Ginger-processed fermented pumpkin polysaccharide and preparation method thereof

Abstract

The application discloses ginger-processed fermented pumpkin polysaccharide and a preparation method thereof, relates to the technical field of natural product extraction, and uses pumpkin pulp and ginger juice as main raw materials, utilizes a natural protease and amylase system in the ginger juice to perform pre-enzymatic hydrolysis on the pumpkin pulp, and then obtains high-purity ginger-processed fermented pumpkin polysaccharide through a combined fermentation process of sequential inoculation of lactic acid bacteria and saccharomyces cerevisiae, alcohol precipitation, deproteinization, dialysis and freeze-drying steps. The obtained polysaccharide has a content of not less than 85%, a protein content of less than 2%, a molecular weight distribution of 50-200 kDa, and a monosaccharide composition mainly composed of glucose, galactose and mannose. The application also determines the monosaccharide composition, molecular weight distribution, functional group characteristics and sugar residue connection mode of the ginger-processed fermented pumpkin polysaccharide. The application has the advantages of mild process conditions, high polysaccharide yield, complete structure reservation and suitability for industrial amplification production.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction technology, specifically to a ginger-fermented pumpkin polysaccharide and its preparation method. Background Technology

[0002] pumpkin( Cucurbita moschata Duch. Pumpkin is a widely cultivated vegetable in my country used for both medicinal and culinary purposes. It is rich in polysaccharides, flavonoids, carotenoids, and other bioactive substances. Pumpkin polysaccharides possess bioactivities such as hypoglycemic effects, antioxidant properties, and immune regulation, and have become a research hotspot in the fields of functional foods and natural medicines.

[0003] Ginger Zingiber officinale Roscoe Ginger is a commonly used pungent and warm exterior-releasing herb in traditional Chinese medicine, containing abundant active ingredients such as protease (gingerase), amylase, gingerol, and shogaol. Studies have shown that endogenous enzymes in ginger promote the release of polysaccharides, and ginger polysaccharides themselves are mainly composed of glucose (accounting for nearly 80%), rich in →4-linked glucans, possessing antioxidant, immunomodulatory, and other biological activities.

[0004] At present, the extraction of pumpkin polysaccharides mainly adopts hot water extraction, alkaline extraction or single enzymatic hydrolysis, which has the following shortcomings: (1) Hot water extraction has a high temperature, and the active polysaccharide structure is easily damaged; (2) Alkaline extraction causes greater damage to the glycosidic bonds of polysaccharides; (3) Single enzymatic hydrolysis has a high cost; (4) Existing fermentation methods are mostly single-strain fermentation, and the fermentation products have a single functional component.

[0005] In traditional Chinese medicine theory, the ginger-processing method for medicinal herbs is believed to enhance efficacy and reduce toxicity. However, there are currently no reports on using ginger juice as a pre-processing agent for pumpkin polysaccharides, followed by the use of natural enzymes from the ginger juice as an endogenous enzyme source, combined with a synergistic approach of dual-strain microbial and dual-substrate fermentation to achieve highly efficient preparation of highly active pumpkin polysaccharides. Furthermore, existing research on pumpkin polysaccharides largely focuses on yield and activity evaluation, with limited research on structural characteristics such as monosaccharide composition, molecular weight distribution, and sugar residue linkage. Summary of the Invention

[0006] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a ginger-fermented pumpkin polysaccharide and its preparation method.

[0007] The technical solution of the present invention is as follows: A method for preparing ginger-fermented pumpkin polysaccharide includes the following steps: S1: Prepare ginger juice and pumpkin puree separately; mix ginger juice and pumpkin puree, use the protease and amylase in ginger juice to enzymatically hydrolyze pumpkin puree, inactivate enzymes, centrifuge and collect supernatant to obtain enzymatic hydrolysate; S2: First, lactic acid bacteria are added to the enzymatic hydrolysate for fermentation, then brewer's yeast is added to continue fermentation. After inactivating the bacteria, the supernatant is collected by centrifugation to obtain ginger and pumpkin dual-substrate fermentation broth. S3: Add anhydrous ethanol to the ginger-pumpkin dual-substrate fermentation broth for alcohol precipitation, collect the precipitate by centrifugation, purify the precipitate by deproteinization and dialysis in sequence, and freeze-dry to obtain ginger-fermented pumpkin polysaccharide.

