Camphor tree seed kernel polysaccharide and its preparation method and application

Abstract

The present invention relates to the technical field of natural extracts, and specifically to a camphor seed kernel polysaccharide, a preparation method thereof, and an application thereof. The camphor seed kernel polysaccharide provided in the present application comprises the following monosaccharides: glucose, mannose, galactose, arabinose, and glucosamine hydrochloride, and the molar ratio is (0.88-0.92): (0.06-0.1): (0.015-0.02): (0.003-0.004): (0.003-0.004); the weight-average molecular weight of the camphor seed kernel polysaccharide is 1000Da-20000Da. The present invention also provides a preparation method for the above-mentioned camphor seed kernel polysaccharide and an application of the polysaccharide. The results showed that camphor seed kernel polysaccharides CCSKP and CCSKP1‑A had good in vitro digestibility, and could promote the growth and reproduction of Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium adolescentis, and produce corresponding SCFAs. They had potential prebiotic activity, which was beneficial to improving the comprehensive utilization efficiency and added value of camphor seed resources.

Description

Technical Field

[0001] The present invention relates to the technical field of natural extracts, and in particular to camphor tree seed kernel polysaccharide, a preparation method and application thereof. Background Art

[0002] Camphor seeds are the fruit of the camphor tree, a member of the Lauraceae family. The kernel is the primary and most valuable part of the seed, rich in oil, protein, and dietary fiber. Existing research on camphor seeds primarily focuses on the extraction and physiological activity of camphor seed kernel oil, as well as the synthesis of structured lipid products using camphor seed kernel oil as a raw material.

[0003] Plant polysaccharides, also known as plant polysaccharides, are a class of naturally occurring high-molecular-weight polymers composed of aldoses or ketoses linked by glycosidic bonds. They are important biomolecules in living organisms and essential for maintaining the normal functioning of life. Scientific research has shown that many plant polysaccharides possess biological activity, including health benefits such as immunomodulation, anti-tumor properties, hypoglycemic and lipid-lowering properties, radiation protection, antibacterial and antiviral properties, and liver protection.

[0004] Currently, there are no published reports on the extraction, structural research and biological activity of camphor seed polysaccharides. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a camphor tree seed kernel polysaccharide.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned camphor seed kernel polysaccharide.

[0007] Another object of the present invention is to provide applications of the camphor tree seed kernel polysaccharide.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A camphor tree seed kernel polysaccharide, comprising the following monosaccharides: glucose, mannose, galactose, arabinose and glucosamine hydrochloride, and the molar ratio is (0.88-0.92): (0.06-0.1): (0.015-0.02): (0.003-0.004): (0.003-0.004);

[0010] The weight average molecular weight of the camphor tree seed kernel polysaccharide is 1000Da to 20000Da.

[0011] Furthermore, the weight average molecular weight of the camphor seed polysaccharide is 4000Da to 15000Da;

[0012] The average particle size of the polysaccharide is 350nm to 1200nm.

[0013] Furthermore, the camphor seed kernel polysaccharide is obtained by deproteinizing and decolorizing the aqueous phase of camphor seed kernel oil extracted from camphor seed kernels by aqueous enzymatic extraction to obtain a decolorized solution, and concentrating, alcohol-precipitating, dialyzing, and freeze-drying the decolorized solution to obtain camphor seed kernel polysaccharide CCSKP.

[0014] Furthermore, the camphor tree seed kernel polysaccharide CCSKP was separated by anion exchange chromatography column and then purified by dextran Sephadex G-50 gel adsorption method to obtain the camphor tree seed kernel polysaccharide CCSKP1-A.

[0015] Furthermore, the camphor seed kernel polysaccharide CCSKP is a polydisperse polysaccharide-protein complex:

[0016] The total sugar, protein, and uronic acid contents were 64.70±1.27%, 11.33±1.12%, and 9.45±0.33%, respectively, with average molecular weights of 14080Da, 7934Da, and 5361Da;

[0017] The molar ratio of glucose, mannose, galactose, arabinose and glucosamine hydrochloride in the monosaccharide composition is 0.911:0.065:0.018:0.004:0.003;

[0018] The particle size is 354.85 nm and the Zeta potential value is -4.64 mV.

[0019] Furthermore, the camphor seed kernel polysaccharide CCSKP1-A is a neutral polysaccharide:

[0020] The total sugar content was 89.50 ± 0.46%, and the protein content was 3.15 ± 0.54%;

[0021] The molar ratio of glucose, mannose, galactose, arabinose and glucosamine hydrochloride in the monosaccharide composition is 0.883:0.094:0.016:0.003:0.004;

[0022] The particle size is 1158.00 nm and the Zeta potential value is -0.03 mV.

[0023] The present invention also provides a method for preparing the camphor tree seed kernel polysaccharide, comprising the following steps:

[0024] (1) Cinnamomum camphora seed kernels were ground using a high-speed grinder, and distilled water was added to prepare a suspension with a volume ratio of 1:6. The pH was adjusted to 5.0-6.0, and cellulase was added to prepare a 3000U / ml solution. The solution was heated in a constant temperature water bath at 55°C for 2 h, and the enzyme was inactivated at high temperature for 10 min. The supernatant was collected by centrifugation and placed in a refrigerator at 4°C for 12 h to further remove free oil.

[0025] (2) Deproteinization and decolorization: Add 2% by weight volume of papain to the treated aqueous phase, hydrolyze at 55°C for 2 h, inactivate the enzyme at high temperature for 10 min, remove the precipitate by centrifugation, collect the supernatant, add 10% by weight volume of pre-treated activated AB-8 macroporous resin to the supernatant, decolorize in a shaker at 40°C for 1 h, remove the macroporous resin by filtration under reduced pressure, and collect the decolorized solution;

[0026] (3) Ethanol precipitation: Use a rotary evaporator to concentrate the decolorized solution to 25% of its original volume at 60°C. Collect the concentrate, add 3 times the volume of anhydrous ethanol, and place in a refrigerator at 4°C overnight. Centrifuge to remove the supernatant, and add appropriate distilled water to the precipitated component for re-dissolution.

[0027] (4) Dialysis and impurity removal: The camphor tree seed kernel polysaccharide solution collected above was placed in a 1000Da dialysis bag and dialyzed with running water for 1 to 2 days. After the dialysis was completed, the camphor tree seed kernel polysaccharide CCSKP was obtained by concentration and freeze drying.

