Method for improving antioxidant activity of pollen pini polysaccharide by utilizing bifidobacterium fermentation
By combining Bifidobacterium fermentation and proteolysis with flash extraction, the problems of low extraction efficiency and difficulty in maintaining the activity of pine pollen polysaccharides have been solved, achieving efficient and environmentally friendly pine pollen polysaccharide extraction and improving the antioxidant activity and extraction rate of polysaccharides.
Patent Information
- Application Number
- CN202511632161.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-10
- Publication Date
- 2026-03-27
AI Technical Summary
Existing methods for extracting pine pollen polysaccharides suffer from problems such as low extraction efficiency, difficulty in maintaining activity, harsh process conditions, poor environmental friendliness, and high energy consumption, which limit their high-value development and large-scale application.
A method combining Bifidobacterium fermentation with proteolysis and flash extraction was adopted. The enzyme system produced by Bifidobacterium during fermentation gently degrades the cell wall of pine pollen, releasing intracellular polysaccharides. The antioxidant activity of the polysaccharides was enhanced by treatment with trichloroacetic acid.
It significantly improves polysaccharide yield and bioactivity, reduces energy consumption, and minimizes environmental pollution, providing an efficient, mild, and environmentally friendly extraction process.
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Figure CN121736128A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of natural product extraction technology and relates to a method for enhancing the antioxidant activity of pine pollen polysaccharides through fermentation with Bifidobacterium. Background Technology
[0002] Pine pollen, a natural product with both medicinal and edible uses, is rich in various bioactive components such as polysaccharides, flavonoids, and phytosterols. Among them, pine pollen polysaccharides have attracted much attention due to their multiple physiological functions, including immunomodulation, anti-tumor activity, and antioxidant effects. Currently, the main extraction methods for pine pollen polysaccharides include hot water extraction, acid-base extraction, enzymatic hydrolysis, and ultrasound-assisted extraction. Hot water extraction is simple to operate, but it suffers from problems such as high extraction temperature, easy degradation of polysaccharides, and limited extraction rate. Although acid-base extraction can improve extraction efficiency, strong acid or alkali environments can easily cause damage to the polysaccharide structure and loss of biological activity, while also leading to environmental pollution and equipment corrosion. Ultrasonic-assisted extraction can improve the extraction rate, but it requires sophisticated equipment, consumes a lot of energy, and may cause polysaccharide denaturation due to localized overheating.
[0003] These traditional methods generally suffer from problems such as difficulty in balancing extraction efficiency and activity retention, demanding process conditions, poor environmental friendliness, high energy consumption, and easy destruction of polysaccharide structure, which seriously restrict the high-value development and large-scale application of pine pollen polysaccharides. Therefore, there is an urgent need to develop a new extraction process that is efficient, mild, environmentally friendly, and can maximize the preservation of the structure and activity of pine pollen polysaccharides. Summary of the Invention
[0004] The purpose of this invention is to overcome the problems of low extraction rate and destruction of activity of pine pollen polysaccharides in the existing technology, and to provide a method for enhancing the antioxidant activity of pine pollen polysaccharides by fermentation with Bifidobacterium.
[0005] The objective of this invention can be achieved through the following technical solutions: The technical solution of the present invention is to provide a method for enhancing the antioxidant activity of pine pollen polysaccharides by fermentation with Bifidobacterium, comprising the following steps: S1. Add Bifidobacterium bacterial solution to pine pollen for fermentation to obtain fermentation product; S2. Adjust the pH of the fermentation product from step S1 to acidic, and add protease for enzymatic hydrolysis to obtain the enzymatic hydrolysis product. S3. The enzymatic hydrolysis product obtained in step S2 is subjected to flash extraction, and the supernatant is collected by centrifugation to obtain crude polysaccharide extract of pine pollen. S4. Add trichloroacetic acid solution to the crude pine pollen polysaccharide extract obtained in step S3, and dialyze to obtain pine pollen polysaccharide with enhanced antioxidant activity.
[0006] In some specific embodiments, in step S1, the fermentation process conditions are: the concentration of Bifidobacterium bacterial culture is (1~2)×10⁻⁶.8 The inoculum concentration was CFU / mL, the inoculum size of Bifidobacterium was 5%–25%, the fermentation temperature was 35℃–39℃, and the fermentation time was 3 days–11 days.
[0007] Preferably, in step S1, the fermentation process conditions are as follows: the concentration of Bifidobacterium bacterial culture is 1.24 × 10⁻⁶. 8 The concentration of CFU / mL was 25% for Bifidobacterium inoculum, the fermentation temperature was 37℃, and the fermentation time was 7 days.
[0008] In some specific embodiments, in step S2, the pH of the fermentation product is adjusted to 4.0~6.0.
[0009] Preferably, in step S2, the solution used to adjust the pH of the fermentation product is a mixture of citric acid and disodium hydrogen phosphate.