[0008] Preferably, in step S1, the ginger juice is prepared as follows: Peel and wash fresh ginger, add distilled water and blend into a paste, centrifuge and collect the supernatant to obtain ginger juice; and / or; The method for preparing the pumpkin puree is as follows: Pumpkin powder or fresh pumpkin is mixed with water to make a pumpkin puree with a mass concentration of 5% to 15%.

[0009] Preferably, in step S1, the volume ratio of ginger juice to pumpkin puree is 1:8 to 1:12, and the mixture is placed at 20 to 35°C for 4 to 6 hours; and / or; The enzymatic hydrolysis temperature is 45–55℃, the enzymatic hydrolysis time is 1–3 h, and the pH of the enzymatic hydrolysate is adjusted to 5.5–6.5. After the enzymatic hydrolysis is completed, the enzyme is inactivated by incubation at 80–90℃ for 10–20 min.

[0010] Preferably, in step S2, lactic acid bacteria are first introduced and fermented at 35-38°C for 12-24 hours; then brewer's yeast is introduced and fermented at 28-32°C for another 12-48 hours. The lactic acid bacteria are *Lactobacillus plantarum*. Lactobacillus plantarum and / or thermophilic streptococci Streptococcus thermophilus The inoculation amount is 2%–4%, in v / v; the brewing yeast is Saccharomyces cerevisiae The inoculation amount is 0.5% to 2%, and the unit is v / v.

[0011] Preferably, in step S3, the alcohol precipitation is performed by adding 2 to 4 times the volume of anhydrous ethanol to the fermentation broth and letting it stand overnight at 4°C; the deproteinization is performed by deproteinizing 3 to 5 times using the Sevag method; and the dialysis purification is performed using a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzing in distilled water for 48 hours, with the water changed every 12 hours.

[0012] The present invention also discloses a ginger-fermented pumpkin polysaccharide, which is prepared by the above-described preparation method.

[0013] Preferably, the ginger-fermented pumpkin polysaccharide content is not less than 85% based on glucose; the protein content is less than 2%; the reducing sugar content is less than 5%; and the uronic acid content is 2% to 5%, where % is a mass percentage. The weight-average molecular weight of the ginger-fermented pumpkin polysaccharide is 50-200 kDa, and the polydispersity index is less than 1.5. The monosaccharide composition of the ginger-fermented pumpkin polysaccharide includes glucose, galactose, mannose, xylose, arabinose, rhamnose, and galacturonic acid.

[0014] Preferably, the monosaccharide composition is: glucose 38%–48%, galactose 22%–32%, mannose 8%–14%, xylose 5%–12%, arabinose 3%–8%, rhamnose 2%–5%, galacturonic acid 2%–5%, where % is molar percentage.

[0015] The beneficial effects of this invention are: (1) This invention utilizes the principle of processing ginger in traditional Chinese medicine to soak and process pumpkin pulp in order to enhance its functional effects.

[0016] (2) This invention makes full use of the natural enzyme system of ginger to replace chemical reagents for pretreatment, thereby reducing costs.

[0017] (3) The ginger-fermented pumpkin polysaccharide obtained by this invention has a polysaccharide content of 88.3% and a protein content of only 1.2%, which is far superior to the products obtained by hot water extraction and alkali extraction.