[0028] Furthermore, the method further comprises:

[0029] (5) Separation by DEAE-52 cellulose anion exchange method

[0030] Pretreatment: The weighed DEAE-52 cellulose was subjected to alkali-acid-alkali pretreatment, and then washed with appropriate amount of distilled water 3-4 times until the pH was 7.0-7.4;

[0031] A 10 mg / mL aqueous solution of camphor seed kernel polysaccharide CCSKP was prepared, the polysaccharide solution was filtered through a 0.45 μm filter membrane, the CCSKP solution was added dropwise to a pre-installed anion exchange chromatography column, and eluted with a gradient NaCl solution of 0 to 0.5 mol / L as the eluent. The flow rate was set to 1 mL / min, and the eluate was automatically collected at a rate of 10 min / tube. 30 tubes of eluate were collected for each gradient, and the absorbance was measured at OD490 nm. An elution curve was drawn, and dialyzed, concentrated, and vacuum freeze-dried according to the elution curve to obtain camphor seed kernel polysaccharide components CCSKP1, CCSKP2, and CCSKP3;

[0032] (6) Purification by Sephadex G-50 gel adsorption

[0033] Accurately weigh the camphor seed kernel polysaccharide component CCSKP1 to prepare a 10 mg / ml polysaccharide solution, filter it with a 0.45 μm filter head, add it to a dextran Sephadex G-50 gel column, and elute it with ultrapure water as the eluent. The eluate is automatically collected in different tubes, and the flow rate is set to 0.2 mL / min, 4 mL / tube. The eluate is collected until all the polysaccharides flow out, and its absorbance is measured and the dextran gel elution curve is drawn. The collected products are combined, concentrated, and freeze-dried to obtain the camphor seed kernel polysaccharide component CCSKP1-A.

[0034] The present invention also provides the use of the camphor tree seed kernel polysaccharide in the preparation of foods and health products that promote the proliferation of probiotics.

[0035] Furthermore, the probiotic is at least one of Lactobacillus acidophilus, Bifidobacterium bifidum, Bifidobacterium infantis and Bifidobacterium adolescentis.

[0036] Compared with the existing technology, the present invention has the following beneficial effects: using process wastewater from the aqueous enzymatic extraction of camphor tree seed kernel oil as raw material, the present invention optimizes the extraction and separation conditions to successfully isolate and obtain camphor tree seed kernel polysaccharides CCSKP and CCSKP1-A. The molecular weight, monosaccharide composition, particle size morphology, and structure of the obtained polysaccharides are analyzed and identified, and their particle size, weight-average molecular weight, and structural composition are determined. In vitro experiments show that the camphor tree seed kernel polysaccharides CCSKP and CCSKP1-A have good in vitro digestibility, can promote the growth and reproduction of Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium adolescentis, and produce corresponding SCFAs, showing potential prebiotic activity and beneficial to improving the comprehensive utilization efficiency and added value of camphor tree seed resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the elution curve of CCSKP on DEAE-52 cellulose anion exchange column;

[0038] Figure 2 is the elution curve of CCSKP1-A on Sephadex G-50 gel column;

[0039] Figure 3 is the molecular weight distribution chromatogram of CCSKP and CCSKP1-A;

[0040] Figure 4 This is the monosaccharide composition chromatogram of CCSKP;

[0041] Figure 5 This is the chromatogram of the monosaccharide composition of CCSKP1-A;

[0042] Figure 6 This is the infrared spectrum analysis diagram of CCSKP and CCSKP1-A;

[0043] Figure 7 The particle size distribution and potential of CCSKP and CCSKP1-A;

[0044] Figure 8 SEM images of CCSKP and CCSKP1-A;

[0045] Among them, A, B, and C are 100x, 500x, and 1000x electron micrographs of CCSKP, respectively; D, E, and F are 100x, 500x, and 1000x electron micrographs of CCSKP1-A, respectively;

[0046] Figure 9 is the hydrolysis degree curve of CCSKP in simulated gastric fluid;

[0047] Figure 10 is the hydrolysis curve of CCSKP1-A in simulated gastric fluid;

[0048] Figure 11 is the hydrolysis degree curve of FOS to simulated gastric fluid;

[0049] Figure 12 This is the hydrolysis degree curve of CCSKP hydrolyzed by α-amylase;

[0050] Figure 13 This is the hydrolysis degree curve of CCSKP1-A by α-amylase;

[0051] Figure 14 is the hydrolysis degree curve of FOS hydrolyzed by α-amylase;

[0052] Figure 15 The effects of CCSKP, CCSKP1-A and FOS on the growth and proliferation of Lactobacillus acidophilus in sugar-free MRS medium and the pH of the culture medium;

[0053] Figure 16 The effects of CCSKP, CCSKP1-A and FOS on the growth and proliferation of Bifidobacterium longum in sugar-free MRS medium and the pH of the culture medium;

[0054] Figure 17 To study the effects of CCSKP, CCSKP1-A and FOS on the growth and proliferation of Bifidobacterium infantis in sugar-free MRS medium and the pH of the culture medium;

[0055] Figure 18To study the effects of CCSKP, CCSKP1-A and FOS on the growth and proliferation of Bifidobacterium adolescentis in sugar-free MRS medium and the pH of the culture medium;

[0056] Figure 19 The effect of CCSKP, CCSKP1-A and FOS on the growth rate of Lactobacillus acidophilus;

[0057] Figure 20 The effect of CCSKP, CCSKP1-A and FOS on the growth rate of Bifidobacterium longum;

[0058] Figure 21 The effects of CCSKP, CCSKP1-A and FOS on the growth rate of Bifidobacterium infantis;

[0059] Figure 22 The effects of CCSKP, CCSKP1-A and FOS on the growth rate of Bifidobacterium adolescentis. DETAILED DESCRIPTION

[0060] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. The embodiments described are only 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 making creative efforts are within the scope of protection of the present invention.

[0061] Camphor tree seed kernels: Fresh, mature camphor tree fruits were collected from the Qingshan Lake Campus of Nanchang University. Papain and macroporous resin AB-8 (analytical grade) were purchased from Beijing Solebeau Technology Co., Ltd. DEAE-52 fiber column and Sephadex G-50 column were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Bovine serum albumin (BSA), d-galacturonic acid, and BCA protein concentration assay kit were purchased from Aladdin Reagent Co., Ltd. Glucose standards, gallic acid, and galacturonic acid (chromatographic grade) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. All other reagents were of HPLC or analytical grade.

[0062] Fructooligosaccharides (FOS) and α-amylase A3178-500KU were purchased from Sigma-Aldrich, USA, and sugar-free MRS broth was from Qingdao Haibo Biological Co., Ltd. Acetic acid, propionic acid, and n-butyric acid were from Aladdin Reagent Co., Ltd., and a lactate assay kit was purchased from Nanjing Jiancheng Bioengineering Institute. Lactobacillus acidophilus (BNCC 336636) and Bifidobacterium adolescentis (CICC6070) were purchased from Shangcheng Beina Chuanglian Biotechnology Co., Ltd., while Bifidobacterium longum (BB 536) and Bifidobacterium infantis (M-63) were obtained from Shanghai Yongsen Dairy Co., Ltd., China. All other reagents were of HPLC or analytical grade.

[0063] Unless otherwise specified, the methods are conventional methods, and the raw materials can be obtained from public commercial channels unless otherwise specified.

[0064] Example 1: Preparation of Cinnamomum camphora seed polysaccharide CCSKP

[0065] Fresh and mature camphor seeds were collected from the Qingshan Lake campus of Nanchang University. The peel and pulp of the seeds were removed and the seeds were dried at (40±1)℃ to constant weight. The seeds were shelled using a shelling machine to obtain camphor seed kernels.

