95.

3. The kiwifruit young fruit pectin polysaccharide according to claim 1 or 2, characterized in that: It is prepared by a deep eutectic solvent-assisted heating extraction method, and its preparation method is as follows: a. Freeze-dry the young kiwifruit, grind into powder and sieve: b. Removal of alcohol-soluble components: Extract the freeze-dried powder of young kiwifruit with 80% (v / v) ethanol, centrifuge and discard the supernatant to obtain a precipitate; c. Deep eutectic solvent-assisted heating extraction: according to a solid-liquid ratio of 1:50 (w / v), the extraction temperature was 95°C, and the extraction time was 4 hours; wherein the deep eutectic solvent was composed of choline chloride, ethylene glycol, and ultrapure water, and the molar ratio of choline chloride to ethylene glycol was 1:3, and the water content of the deep eutectic solvent was 40%. After the extraction was completed, the supernatant was retained by centrifugation; d. Alcohol precipitation: Use 75% (v / v) ethanol (final concentration) for graded precipitation, then centrifuge and collect the precipitate; e. Redissolve: Redissolve the precipitate from step d with ultrapure water; f. Starch removal: Use α-amylase and saccharifying enzyme to remove starch in the reconstituted solution, centrifuge, and reserve the supernatant; g. Ultrafiltration: The supernatant is subjected to ultrafiltration centrifugation to collect purified fractions with molecular weights between 3 kDa and 100 kDa, and then freeze-dried to obtain high-purity kiwifruit young fruit pectin polysaccharide (YKDHP).

4. A modified pectin polysaccharide from young kiwifruit, characterized by: The invention relates to a method for structurally modifying the pectin polysaccharide of kiwifruit young fruit prepared by any one of claims 1 to 3 through low-temperature alkaline deesterification technology, ultrasonic Fenton degradation technology or controlled partial acid hydrolysis technology. The modified pectin polysaccharide of kiwifruit young fruit contains 90.11mg±1.74mg-90.80mg±1.75mg of total polysaccharide, 23.23mg±1.37mg-58.52mg±1.24mg of total uronic acid, 2.34mg±0.08mg-2.68mg±0.04mg of total protein, and 3.21mg GAE±0.14mg GAE-6.78mg GAE±0.10mg GAE (gallic acid equivalent) of total bound phenols per 100mg of the modified pectin polysaccharide of kiwifruit young fruit; the esterification degree of the modified pectin polysaccharide of kiwifruit young fruit is 5.59%±0.46%-47.48%±0.43%, and the molecular weight (M w ) is (0.609±0.021)×10 4 Da–(8.133±0.068)×10 4 Da, polydispersity coefficient (M w / M n ) is 1.361–1.

781.

5. The modified pectin polysaccharide of kiwifruit young fruit according to claim 4, characterized in that: It contains the following monosaccharides in the following molar percentages: Mannose (Man) 2.08 mol%–5.66 mol%, rhamnose (Rha) 4.99 mol%–16.02 mol%, glucuronic acid (GlcA) 1.49 mol%–5.44 mol%, galacturonic acid (GalA) 14.97 mol%–57.62 mol%, glucose (Glc) 1.72 mol%–3.33 mol%, galactose (Gal) 13.54 mol%–52.55 mol%, arabinose (Ara) 0.00 mol%–19.90 mol%; the molar proportion of HG is 9.29 mol%–41.60 mol%, the molar proportion of RG-I is 45.58 mol%–83.81 mol%, and the side chain length of RG-I is 0.84–12.

76.

6. The modified pectin polysaccharide of young kiwifruit according to claim 4 or 5, characterized in that: The modified pectin polysaccharide of kiwifruit young fruit contains 90.36mg±1.81mg of total polysaccharide, 23.23mg±1.37mg of total uronic acid, 2.68mg±0.04mg of total protein, and 6.78mg GAE±0.10mg GAE (gallic acid equivalent) of total bound phenol per 100mg; the degree of esterification of the modified pectin polysaccharide of kiwifruit young fruit is 16.76%±0.09%, and the molecular weight (M w ) is: (8.133±0.068)×10 4 Da, polydispersity coefficient (M w / M n ) is 1.781; It contains the following monosaccharides in the following molar percentages: Mannose (Man) 2.08 mol%, rhamnose (Rha) 5.68 mol%, glucuronic acid (GlcA) 1.49 mol%, galacturonic acid (GalA) 14.97 mol%, glucose (Glc) 3.33 mol%, galactose (Gal) 52.55 mol%, arabinose (Ara) 19.90 mol%; the molar proportion of HG is 9.29 mol%, the molar proportion of RG-I is 83.81 mol%, and the side chain length of RG-I is 12.

76.

7. A method for preparing modified pectin polysaccharides from young kiwifruit according to any one of claims 4 to 6, characterized in that: It modifies the pectin polysaccharide of young kiwifruit through low-temperature alkaline deesterification technology, ultrasonic Fenton degradation technology or controlled partial acid hydrolysis technology; Wherein, the preparation method of the low-temperature alkaline deesterification technology is: a. Take kiwifruit pectin polysaccharide YKDHP and add it to NaOH solution with pH value of 11.0, and stir at 4℃ for 30min; b. Add hydrochloric acid to the solution of step a until the pH value is neutral 7.0, and ultrafiltration is performed through an ultrafiltration centrifuge tube with a molecular weight limit of 3 kDa to retain the upper retained component, and then freeze-dry to obtain a kiwifruit young fruit modified pectin polysaccharide (YKDHP-D) with a reduced degree of esterification; The preparation method of the ultrasonic Fenton degradation technology is: a. Take young kiwifruit pectin polysaccharide YKDHP, add it to Vc solution and H2O2 solution, and ultrasonicate it at 520W and 24kHZ for 2h; b. The mixed solution obtained in step a was ultrafiltered through an ultrafiltration centrifuge tube with a molecular weight limit of 3 kDa, and the upper retained component was retained, followed by freeze-drying to obtain a kiwifruit young fruit modified pectin polysaccharide (YKDHP-U) with reduced molecular weight; The preparation method of the controllable partial acid hydrolysis technology is: a. Take kiwifruit pectin polysaccharide YKDHP and add it to trifluoroacetic acid solution to make the final concentration of trifluoroacetic acid in the system 0.25M, then place it at 95℃ for 3 hours; b. NaOH was added to the solution in step a until the pH value was neutral 7.0, and the solution was ultrafiltered through an ultrafiltration centrifuge tube with a molecular weight limit of 3 kDa, retaining the upper retained component, and then freeze-dried to obtain a kiwifruit young fruit modified pectin polysaccharide (YKDHP-A) with reduced side chain length.

8. The method for preparing modified pectin polysaccharides from young kiwifruit according to claim 7, wherein: In the preparation method using ultrasonic Fenton degradation technology, the molar ratio of Vc to H2O2 is 1:

2.

9. Use of the pectin polysaccharide from young kiwifruit fruits according to any one of claims 1 to 3 or the modified pectin polysaccharide from young kiwifruit fruits according to any one of claims 4 to 6 in the preparation of health foods or medicines that help enhance immunity.

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