[0018] (4) Through systematic structural characterization methods, this invention has clarified that the monosaccharide composition of polysaccharides is mainly glucose (45.2 mol%), and the sugar residues are mainly linked by →4)-D-Glcp(1→ and tD-Glcp, which confirms that ginger juice enzymatic hydrolysis has a remodeling effect on polysaccharide glycosidic bonds.

[0019] (5) The process conditions of this invention are mild and the polysaccharide structure is well preserved, making it suitable for industrial-scale production. Attached Figure Description

[0020] Figure 1 A bar chart comparing the monosaccharide composition of GFPP-1 (ginger fermentation), Comparative Example 1 (hot water extraction control), and Comparative Example 3 (ginger-based control only) (HPIC-PAD analysis). Data in the figure are expressed as mean ± standard deviation (n=3). P<0.01, P<0.05, compared with the GFPP-1 group.

[0021] Figure 2 This is a comparative graph (HPGPC analysis) showing the molecular weight distribution of GFPP-1 (ginger fermentation), Comparative Example 1 (hot water extraction), and Comparative Example 4 (combined fermentation only), including weight-average molecular weight (Mw) and polydispersity index (PDI). Data in the graph are expressed as mean ± standard deviation (n=3). P<0.01, P<0.05, compared with the GFPP-1 group.

[0022] Figure 3The Fourier Transform Infrared (FT-IR) spectra of ginger-fermented pumpkin polysaccharide.

[0023] Figure 4 The figures show a comparison of the physicochemical properties of GFPP-1 and Comparative Examples 1-6. (a) compares the polysaccharide yield, (b) compares the polysaccharide purity, (c) compares the protein residue, and (d) compares the uronic acid content. Data in the figures are expressed as mean ± standard deviation (n=3). P<0.01, compared with Comparative Example 1 (hot water extraction). Detailed Implementation

[0024] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0025] Example 1: Preparation of ginger-fermented pumpkin polysaccharide (1) Raw material pretreatment: Take 2kg of fresh pumpkin (variety: honey pumpkin, purchased from local farmers market), remove the seeds, wash and slice (about 5mm thick), dry in a 65℃ hot air oven until the moisture content is less than 8%, grind with a grinder and pass through a 60-mesh sieve to obtain pumpkin powder for later use. Take 500g of fresh ginger (variety: yellow ginger, purchased from farmers market), wash and peel, soak in 4℃ ice water for 30min, add distilled water at a material-to-liquid ratio of 1:3 and grind into a paste, centrifuge at 3000rpm for 10min, take about 350mL of supernatant, which is ginger juice. The protease activity of ginger juice was measured to be about 132U / mL and the amylase activity was about 28U / mL.

[0026] (2) Soaking and processing with ginger juice: Weigh 100g of pumpkin powder, add it to 900mL of purified water, stir and disperse to obtain pumpkin pulp, add 100mL of ginger juice to the pumpkin pulp (the volume ratio of ginger juice to pumpkin pulp is 1:9), mix at room temperature and let stand for 5 hours to obtain a mixture; (3) Preparation of enzymatic hydrolysate: Adjust the pH of the mixture to 6.0, and enzymatically hydrolyze the pumpkin pulp with protease and amylase in ginger juice for 2 hours under constant temperature and magnetic stirring at 50℃. After the enzymatic hydrolysis is completed, inactivate the enzyme in a water bath at 85℃ for 15 minutes, cool, centrifuge at 4000 rpm for 15 minutes, and take about 800 mL of supernatant for later use.

[0027] (4) Dual substrate co-fermentation: Lactobacillus plantarum ( Lactobacillus plantarum ATCC 14917, purchased from China General Microbiological Culture Collection Center, CGMCC, Beijing) mother liquor and brewer's yeast ( Saccharomyces cerevisiaeATCC 9763 (purchased from China General Microbiological Culture Collection Center, CGMCC, Beijing) was used to prepare mother liquors to an OD600 ≈ 1.0. Lactic acid bacteria were first inoculated into the supernatant at 3% (v / v) and fermented at 37℃ and 120 rpm for 18 h. Then, *Saccharomyces cerevisiae* was inoculated at 1% (v / v) and fermentation continued for 24 h. After fermentation, the supernatant was incubated at 85℃ for 15 min, cooled, and centrifuged at 6000 rpm for 20 min, collecting approximately 700 mL of the fermentation supernatant.