[0066] (1) First, the camphor tree seed kernels were ground using a high-speed grinder. Then, distilled water was added to prepare a 1:6 (v / v) suspension. The pH was adjusted to 5.0-6.0. Cellulase was then added to prepare a 3000U / ml solution. The solution was heated in a constant temperature water bath at 55°C for 2 hours. After high temperature inactivation of the enzyme for 10 minutes, the supernatant was collected by centrifugation. The collected supernatant was placed in a 4°C refrigerator for 12 hours. The free oil was further removed to obtain the aqueous phase. The protein content of the solid phase in the aqueous phase was analyzed to be 46.12±0.31%, the total sugar content was 26.54±0.48%, the uronic acid content was 11.01±0.64%, and the polyphenol content was 4.21±0.24%.

[0067] (2) Deproteinization and decolorization: Papain (2%, w / v) was added to the treated aqueous phase and enzymatically hydrolyzed at 55°C for 2 h. After high-temperature enzyme inactivation for 10 min, the precipitate was removed by centrifugation and the supernatant was collected. Pre-treated activated AB-8 macroporous resin (10%, w / v) was added to the supernatant and decolorized in a shaker at 40°C for 1 h. The macroporous resin was removed by vacuum filtration and the decolorized solution was collected.

[0068] (3) Ethanol precipitation: Concentrate the decolorized solution to 25% of its original volume using a rotary evaporator at 60°C. Collect the concentrate, add 3 times the volume of anhydrous ethanol, and place in a refrigerator at 4°C overnight. Centrifuge to remove the supernatant, and add an appropriate amount of distilled water to the precipitated fraction for reconstitution.

[0069] (4) Dialysis: The collected camphor seed kernel polysaccharide solution was placed in a 1000 Da dialysis bag and dialyzed against flowing water for 1-2 days. In special cases, it could be dialyzed against distilled water. After dialysis, the solution was concentrated and freeze-dried to obtain a camphor seed kernel polysaccharide CCSKP sample with a calculated yield of 5.34%.

[0070] Example 2: Preparation of Cinnamomum camphora seed polysaccharide CCSKP1-A

[0071] The camphor tree seed kernel polysaccharide CCSKP sample prepared in Example 1 was used as a raw material to prepare camphor tree seed kernel polysaccharide CCSKP1-A. The specific steps are as follows:

[0072] (1) Separation by DEAE-52 cellulose anion exchange method

[0073] Pretreatment: Weigh the DEAE-52 cellulose and perform an alkali-acid-alkali pretreatment. Finally, wash the DEAE-52 cellulose with an appropriate amount of distilled water 3-4 times until the pH reaches 7.0-7.4.

[0074] Prepare 10mL of camphor tree seed kernel polysaccharide CCSKP aqueous solution (10mg / mL), and filter the polysaccharide solution through a 0.45μm filter membrane. Add the CCSKP solution dropwise to a pre-installed anion exchange chromatography column (2.6×30cm), and elute with a gradient of 0-0.5mol / L NaCl solution as the eluent. Set the flow rate to 1mL / min, and automatically collect the eluate at a rate of 10min / tube. Collect 30 tubes of eluate for each gradient, measure its absorbance at OD490nm, and draw an elution curve. The elution curve is as follows: Figure 1 As shown, from Figure 1 It can be seen that the elution curve presents three peaks, among which the distilled water elution component accounts for the largest proportion, followed by the components eluted with 0.10 and 0.20 mol / LNaCl. The eluates of the three components were collected respectively, and then dialyzed, concentrated and freeze-dried to obtain the three components CCSKP1, CCSKP2 and CCSKP3. The yields of each component were calculated to be 25.65%, 2.53% and 1.08%, respectively.

[0075] (2) Purification by Sephadex G-50 gel adsorption

[0076] Accurately weigh about 10 mg of camphor seed kernel polysaccharide component CCSKP1, prepare it into a 10 mg / ml polysaccharide solution, filter it with a 0.45 μm filter head, add it to a dextran Sephadex G-50 gel column (1.0×60 cm), elute it with ultrapure water as the eluent, and automatically collect the eluate in the tube. Set the flow rate to 0.2 mL / min, 4 mL / tube, and collect the eluate until all the polysaccharides flow out. Measure its absorbance value and draw the dextran gel elution curve. The elution curve is as follows: Figure 2 As shown, the peak shape is symmetrical, indicating that the component does not contain obvious impurities and has high purity. The eluted peak is collected and concentrated, freeze-dried, and other steps to obtain camphor seed kernel polysaccharide CCSKP1-A.

[0077] Example 3: Chemical composition determination and analysis of camphor seed kernel polysaccharide CCSKP sample

[0078] 1. Determination method

[0079] 1) The camphor tree seed kernel polysaccharide CCSKP sample obtained in Example 1 and the camphor tree seed kernel polysaccharide CCSKP1-A obtained in Example 2 were subjected to the phenol-sulfuric acid method for determining the polysaccharide content, the BCA protein concentration detection kit was used to detect the protein content in the polysaccharide sample, and the carbazole-sulfuric acid method was used to determine the uronic acid content in the polysaccharide sample.

[0080] 2) Molecular weight determination of camphor seed polysaccharide

[0081] The molecular weight distribution of polysaccharide samples was determined by high performance liquid gel permeation chromatography.

[0082] Accurately weigh the sample and standard, prepare the sample into a 5 mg / ml solution, centrifuge at 12000 rpm for 10 min, filter the supernatant with a 0.22 μm microporous filter membrane, and then transfer the sample to a 1.8 ml injection vial.

[0083] Chromatographic column: BRT105-104-102 gel column in series (8×300 mm); mobile phase: 0.05 M NaCl solution; flow rate: 0.6 ml / min, column temperature: 40°C; injection volume: 20 μl; detector: differential detector RI-10A.

[0084] 3) Determination and analysis of monosaccharide composition of camphor seed polysaccharides

[0085] Accurately weigh 5 mg of sample into an ampoule, add 2 mL of 3M TFA, and hydrolyze at 120°C for 3 h. Drain the acid hydrolysis solution with nitrogen, add 5 mL of water, and vortex to mix thoroughly. Aspirate 50 μL of the sample and add 950 μL of deionized water. Centrifuge at 12,000 rpm for 5 min. Collect the supernatant for IC analysis.

[0086] Chromatographic column: Dionex Carbopac TMPA20 (3*150mm); mobile phase: A (H2O); B (15mMNaOHC: 15mM NaOH & 100mMNaOAC); flow rate: 0.3ml / min; injection volume: 5μL; column temperature: 30℃; detector: electrochemical detector.

[0087] 4) Spectroscopic determination and analysis of the group structure of camphor seed polysaccharides

[0088] Cinnamomum camphora seed kernel polysaccharides CCSKP and CCSKP1-A were dissolved in distilled water to prepare a test solution with a sample concentration of 1 mg / mL.

[0089] The organic functional groups of camphor seed polysaccharides CCSKP and CCSKP1-A were identified by Fourier transform infrared spectroscopy (FT-IR). The samples were mixed with KBr at a ratio of 1:100 (w / w) and the fluorescence was detected by Nicolet 5700 FT-IR spectrometer at 4000-400 cm -1 The infrared spectra of camphor seed kernel polysaccharides CCSKP and CCSKP1-A were measured and recorded at the wavelength, and their infrared spectra were analyzed.

[0090] 5) Particle size and potential determination of camphor seed polysaccharide

[0091] The particle size distribution and zeta potential (1 mg / mL) of camphor seed kernel polysaccharides CCSKP and CCSKP1-A were measured using a Zetasizer Nano-ZS90 at 25° C. The dispersion refractive index of the sample and distilled water were 1.45 and 1.33, respectively.

[0092] 6) Microscopic morphology determination and analysis of polysaccharides from camphor tree seed kernels using scanning electron microscopy

[0093] The freeze-dried camphor tree seed kernel polysaccharides CCSKP and CCSKP1-A samples prepared by scanning gold film were scanned using a SU8020 scanning electron microscope. The microscopic morphology of the samples was observed and recorded at different magnifications (×100, ×500 and ×1000).

[0094] 2. Measurement results

[0095] Excel was used to analyze the experimental data in this experiment, and all values are expressed as mean ± SD. OriginPro 9.0 software was used to process the charts, and the results are as follows:

[0096] 1) Total sugar, protein and uronic acid content of camphor seed polysaccharides CCSKP and CCSKP1-A

[0097] The total sugar, protein, and uronic acid contents of CCSKP were 64.70±1.27%, 11.33±1.12%, and 9.45±0.33%, respectively, suggesting that CCSKP may be a polysaccharide-protein complex. The polysaccharide fraction CCSKP1-A had a total sugar content of 89.50±0.46%, a protein content of 3.15±0.54%, and no detectable uronic acid content, indicating that CCSKP1-A is a neutral sugar.

[0098] 2) Molecular mass of camphor seed polysaccharides CCSKP and CCSKP1-A

[0099] The molecular weight of the polysaccharides CCSKP and CCSKP1-A was determined by high performance gel permeation chromatography. The standard curve was drawn with the logarithm of molecular weight (logMw) versus retention time (t). The linear regression equation was logMw = -0.1932x -12.21, R 2 =0.9934. The molecular weights of CCSKP and CCSKP1-A were determined under the same conditions. The molecular weight standard chromatogram is shown in FIG. Figure 3 As shown, from Figure 3 It can be seen that the chromatogram of CCSKP has three peaks with retention times (t) of 40.99 min, 42.38 min and 43.33 min. Substituting them into the regression equation, the molecular weights were calculated to be 14080 Da, 7934 Da and 5361 Da, respectively, indicating that CCSKP is a non-uniform component; the chromatogram of CCSKP1-A has only a single chromatographic peak with good symmetry, indicating that the purity of the refined polysaccharide CCSKP1-A is relatively high. Its molecular weight is calculated to be 4219 Da, and the polydispersity index (Mw / Mn) is 1.26, indicating that CCSKP1-A is a relatively uniform polysaccharide in terms of molecular weight.

[0100] 3) Monosaccharide composition of camphor seed polysaccharides CCSKP and CCSKP1-A

[0101] Ion exchange chromatography (IC) was used to detect the monosaccharide composition of camphor seed kernel polysaccharides CCSKP and CCSKP1-A. The retention time was compared with that of the control monosaccharide mixed standard. Figures 4-5 As shown, Figure 4 This is the chromatogram of the monosaccharide composition of camphor seed polysaccharide CCSKP. Figure 5 This is the chromatogram of the monosaccharide composition of camphor seed kernel polysaccharide CCSKP1-A.

[0102] It can be seen that the monosaccharide compositions of CCSKP and CCSKP1-A are consistent, both containing arabinose (Ara), glucosamine hydrochloride (GlcN), galactose (Gal), glucose (Glc), and mannose (Man). Area normalization calculations revealed that the monosaccharide composition of CCSKP is glucose, mannose, galactose, arabinose, and glucosamine hydrochloride, with a molar ratio of 0.911:0.065:0.018:0.004:0.003; the monosaccharide composition of CCSKP1-A is glucose, mannose, galactose, arabinose, and glucosamine hydrochloride, with a molar ratio of 0.883:0.094:0.016:0.003:0.004. Preliminary inference suggests that the carbon chain backbone of camphor seed kernel polysaccharides CCSKP and CCSKP1-A is primarily composed of glucose, with small amounts of arabinose, galactose, and mannose possibly interspersed within the main chain or present as side chains. Furthermore, it is worth noting that the uronic acid content of CCSKP, as determined by basic chemical composition, was 9.45±0.33%, while no monosaccharide components containing uronic acid were detected in the monosaccharide composition analysis. This may be because the sulfuric acid-carbazole method is significantly affected by interference from neutral sugars, particularly glucose and galactose. CCSKP contains a high glucose content, leading to errors in the chemical measurement results.

[0103] 4) Infrared spectroscopy analysis of camphor seed polysaccharides

[0104] Infrared spectra of camphor seed polysaccharides CCSKP and CCSKP1-A Figure 6 , at 4000-500cm -1 In the range of 3600 cm, CCSKP and CCSKP1-A have similar polysaccharide characteristic absorption peaks, indicating that they have similar characteristic structures. -1 ~3300cm -1 The strong and broad absorption peaks in the range of 2920 cm-1 are the -OH stretching vibrations within and between molecules. -1 The absorption peaks on the left and right are generally considered to be characteristic absorption peaks of polysaccharides (attributed to the stretching vibration of CH), indicating that both are carbohydrate compounds.

[69] At 1650cm -1 ~1600cm -1 The absorption peaks in the range of 1400 cm-1 are considered to be the stretching vibration of carbonyl groups, and the absorption peaks at 1400 cm-1 are considered to be the stretching vibration of carbonyl groups. -1 The absorption peaks observed at 1020 cm are attributed to the asymmetric and symmetric stretching vibrations of C=O, which indicates the possible presence of aldehyde groups. -1The absorption peaks on the left and right are the molecular fingerprints of polysaccharides, which are formed by the CO stretching vibration of the pyranose ring COC, indicating the presence of pyranose in CCSKP and CCSKP1-A, representing glucose, galactose and mannose respectively. In addition, 932cm -1 / 846cm -1 and 931cm -1 / 848cm -1 The absorption peaks at 100 nm and 100 nm confirmed that CCSKP and CCSKP1-A contained both β- and α-glycosidic bonds.

[0105] 5) Particle size and potential of camphor seed polysaccharide

[0106] like Figure 7 As shown, the particle size of CCSKP1-A increased significantly from 354.85 nm for CCSKP to 1158.00 nm, with a particle dispersion index (PDI) of 0.55, less than 0.6. This indicates that CCSKP1-A does not exist as a single molecule in water, but rather as aggregates formed by intermolecular aggregation. This is presumably because the purification of CCSKP1-A enhances the intermolecular crosslinking between polysaccharide molecules, enhancing the interactions between large particles in aqueous solution. The presence of impurities in CCSKP may interfere with these interactions. Since the majority of CCSKP and CCSKP1-A particles are larger than 100 nm, the particle size intensity accurately represents the size of camphor seed kernel polysaccharides in aqueous solution. The zeta potential values of CCSKP1-A and CCSKP, respectively, are -0.03 mV and -4.64 mV, confirming that camphor seed kernel polysaccharides are neutral polysaccharides, consistent with the conclusions of the monosaccharide composition analysis. Due to the low potential, it has poor stability in solution and is prone to aggregation.