[0028] (5) Alcohol precipitation purification: Slowly add 3 volumes of pre-cooled anhydrous ethanol (final concentration approximately 75%, v / v, referring to the volume fraction of ethanol in the alcohol precipitation system) to the fermentation broth and let it stand overnight at 4°C. Centrifuge at 6000 rpm for 15 min, collect the precipitate, dissolve it in a small amount of distilled water, and deproteinize it 4 times with Sevag reagent (chloroform: n-butanol volume ratio = 4:1). Each deproteinization process involves mixing the precipitate with Sevag reagent at a volume ratio of 4:1, shaking for 20 min, centrifuging at 3000 rpm for 15 min, discarding the interfacial protein layer, and repeating this process 4 times. Dialyze the solution in distilled water using a 3500 Da dialysis bag for 48 h, changing the water every 12 h. Freeze-dry the dialysate to obtain ginger-fermented pumpkin polysaccharide (GFPP-1), with a yield of 4.8%.

[0029] Comparative Example 1 (Hot Water Extraction Control Group) Weigh 100g of pumpkin powder and add 2000mL of purified water (solid-to-liquid ratio 1:20, g / mL). Stir and extract in a 90℃ constant temperature water bath for 2 hours. After extraction, cool to room temperature, centrifuge at 6000rpm for 15 minutes, collect the supernatant, concentrate under reduced pressure to 1 / 3 of the original volume, add 3 times the volume of pre-cooled anhydrous ethanol, and precipitate overnight at 4℃. Subsequent deproteinization, dialysis, and freeze-drying steps are the same as in Example 1. This group does not undergo ginger juice enzymatic hydrolysis and microbial fermentation.

[0030] Comparative Example 2 (Alkali Extraction Control Group) Weigh 100g of pumpkin powder and add 2000mL of 0.1mol / L NaOH solution (solid-to-liquid ratio 1:20, g / mL). Extract at room temperature with magnetic stirring for 2h. After extraction, adjust the pH to 7.0 with 1mol / L HCl, centrifuge at 6000rpm for 15min, collect the supernatant, concentrate under reduced pressure to 1 / 3 of the original volume, add 3 times the volume of pre-cooled anhydrous ethanol, and precipitate overnight at 4℃. Subsequent deproteinization, dialysis, and freeze-drying steps are the same as in Example 1. This group does not undergo ginger juice enzymatic hydrolysis and microbial fermentation.

[0031] Comparative Example 3 (Ginger-based only) Compared with Example 1, the difference is that step (4) is omitted, that is, no Lactobacillus plantarum and Saccharomyces cerevisiae are added, and the rest is the same as Example 1.

[0032] Comparative Example 4 (fermentation with compound bacteria only) Compared with Example 1, the difference is that no ginger juice was added, and pumpkin puree was directly fermented with Lactobacillus plantarum and Saccharomyces cerevisiae, while the rest is the same as in Example 1.

[0033] Comparative Example 5 (Ginger-based and Lactobacillus plantarum fermentation alone) The difference from Example 1 is that only Lactobacillus plantarum was used for fermentation, while the rest is the same as in Example 1.

[0034] Comparative Example 6 (Ginger-based and single brewing yeast fermentation) The difference from Example 1 is that only brewer's yeast is used for fermentation, while the rest is the same as in Example 1.

[0035] Example 2: Determination of Physicochemical Properties Sugar content (calculated as glucose) was determined by the phenol-sulfuric acid method, protein content (calculated as bovine serum albumin) was determined by the Coomassie Brilliant Blue G-250 method, and uronic acid content (calculated as galacturonic acid) was determined by the m-hydroxybiphenyl method; the results are shown in Table 1.

[0036] Table 1. Content of GFPP-1 obtained in Example 1 and Comparative Examples 1-6

[0037] Table 1 shows that, compared with the hot water extraction control group of Comparative Example 1 and the alkali extraction control group of Comparative Example 2, the purity of GFPP-1 prepared in Example 1 increased from 76.5% to 88.3% and the protein residue decreased from 3.8% to 1.2%. Compared with Comparative Example 3 (ginger-only) and Comparative Example 4 (combined fermentation only), the polysaccharide purity of ginger-only or single-strain fermentation was lower than that of the combined process (88.3%), demonstrating a synergistic effect between ginger-only fermentation and fermentation. The results of Comparative Examples 5 and 6 indicate that combined microbial fermentation is superior to single-strain fermentation.

[0038] Example 3: Monosaccharide composition analysis (HPIC-PAD) Approximately 10 mg of each of Example 1 (GFPP-1), Comparative Example 1, and Comparative Example 3 were added to 2 mL of 4 mol / L trifluoroacetic acid (TFA) and hydrolyzed at 110 °C under sealed conditions for 6 h. After hydrolysis, the mixture was dried under nitrogen and washed three times with methanol to remove residual TFA. The residue was dissolved in 1 mL of ultrapure water and filtered through a 0.22 μm filter membrane. A Dionex CarboPac PA20 analytical column (3 mm × 150 mm) was used at 30 °C; mobile phase A was 0.25 mol / L NaOH, and mobile phase B was 1.0 mol / L NaAc, with gradient elution at a flow rate of 0.25 mL / min.

[0039] Gradient elution program: 0–25 min, mobile phase A is NaOH, mobile phase B is NaAc, A:B = 100:0 (v / v), isocratic elution of neutral monosaccharides (rhamnose, arabinose, galactose, glucose, xylose, mannose); 25–35 min, B linearly increases from 0% to 20% (i.e., NaAc 0 increases to 0.20 mol / L), eluting acidic sugars such as galacturonic acid; 35–40 min, B linearly increases from 20% to 100% (NaAc 0.20 increases to 1.0 mol / L), washing the column; 40–45 min, B is maintained at 100%; 45–65 min, the column is restored to A:B = 100:0 equilibrium. Column temperature: 30℃; injection volume: 10 μL; detector: pulsed amperometric detector (PAD), Au as working electrode, Ag / AgCl as reference electrode, four-potential waveform.

[0040] The results are as follows Figure 1 As shown, the GFPP-1 prepared in Example 1 mainly consists of glucose (Glc, 45.2 mol%), galactose (Gal, 28.6 mol%), mannose (Man, 12.1 mol%), xylose (Xyl, 8.7 mol%), and arabinose (Ara, 5.4 mol%), while also containing small amounts of rhamnose (Rha, 3.4 mol%) and galacturonic acid (GalA, 3.2 mol%). Glucose is the dominant monosaccharide in ginger polysaccharides (approximately 80 mol%). After ginger fermentation, the glucose content in pumpkin polysaccharides was significantly higher than that of the hot water extraction control in Comparative Example 1 (38.5 mol%) and the ginger-only control in Comparative Example 3 (42.1 mol%). During the enzymatic hydrolysis of ginger juice, ginger-derived glucans were introduced into the pumpkin polysaccharide system, indicating that the ginger juice enzymatic hydrolysis-combined fermentation process is superior to traditional methods in regulating polysaccharide composition.

[0041] Example 4: Molecular weight determination (HPGPC) A TSK-GEL G5000PWXL tandem G3000PWXL gel chromatography column was used at a column temperature of 35℃; the mobile phase was 0.1 mol / L NaNO3 (containing 0.02% NaN3), and the flow rate was 0.6 mL / min; a differential refractive index detector (RID) was used. Calibration curves were established using dextran standards (580, 48.6, 12.4, 1.8 kDa).