[0107] 6) Scanning electron microscopy of the microscopic morphology of polysaccharides from camphor tree seed kernels

[0108] Scanning electron microscopy can be used to observe the surface morphology of camphor seed polysaccharides CCSKP and CCSKP1-A, such as Figure 8 As shown, Figure 8 A, B, and C are electron micrographs of CCSKP at 100x, 500x, and 1000x magnification, respectively; D, E, and F are electron micrographs of CCSKP1-A at 100x, 500x, and 1000x magnification, respectively. It can be seen that at low magnification (×100), CCSKP exhibits an irregular, rough structure with highly branched regions in some areas, unlike the granular structure of most polysaccharides. At higher magnification (×1000), numerous polygonal polymer fragments are observed on the CCSKP surface, while CCSKP1-A exhibits a larger, flake-like structure with a relatively smooth surface.

[0109] Example 4: Effects of Cinnamomum camphora seed polysaccharides CCSKP and CCSKP1-A on the proliferation of probiotics in vitro

[0110] 1.1 Experimental methods

[0111] 1.1.1 Preparation of standard curve for reducing sugar determination

[0112] Reducing sugars were determined by the 3,5-dinitrosalicylic acid (DNS) colorimetric method using glucose as the standard.

[0113] Preparation of DNS reagent: Accurately weigh 6.35 g of 3,5-dinitrosalicylic acid, dissolve it with a small amount of distilled water, add 262 ml of 2M NaOH solution and 185 g of sodium tartrate solution (use hot water to dissolve it as appropriate), then add 5 g of phenol and anhydrous sodium sulfate respectively, stir to dissolve, transfer to a 1000 ml brown volumetric flask to make up the volume, mix thoroughly, and let it stand for one week before use.

[0114] Preparation of standard curve: Pipette 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL of glucose standard solution into 50mL centrifuge tubes respectively and add ultrapure water to make up to 2.0mL, then add 2.0mL of DNS reagent, mix and shake well, incubate in boiling water for 5min, take out, let stand and cool to room temperature, add ultrapure water to make up to 25mL, shake well, and measure the OD of the solution using a UV spectrophotometer. 540 , and draw a standard curve.

[0115] 1.1.2 In vitro simulated digestion of camphor seed polysaccharides

[0116] 1.1.2.1 Analysis of the digestibility of camphor seed polysaccharide in simulated gastric juice

[0117] The pH of SGF was adjusted to 1, 2, 3, 4, and 5 using 5 M HCl. Subsequently, 5.0 mL of CCSKP, CCSKP1-A, and FOS solutions (10 mg / mL) were mixed with 5 mL of SGF at different pH values. The mixtures were incubated in a shaking incubator at 37°C for 6 hours (130 rpm). The reducing sugar and total sugar contents of the mixtures were measured at 0, 1, 2, 4, and 6 hours. Reducing sugars were determined using the DNS method, and total sugars were determined using the phenol-sulfuric acid method. The degree of hydrolysis of the samples was calculated according to the following formula.

[0118]

[0119] Where T0 is the initial mass of reducing sugar, T i It refers to the difference between the mass of reducing sugar measured at a certain time point and the initial mass of reducing sugar.

[0120] 1.1.2.2 Analysis of the digestibility of camphor seed polysaccharides to α-amylase

[0121] The resistance of CCSKP and CCSKP1-A samples of camphor seed kernel to α-amylase was analyzed, and FOS was used as a positive control group. First, 20 mg of α-amylase was dissolved in 500 ml of sodium phosphate buffer (20 mM, pH 7.4) to prepare a 2 U / ml enzyme solution, and the pH values were adjusted to 5, 6, 7, and 8, respectively. Then, 5 mL of sodium phosphate buffer solution (10 mg / mL) of CCSKP and CCSKP1-A were taken and mixed with 5 mL of enzyme solutions of different pH values. Finally, the mixed solutions of different pH values were incubated in a 37°C water bath for 6 hours, and samples were taken out at reaction times of 0, 1, 2, 4, and 6 hours, and the degree of hydrolysis was determined according to the above formula (3-1).

[0122] 1.1.3 Effect of Cinnamomum camphora seed polysaccharide on the proliferation of probiotics in vitro

[0123] 1.1.3.1 Bacteria Activation

[0124] Four freeze-dried bacterial powders—B. longum, B. infantis, B. adolescentis, and L. acidophilus—were dissolved in a 1.0% sterile aqueous protein solution and inoculated into activated culture media sterilized at 121°C for 20 minutes. The strains were cultured for two generations at 37°C for 48 hours. Culture conditions were anaerobic at 37°C (85% N2, 10% CO2, and 5% H2) in an anaerobic chamber.

[0125] 1.1.3.2 Effect of Cinnamomum camphora seed polysaccharide on the proliferation of probiotics in vitro

[0126] The growth characteristics of probiotics (Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium adolescentis and Lactobacillus acidophilus) were studied in the presence of CCSKP, CCSKP1-A and FOS. 600 ) Measure the bacterial concentration to evaluate the bacterial growth of CCSKP, CCSKP1-A and FOS, and determine the pH value of the culture medium after 48 hours of fermentation. First, take an appropriate amount of test tubes, fill them with 10 ml of liquid culture medium, and then add 0, 0.5, 1.0, 1.5, 2.0 and 3.0% CCSKP, CCSKP1-A and FOS respectively, and dissolve them with slight heat. Then, centrifuge at 4000r / min for 10 minutes. Take the supernatant and prepare it into a proliferation medium. The culture medium is sterilized at 121°C for 20 minutes, and the four activated bacterial species are inoculated at a ratio of 5%. After constant temperature anaerobic culture at 37°C for 48 hours. Determine the DO of each culture solution. 600 and culture medium pH.

[0127] 1.1.3.3 Effect of Cinnamomum camphora seed polysaccharide on the growth rate of probiotics

[0128] 2% (w / v) polysaccharide samples were added to the sugar-free MRS medium, and then 2% (v / v) probiotic suspension was inoculated and cultured anaerobically at 37°C for 48 hours. Samples were taken at 0h, 6h, 12h, 24h, 30h, 36h and 48h, and the OD of the culture medium was measured. 600 The growth curves of four beneficial bacteria in the culture medium supplemented with CCSKP, CCSKP1-A and FOS were drawn to investigate the effects of camphor seed polysaccharide on the growth rates of Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium adolescentis.

[0129] 1.1.4 Effect of Cinnamomum camphora seed polysaccharide on the production of short-chain fatty acids by probiotic fermentation

[0130] Sugar-free MRS medium was supplemented with 2% (w / v) of various carbon sources and then inoculated with a 2% (v / v) probiotic suspension culture for anaerobically incubated at 37°C for 48 h. After fermentation, the culture was centrifuged at 4000 rpm for 20 min, and the supernatant was filtered through a 0.22 μm membrane and analyzed by gas chromatography (GC).