[0042] The results are as follows Figure 2As shown, GFPP-1 exhibits two main chromatographic peaks with weight-average molecular weights (Mw) of 82 kDa (peak area percentage 78%) and 15 kDa (22%), respectively, and a polydispersity index (PDI) of 1.28. The molecular weight of the pumpkin polysaccharide prepared in this invention is significantly increased. This is because pumpkin polysaccharide itself is mainly composed of high molecular weight glucans, and the ginger juice enzymatic hydrolysis-combined fermentation process does not destroy the polysaccharide backbone structure. Ginger polysaccharide is mainly composed of low molecular weight oligosaccharides (approximately 5 kDa). During fermentation, some low molecular weight oligosaccharides are introduced, corresponding to the appearance of the 15 kDa secondary peak in GFPP-1. HPGPC analysis of Comparative Example 1 (hot water extraction) showed a weight-average molecular weight of 46 kDa and a PDI of 1.42. The weight-average molecular weight of Comparative Example 4 (combined microbial fermentation only) was 68 kDa, with a PDI of 1.35. The above comparisons indicate that the molecular weight of the ginger fermentation product is significantly higher than that of the hot water extraction method and the combined fermentation method alone, suggesting that the combined ginger juice enzymatic hydrolysis-combined fermentation process is beneficial for retaining high molecular weight polysaccharide components.

[0043] Example 5: Fourier Transmission Infrared Spectroscopy (FT-IR) Take approximately 2 mg of dried GFPP-1 powder, mix it with approximately 200 mg of dried KBr, grind them evenly, and compress into tablets. Use a Fourier transform infrared spectroscopy system (FTIR) at 4000–400 cm⁻¹. -1 Scan within range, resolution 4cm -1 The number of scans was 32.

[0044] The results are as follows Figure 3 As shown, GFPP-1 at 3380 cm⁻¹ -1 There is a strong and broad absorption peak for the OH stretching vibration at 2920 cm⁻¹. -1 The peak at 1640 cm⁻¹ represents the CH stretching vibration peak. -1 An absorption peak of uronic acid carbonyl group (C=O) appeared at 1410 cm⁻¹, consistent with the determination result of uronic acid content of 3.16%; -1 The peak at 1075 cm⁻¹ represents the CH bending vibration peak. -1 and 1040cm -1 There is strong absorption nearby, attributed to COC glycosidic bonds and CO stretching vibrations; 890 cm⁻¹ -1 Characteristic absorption of β-glycosidic bond is present at 810 cm⁻¹. -1 The presence of α-glycosidic bond characteristic absorption indicates that GFPP-1 possesses a typical polysaccharide backbone, containing both α- and β-glycosidic bond configurations, suggesting that ginger juice processing and fermentation did not disrupt the basic molecular backbone of pumpkin polysaccharides.

[0045] Example 6: Analysis of sugar residue methylation (PMAA-GC-MS) Approximately 10 mg of each sample (GFPP-1, hot water extract of Comparative Example 1, and a simple mixture sample, the simple mixture being a 1:9 volume ratio of ginger juice and pumpkin puree) were taken and methylated according to the Ciacanu-Kerek method. The polysaccharide sample was first dissolved in DMSO, then NaOH powder and methyl iodide were added, and the mixture was stirred at room temperature for 2 hours. The methylation process was repeated until no infrared detection was observed in the 3200–3600 cm⁻¹ range. -1 Hydroxyl characteristic absorption peak. The fully methylated polysaccharide was hydrolyzed with 2 mol / L TFA at 110℃ for 4 h, reduced with NaBH4, and acetylated with pyridine-acetic anhydride (1:1) at 90℃ for 1 h to obtain the PMAA derivative, which was then analyzed by GC-MS.

[0046] GC-MS conditions: DB-5MS capillary column (30m × 0.25mm, 0.25μm); temperature program: 140℃ for 2 min, increased to 180℃ at 3℃ / min, then increased to 280℃ at 8℃ / min and held for 5 min; helium carrier gas, flow rate 1.0 mL / min; EI ion source, ion source temperature 230℃. The linkage mode of major sugar residues of GFPP-1 is shown in Table 2.

[0047] Table 2. Linkage of major sugar residues in GFPP-1

[0048] Table 2 shows that ginger juice enzymatic hydrolysis has a common glycosidic bond remodeling effect in polysaccharide structure modification (not a simple physical introduction, but rather refers to the cleavage and recombination of glycosidic bonds in pumpkin and ginger polysaccharides by endogenous enzymes such as proteases and amylases in ginger juice during enzymatic hydrolysis, allowing polysaccharide fragments from different sources to form new linkages through reassembly, which belongs to structural remodeling at the glycosidic bond level); that is, →4)-linked glucans in ginger are introduced into the pumpkin polysaccharide system, significantly increasing the proportion of →4)-D-Glcp(1→. This sugar residue linkage is a common active structural unit in plant polysaccharides, with multiple functions such as antioxidation and immunomodulation. The bioactivity was observed. Specifically, the proportion of →4)-D-Glucp(1→) in GFPP-1 was 45.4%, significantly higher than that in Comparative Example 1 (hot water extraction, 31.2%) and the simple mixture sample (37.8%). The sugar residue composition of the simple mixture sample was between that of GFPP-1 and Comparative Example 1, indicating that →4)-linked glucans from ginger were indeed partially introduced through physical mixing. However, its proportion (37.8%) was still significantly lower than that of GFPP-1 treated with enzymatic hydrolysis and fermentation (45.4%), confirming that ginger juice hydrolysis and combined fermentation further remodeled the glycosidic bonds of the polysaccharides, achieving a synergistic effect.

[0049] Example 7: Comparison of Physicochemical Properties of Different Extraction Methods Various physicochemical properties were measured for the samples of Example 1 and Comparative Examples 1-6.

[0050] The results are as follows Figure 4 As shown, compared with Comparative Example 1 (hot water extraction), the yield of GFPP-1 polysaccharides increased by 54.8% (4.8%, 3.1%), the polysaccharide content increased by 15.4% (88.3%, 76.5%), and the protein residue decreased by 68.4% (1.2%, 3.8%), all of which were statistically significant (P<0.01). Compared with Comparative Example 2 (alkali extraction), the polysaccharide content of GFPP-1 increased by 8.7% (88.3%, 81.2%), and the protein residue decreased by 52.0% (1.2%, 2.5%). Compared with Comparative Example 3 (ginger-processed only), the polysaccharide content of GFPP-1 increased by 7.5% (88.3%, 82.1%), indicating that adding compound fermentation to the ginger-processed method can further improve the purity. Compared with Comparative Example 4 (compound fermentation only), the polysaccharide content of GFPP-1 increased by 4.5% (88.3%, 84.5%), indicating that the pretreatment of ginger juice by enzymatic hydrolysis has an additional contribution to the purity of polysaccharides. The polysaccharide contents of Comparative Example 5 (fermented with lactic acid bacteria only) and Comparative Example 6 (fermented with yeast only) were 83.2% and 82.8%, respectively, both lower than GFPP-1, confirming that dual-strain fermentation is superior to single-strain fermentation. Regarding uronic acid content, GFPP-1 (3.16%) was higher than all comparative examples (1.85%–2.78%), indicating that the ginger fermentation process helps retain acidic polysaccharide components. These data show that ginger juice enzymatic hydrolysis combined with dual-strain fermentation is superior to traditional extraction methods and single processes in terms of polysaccharide yield, purity, protein removal, and uronic acid retention.