[0131] Chromatographic analysis conditions were as follows: an Agilent 7890A gas chromatograph equipped with a DB-WAX column (30 m × 0.25 mm × 0.25 μm) and a flame ionization detector (FID). The carrier gas (nitrogen) flow rate was 5 mL / min; the air, hydrogen, and nitrogen flow rates in the detector were 400, 30, and 30 mL / min, respectively; the injection volume was 0.5 μL; the injector temperature was 250°C; and the detector temperature was 260°C. The temperature program was as follows: an initial temperature of 90°C for 0.5 min, then increased to 140°C at a rate of 3°C / min, and then to 250°C at a rate of 4°C / min. Lactic acid content was determined using a lactic acid assay kit according to the kit instructions.

[0132] 1.2 Data processing and analysis

[0133] All values obtained in this experiment are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 22.0. Details on the method for indicating significant differences are provided in the figure or table legends. Charts and graphs were processed using Origin Pro 9.0 software.

[0134] 1.3 Results and Analysis

[0135] 1.3.1 Digestive resistance of camphor seed polysaccharide in simulated gastric acid

[0136] The hydrolysis degree of CCSKP, CCSKP1-A and FOS in simulated gastric fluid with different pH values were analyzed as follows: Figure 9 、 Figure 10 and Figure 11 As shown in the figure, the degree of hydrolysis of CCSKP, CCSKP1-A, and FOS in simulated gastric fluid increases with increasing hydrolysis time. This is because a longer reaction time facilitates the hydrolysis of polysaccharides into monosaccharides and disaccharides under acidic conditions. pH has a significant effect on the degree of hydrolysis of polysaccharides, with the hydrolysis rate increasing at lower pH. After incubation for 6 h at pH 1-5, the hydrolysis degrees of FOS were 2.13±0.08%, 1.75±0.04%, 1.40±0.06%, 1.18±0.08%, and 1.07±0.06%, respectively; the hydrolysis degrees of CCSKP were 4.45±0.18%, 4.66±0.13%, 3.36±0.06%, 3.32±0.20%, and 2.84±0.03%, respectively; the hydrolysis degrees of CCSKP1-A were 1.69±0.03%, 1.66±0.03%, 1.47±0.05%, 1.32±0.02%, and 1.09±0.03%. After incubation for 6 hours at pH 1, the hydrolysis degrees of camphor seed kernel polysaccharides CCSKP and CCSKP1-A reached maximum values of 4.66±0.13% and 1.69±0.03%, respectively. This may be because the molecular weight distribution of the polysaccharide CCSKP is relatively dispersed, and the glycosidic bond structure of some polysaccharides is unstable and easily decomposed at low pH values. The maximum hydrolysis degrees of camphor seed kernel polysaccharides CCSKP and CCSKP1-A in simulated gastric fluid were both less than 5%, indicating that CCSKP and CCSKP1-A have good stability in gastric acid and may be able to reach the intestine. The resistance of CCSKP and CCSKP1-A to simulated gastric fluid is both over 95%, which means that they can be considered as potential prebiotics.

[0137] 1.3.2 Digestion resistance of camphor seed polysaccharides to α-amylase

[0138] The changes in the resistance of CCSKP, CCSKP1-A and FOS to α-amylase hydrolysis at different pH values are shown in Figure 2. Figure 12 、 Figure 13 and Figure 14As shown in the figure, both hydrolysis time and pH significantly affected the degree of hydrolysis of CCSKP and CCSKP1-A, with the degree of hydrolysis increasing with increasing incubation time. After 6 h of incubation at pH 7, the maximum degrees of hydrolysis for CCSKP and CCSKP1-A were 5.90±0.13% and 5.74±0.23%, respectively, slightly higher than the 3.56% for FOS, indicating that both CCSKPs possessed high resistance to α-amylase. CCSKP and CCSKP1-A were well-suited to digestion in the upper gastrointestinal tract, reaching the large intestine and being utilized by intestinal flora. Furthermore, there was no significant difference in the hydrolysis rates between the two samples (p>0.05), indicating that the resistance of the two CCSKP samples to α-amylase was not affected by the purification process used in this study. This strong enzymatic resistance may be due to the configuration of β-glycosidic bonds within the camphor seed polysaccharide molecules.

[0139] 1.3.3 Effect of Cinnamomum camphora seed polysaccharide on the proliferation of probiotics

[0140] In order to study the effect of camphor seed kernel polysaccharides on the growth and reproduction of probiotics, concentration dependence analysis and acidification activity study were conducted. Bifidobacterium and Lactobacillus acidophilus were cultured anaerobicly using camphor seed kernel polysaccharides CCSKP and CCSKP1-A as the sole carbon source, and FOS was used as negative or positive control. The effects of CCSKP, CCSKP1-A and FOS in sugar-free MRS medium on the growth and proliferation of different probiotics and the base pH of the culture medium were shown in Figure 2. Figure 15 、 Figure 16 、 Figure 17 、 Figure 18 shown. Figures 15 to 18 The probiotics shown are Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium adolescentis; a, b, c, d, e, f represent significant differences in each column of the same color (n=3).

[0141] Obviously, after incubation for 48 hours, the proliferation effects of CCSKP, CCSKP1-A and FOS were significantly higher than those of the blank control, indicating that probiotics can use camphor seed polysaccharides CCSKP and CCSKP1-A as carbon sources to promote their own growth and proliferation. Figure 15 ), with the increase of CCSKP, CCSKP1-A and FOS concentrations, the OD of Lactobacillus acidophilus 600 The values also gradually increased. At a concentration of 3%, FOS had the strongest stimulating effect on the proliferation of Lactobacillus acidophilus, followed by CCSKP and CCSKP1-A (p < 0.05). Similarly, CCSKP and CCSKP1-A had a positive growth effect on Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium adolescentis within the tested concentration range ( Figure 16-18However, as the FOS concentration increased from 1.5% to 3.0%, the OD 600 The values decreased significantly (p<0.05). When the concentrations of FOS and CCSKP were 2.0% to 3.0%, the OD values of Bifidobacterium adolescentis were significantly decreased (p<0.05). 600 Overall, FOS had the most significant effect on the proliferation of Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium adolescentis, followed by CCSKP (p<0.05).

[0142] like Figure 15-18 As shown, CCSKP, CCSKP1-A, and FOS samples all enhanced the acidification activity of the tested probiotics (p < 0.05). The pH of the culture medium supplemented with CCSKP decreased, demonstrating a greater effect than that of the culture medium supplemented with CCSKP1-A. These results indicate that the four probiotics consumed CCSKP and CCSKP1-A at varying efficiencies, resulting in varying degrees of pH reduction. However, for all tested strains, the culture medium supplemented with FOS exhibited superior proliferation and acidification, a finding consistent with that of Macroglobulus polysaccharides. This effect may be related to the diverse metabolic pathways involved in substrate digestion in different growth media, perhaps because the strains preferentially selected simpler monosaccharides as carbon sources, which were then metabolized to produce more organic acids.

[0143] Overall, the decrease in pH reflects the ability of probiotics to utilize CCSKP and CCSKP1-A, the polysaccharides from camphor tree kernels, as a carbon source, leading to the production of short-chain fatty acids, which inevitably decreases the pH of the culture medium. In summary, the proliferation and acidification activity results of the four tested strains suggest that CCSKP and CCSKP1-A are potential prebiotics, but CCSKP exhibits stronger prebiotic properties than CCSKP1-A.

[0144] 1.3.4 Effect of Cinnamomum camphora seed polysaccharide on the growth rate of probiotics

[0145] In order to investigate the effect of camphor seed polysaccharide on the growth rate of Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium adolescentis, the strains were inoculated into culture medium supplemented with 2% (w / v) sample, and cultured anaerobically at 37°C. The absorbance and pH value of the culture medium were measured at different time points, and the growth curves of the strains were drawn. The results are shown in Table 1. Figure 19 、 Figure 20 、 Figure 21 、 Figure 22 Effects of CCSKP, CCSKP1-A, and FOS on the growth rate of probiotics; Figure 19 :Lactobacillus acidophilus, Figure 20 :Bifidobacterium longum, Figure 21 Bifidobacterium infantis, Figure 22: Bifidobacterium adolescentis;

[0146] Depend on Figures 19-22 It can be seen that CCSKP, CCSKP1-A and FOS have a promoting effect on the growth and proliferation of the four strains. In the medium supplemented with CCSKP, after culturing Lactobacillus acidophilus for 24 hours, OD 600 The OD value reached the maximum value (0.97±0.03) and the pH value decreased to the minimum value (4.78±0.04). Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium adolescentis entered the stable phase after 30h of culture in CCSKP medium. With the continuous increase of culture time, the number of bacteria and pH in the culture medium remained basically unchanged. In the culture medium supplemented with CCSKP1-A, Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium adolescentis entered the stable phase after 24h, 30h, 30h and 36h of culture respectively. After 30h of culture of Lactobacillus acidophilus, OD value decreased to the minimum value (4.78±0.04). 600 The value reached the maximum value (0.43±0.04), and the pH value decreased to the minimum value (5.33±0.02). At the same time, in the culture medium with FOS added, the adaptability of probiotics to FOS was better than that of camphor seed polysaccharides CCSKP and CCSKP1-A. The OD 600 The values were all larger, and the corresponding pH values were lower. In summary, the absorbance values of CCSKP, CCSKP1-A, and FOS cultures increased and the pH decreased over time, indicating that probiotics can utilize CCSKP, CCSKP1-A, and FOS to promote their own growth and reproduction, while also producing acidic metabolites. However, under the same concentrations of CCSKP and CCSKP1-A, the time it took for probiotics in CCSKP culture medium to reach a stable stage was shortened from 30-36 hours to 24-30 hours, indicating that CCSKP1-A has a weaker promoting effect on probiotics than CCSKP. These results confirm that CCSKP and CCSKP1-A, polysaccharides from camphor tree seeds, are potential prebiotics.

[0147] 1.3.5 Results of probiotic fermentation of camphor seed polysaccharide to produce short-chain fatty acids

[0148] Short-chain fatty acids are the main end products of probiotic metabolism. The levels of short-chain fatty acids can reflect not only the probiotic's proliferative activity but also the utilization rate of CCSKP, CCSKP1-A, and FOS, a polysaccharide from the camphor tree seed kernel. As shown in Table 1, the concentrations of lactic acid, acetic acid, propionic acid, and butyric acid in CCSKP, CCSKP1-A, and FOS, a polysaccharide from the camphor tree seed kernel, after 48 hours of in vitro fermentation, were significantly higher than those in the control group (p < 0.05). This indicates that CCSKP and CCSKP1-A, a polysaccharide from the camphor tree seed kernel, can promote the production of SCFAs to a certain extent. Specifically, CCSKP and CCSKP1-A components can be utilized or fermented as carbon sources by Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium adolescentis. Furthermore, during the 48-hour fermentation period of the test probiotics, CCSKP, CCSKP1-A, and FOS all significantly increased the concentrations of SCFAs, especially acetic and lactic acid concentrations (p < 0.05), while propionic and butyric acid concentrations showed no significant difference (p > 0.05). Furthermore, the total SCFA concentration in the FOS group was significantly higher than that in the camphor seed kernel polysaccharides CCSKP and CCSKP1-A (p < 0.05), and the total SCFA concentration in the CCSKP1-A group was lower than that in the CCSKP group (p < 0.05). The total SCFA concentrations produced in the culture medium were in the order of FOS > CCSKP > CCSKP1-A. This result is consistent with the results of the in vitro proliferation experiment, in which CCSKP exhibited a stronger proliferative effect than CCSKP1-A, but not as strong as FOS.

[0149] Table 1 Distribution of short-chain fatty acids in liquid culture medium with different carbon sources

[0150]

[0151]

[0152] Note: (Aa: acetic acid; Pa: propionic acid; Ba: butyric acid; La: lactic acid); Control: culture without carbon source (sugar-free MRS-based medium); abcdefg indicates significant differences in the same column (p<0.05); ABCD indicates significant differences in the same row (p>0.05); mean ± SD (n = 3).

[0153] 1.4 Chapter Summary

[0154] 1. Study on the digestibility of camphor seed kernel polysaccharides CCSKP and CCSKP1-A. The results showed that after 6 hours of hydrolysis in simulated gastric fluid at pH 1-5, the maximum hydrolysis degrees of CCSKP and CCSKP1-A were 4.66±0.13% and 1.69±0.03%, respectively. The maximum hydrolysis degrees by α-amylase were 5.90±0.13% and 5.74±0.23%, respectively. This indicates that both CCSKP and CCSKP1-A are well resistant to hydrolysis by gastric acid and α-amylase and are not easily digested.

[0155] 2. Study on the in vitro proliferation effects of camphor seed kernel polysaccharides CCSKP and CCSKP1-A on probiotic bacteria. The results showed that the proliferation effects of CCSKP and CCSKP1-A on Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium adolescentis were significantly higher than those of the blank control after 48 hours of anaerobic culture, indicating that both CCSKP and CCSKP promote the growth and proliferation of probiotic strains, but CCSKP has a stronger promoting effect on probiotics.

[0156] 3. Investigate the effects of CCSKP and CCSKP1-A, a polysaccharide from camphor tree seed kernel, on the growth rate of the test strain and the pH change of the culture medium. The results showed that after culturing Lactobacillus acidophilus in the culture medium containing camphor tree seed kernel polysaccharide for 24 hours, the OD 600 The values reached their maximum values, and the three strains of Bifidobacterium reached their maximum growth at 32-36 hours. In addition, the probiotics had better adaptability to CCSKP.

[0157] 4. We investigated the effects of camphor seed kernel polysaccharides CCSKP and CCSKP1-A on short-chain fatty acid production by probiotics. Results showed that CCSKP and CCSKP1-A produced acetic acid, sodium propionate, butyric acid, and lactic acid in the four probiotic strains. Acetic acid and lactic acid were the primary components, with acetic acid being the most abundant. This suggests that the four probiotic strains can utilize CCSKP and CCSKP1-A components to promote their proliferation and produce organic acids, making them potential prebiotics.