[0051] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various other corresponding changes and modifications based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing ginger-fermented pumpkin polysaccharide, characterized in that, Includes the following steps: S1: Prepare ginger juice and pumpkin puree separately; mix ginger juice and pumpkin puree, use the protease and amylase in ginger juice to enzymatically hydrolyze pumpkin puree, inactivate enzymes, centrifuge and collect supernatant to obtain enzymatic hydrolysate; S2: First, lactic acid bacteria are added to the enzymatic hydrolysate for fermentation, then brewer's yeast is added to continue fermentation. After inactivating the bacteria, the supernatant is collected by centrifugation to obtain ginger and pumpkin dual-substrate fermentation broth. S3: Add anhydrous ethanol to the ginger-pumpkin dual-substrate fermentation broth for alcohol precipitation, collect the precipitate by centrifugation, and sequentially remove proteins from the precipitate, dialyze and purify it, and then freeze-dry it to obtain ginger-fermented pumpkin polysaccharide.

2. The preparation method according to claim 1, characterized in that, In step S1, the ginger juice is prepared as follows: Peel and wash fresh ginger, add distilled water and blend into a paste, centrifuge and collect the supernatant to obtain ginger juice; and / or; The method for preparing the pumpkin puree is as follows: Pumpkin powder or fresh pumpkin is mixed with water to make a pumpkin puree with a mass concentration of 5% to 15%.

3. The preparation method according to claim 1, characterized in that, In step S1, the volume ratio of ginger juice to pumpkin puree is 1:8 to 1:12, and the mixture is placed at 20 to 35°C for 4 to 6 hours; and / or; The enzymatic hydrolysis temperature is 45–55℃, the enzymatic hydrolysis time is 1–3 h, and the pH of the enzymatic hydrolysate is adjusted to 5.5–6.

5. After the enzymatic hydrolysis is completed, the enzyme is inactivated by incubation at 80–90℃ for 10–20 min.

4. The preparation method according to claim 1, characterized in that, In step S2, lactic acid bacteria are first introduced and fermented at 35-38℃ for 12-24 hours; then brewer's yeast is introduced and fermented at 28-32℃ for another 12-48 hours. The lactic acid bacteria are *Lactobacillus plantarum*. Lactobacillus plantarum and / or thermophilic streptococci Streptococcus thermophilus The inoculation amount is 2%–4%, in v / v; the brewing yeast is Saccharomyces cerevisiae The inoculation amount is 0.5% to 2%, and the unit is v / v.

5. The preparation method according to claim 1, characterized in that, In step S3, the alcohol precipitation involves adding 2 to 4 times the volume of anhydrous ethanol to the fermentation broth and letting it stand overnight at 4°C; the deproteinization involves deproteinizing the protein 3 to 5 times using the Sevag method; and the dialysis purification uses a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzing in distilled water for 48 hours, with the water changed every 12 hours.

6. A ginger-based fermented pumpkin polysaccharide, characterized in that, It is prepared by any one of the preparation methods described in claims 1 to 5.

7. The ginger-fermented pumpkin polysaccharide according to claim 6, characterized in that, The ginger-fermented pumpkin polysaccharide content is not less than 85% based on glucose; the protein content is less than 2%; the reducing sugar content is less than 5%; and the uronic acid content is 2% to 5%, where % is by mass. The weight-average molecular weight of the ginger-fermented pumpkin polysaccharide is 50-200 kDa, and the polydispersity index is less than 1.

5. The monosaccharide composition of the ginger-fermented pumpkin polysaccharide includes glucose, galactose, mannose, xylose, arabinose, rhamnose, and galacturonic acid.

8. The ginger-fermented pumpkin polysaccharide according to claim 7, characterized in that, The monosaccharide composition is as follows: glucose 38%–48%, galactose 22%–32%, mannose 8%–14%, xylose 5%–12%, arabinose 3%–8%, rhamnose 2%–5%, galacturonic acid 2%–5%, where % is molar percentage.

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