[0158] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and are not intended to limit them. Although the present application has been described in detail with reference to preferred embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present application can still be modified or some technical features can be replaced by equivalents, which should all be included in the scope of the technical solutions requested for protection in this application.

Claims

1. A camphor tree seed kernel polysaccharide, characterized in that The camphor tree seed kernel polysaccharide includes the following monosaccharides: Glucose, mannose, galactose, arabinose and glucosamine hydrochloride, and the molar ratio is (0.88-0.92): (0.06-0.1): (0.015-0.02): (0.003-0.004): (0.003-0.004); The weight average molecular weight of the camphor tree seed kernel polysaccharide is 1000Da to 20000Da.

2. The camphor seed polysaccharide according to claim 1, characterized in that The weight average molecular weight of the camphor seed polysaccharide is 4000Da to 15000Da; The average particle size of the polysaccharide is 350nm to 1200nm.

3. The camphor tree seed kernel polysaccharide according to claim 1, characterized in that The camphor tree seed kernel polysaccharide is prepared by deproteinizing and decolorizing the aqueous phase of camphor tree seed kernel oil extracted from camphor tree seed kernels by aqueous enzymatic method to obtain a decolorized solution, and concentrating, alcohol-precipitating, dialyzing, and freeze-drying the decolorized solution to obtain camphor tree seed kernel polysaccharide CCSKP.

4. The camphor tree seed kernel polysaccharide according to claim 3, characterized in that The camphor tree seed kernel polysaccharide CCSKP was separated by anion exchange chromatography column, and the eluate of the distilled water elution component was collected, dialyzed, concentrated, and freeze-dried. The camphor tree seed kernel polysaccharide CCSKP1-A was purified by dextran Sephadex G-50 gel adsorption method.

5. The camphor seed polysaccharide according to claim 3, characterized in that The total sugar, protein and uronic acid contents of the camphor seed kernel polysaccharide CCSKP were 64.70±1.27%, 11.33±1.12% and 9.45±0.33%, respectively. The molecular weight of the polysaccharide CCSKP was determined by high performance gel permeation chromatography. The chromatogram of CCSKP showed three peaks, and the calculated molecular weights were 14080Da, 7934Da and 5361Da, respectively. The molar ratio of glucose, mannose, galactose, arabinose and glucosamine hydrochloride in the monosaccharide composition is 0.911:0.065:0.018:0.004:0.003; The particle size is 354.85 nm and the Zeta potential value is -4.64 mV.

6. The camphor seed polysaccharide according to claim 4, characterized in that The camphor seed kernel polysaccharide CCSKP1-A is a neutral polysaccharide: The total sugar content was 89.50 ± 0.46%, and the protein content was 3.15 ± 0.54%; The molar ratio of glucose, mannose, galactose, arabinose and glucosamine hydrochloride in the monosaccharide composition is 0.883:0.094:0.016:0.003:0.004; The particle size is 1158.00 nm and the Zeta potential value is -0.03 mV.

7. A method for preparing camphor seed polysaccharide according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) Cinnamomum camphora seed kernels were ground using a high-speed grinder, and distilled water was added to prepare a suspension with a volume ratio of 1:

6. The pH was adjusted to 5.0-6.0, and cellulase was added to prepare a 3000U / ml solution. The solution was heated in a constant temperature water bath at 55°C for 2 h. After the enzyme was inactivated at high temperature for 10 min, the supernatant was collected by centrifugation and placed in a refrigerator at 4°C for 12 h to further remove free oil. (2) Deproteinization and decolorization: add 2% by weight volume of papain to the treated aqueous phase, hydrolyze at 55°C for 2 h, inactivate the enzyme at high temperature for 10 min, remove the precipitate by centrifugation, collect the supernatant, add 10% by weight volume of pre-treated activated AB-8 macroporous resin to the supernatant, decolorize in a shaker at 40°C for 1 h, remove the macroporous resin by filtration under reduced pressure, and collect the decolorized solution; (3) Ethanol precipitation: Use a rotary evaporator to concentrate the decolorized solution to 25% of its original volume at 60°C. Collect the concentrate, add 3 times the volume of anhydrous ethanol, and place it in a refrigerator at 4°C overnight. Centrifuge to remove the supernatant, and add appropriate distilled water to the precipitated components for re-dissolution. (4) Dialysis and impurity removal: The reconstituted camphor seed kernel polysaccharide solution is placed in a 1000Da dialysis bag and dialyzed with running water for 1 to 2 days. After the dialysis is completed, the camphor seed kernel polysaccharide CCSKP is obtained by concentration and freeze drying.

8. The method for preparing camphor seed kernel polysaccharide according to claim 7, wherein The method further comprises: (5) DEAE-52 cellulose anion exchange separation Pretreatment: The weighed DEAE-52 cellulose was subjected to alkali-acid-alkali pretreatment, and then washed with appropriate amount of distilled water 3-4 times until the pH was 7.0-7.4; A 10 mg / mL aqueous solution of camphor seed kernel polysaccharide CCSKP was prepared, the polysaccharide solution was filtered through a 0.45 μm filter membrane, the CCSKP solution was added dropwise to a pre-installed anion exchange chromatography column, and eluted with a gradient NaCl solution of 0 to 0.5 mol / L as the eluent, the flow rate was set to 1 mL / min, the eluate flowing out was automatically collected at a speed of 10 min / tube, 30 tubes of eluate for each gradient were collected, the absorbance was measured at OD490 nm, and an elution curve was drawn. According to the elution curve, the eluates of the distilled water elution fraction, the 0.1 mol / L NaCl elution fraction, and the 0.2 mol / L NaCl elution fraction were respectively collected for dialysis, concentration, and vacuum freeze-drying to obtain camphor seed kernel polysaccharide components CCSKP1, CCSKP2, and CCSKP3; (6) Purification by Sephadex G-50 gel adsorption method Accurately weigh the camphor seed kernel polysaccharide component CCSKP1 to prepare a 10 mg / ml polysaccharide solution, filter it with a 0.45 μm filter head, add it to a dextran Sephadex G-50 gel column, and elute it with ultrapure water as the eluent. The eluate is automatically collected in different tubes, and the flow rate is set to 0.2 mL / min, 4 mL / tube. The eluate is collected until all the polysaccharides flow out, and its absorbance is measured and the dextran gel elution curve is drawn. The collected products are combined, concentrated, and freeze-dried to obtain the camphor seed kernel polysaccharide component CCSKP1-A.

9. Use of the camphor tree seed kernel polysaccharide according to any one of claims 1 to 6 in the preparation of food for promoting the proliferation of probiotics.

10. The use according to claim 9, characterized in that The food is a health product.

11. The use according to claim 9, wherein: The probiotic is Lactobacillus acidophilus Lactobacillus acidophilus , Bifidobacterium longum Bifidobacterium longum , Bifidobacterium infantis Bifidobacterium infantis and Bifidobacterium adolescentis Bifidobacterium adolescentis At least one of them.

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