[0010] Preferably, in step S2, before adjusting the pH of the fermentation product, the fermentation product is sterilized for a time of (10~20) min at a temperature of 85℃.
[0011] As a more preferred step, in step S2, before adjusting the pH of the fermentation product, the fermentation product is sterilized for 15 minutes at a temperature of 85°C.
[0012] In some specific embodiments, in step S2, the protease is papain. The conditions for enzymatic hydrolysis are: the amount of enzyme added is (2~3)%, the hydrolysis time is (2~3) h, and the hydrolysis temperature is (45~50)℃.
[0013] Preferred conditions for enzymatic hydrolysis are: enzyme addition of 2.5%, hydrolysis time of 2.5 h, and hydrolysis temperature of 48℃.
[0014] Preferably, after enzymatic hydrolysis, an enzyme inactivation treatment is performed for 15 minutes at a temperature of 95°C.
[0015] In some specific embodiments, in step S3, the flash extraction process conditions are: flash voltage of (90~110) V and flash time of (80~100) s; The centrifugation process conditions are as follows: centrifugation speed is (11000~13000) r / min, and centrifugation time is (5~15) min.
[0016] As a preferred embodiment, in step S3, the flash extraction process conditions are: flash voltage of 100 V and flash time of 90 s. The centrifugation process conditions were: centrifugation speed of 12000 r / min and centrifugation time of 10 min.
[0017] In some specific embodiments, in step S4, the volume ratio of trichloroacetic acid solution to crude pine pollen polysaccharide extract is 1:1, wherein the trichloroacetic acid solution is selected as having a volume percentage of 5% to 15% trichloroacetic acid.
[0018] In some specific embodiments, in step S4, the dialysis process conditions are: the molecular weight cutoff of the dialysis bag used is ≥3500 Da, and the dialysis time is 8~10 h.
[0019] Compared with the prior art, the present invention has the following advantages: (1) The Bifidobacterium introduced in this invention is a probiotic that produces various metabolites, such as proteases, during its metabolism. These metabolites help break down large active molecules into smaller active molecules, thereby increasing the activity of the active ingredients. Therefore, this invention utilizes the multiple enzyme systems produced by Bifidobacterium during fermentation to gently degrade the cell walls of pine pollen, thereby efficiently releasing intracellular polysaccharides. This not only significantly improves the polysaccharide yield but also effectively avoids the damage to the polysaccharide structure caused by harsh conditions such as high temperature and strong acid / alkali, thus better maintaining its antioxidant activity. At the same time, this method has the advantages of mild conditions, environmental friendliness, low energy consumption, and the ability to improve polysaccharide yield and biological activity, providing a new approach for the green and sustainable extraction of pine pollen polysaccharides.
[0020] (2) The Bifidobacterium microbial fermentation of pine pollen used in this invention saves energy consumption, is environmentally friendly, has lower cost, makes the product easier to separate, has a high polysaccharide conversion rate, and the fermentation process is less likely to cause pollution. Attached Figure Description
[0021] Figure 1 A schematic diagram illustrating the effect of different fermentation temperatures on the fermentation of pine pollen polysaccharides.
[0022] Figure 2 A schematic diagram illustrating the effect of different fermentation times on the fermentation of pine pollen polysaccharides.
[0023] Figure 3 This is a schematic diagram illustrating the effect of different inoculum ratios on the fermentation of pine pollen polysaccharides.
[0024] Figure 4 The DPPH free radical scavenging rate of pine pollen polysaccharides was obtained in Example 2 and Comparative Example 1.
[0025] Figure 5 The ABTS free radical scavenging rate of pine pollen polysaccharides was obtained in Example 2 and Comparative Example 1.
[0026] Figure 6 The hydroxyl radical scavenging rate of pine pollen polysaccharides was obtained for Example 2 and Comparative Example 1.
[0027] Figure 7 This is the standard curve for glucose content.
[0028] Figure 8 This is a standard curve for protein content. Detailed Implementation
[0029] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.
[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
[0031] Unless otherwise specified, all steps of this application may be performed sequentially or randomly, but sequentially is preferred.
[0032] The sources of some of the materials used in the following embodiments and comparative examples: (1) Pine pollen comes from the Haozhou Traditional Chinese Medicine Market; (2) Bifidobacterium longum was purchased from Taisto Biotechnology Co., Ltd., with the serial number ATCC15707.
[0033] Example 1: This embodiment provides a method for enhancing the antioxidant activity of pine pollen polysaccharides through fermentation with Bifidobacterium longum, comprising the following steps: (1) After anaerobic activation of the Bifidobacterium longum strain, the bacterial concentration was adjusted to 1.24 × 10⁻⁶. 8 The bacterial solution was sprayed evenly onto pine pollen at a concentration of CFU / mL. The inoculation rate was set at 5%~25% (v:g). The mixture was stirred evenly and fermented at a temperature of 35℃~39℃ for 3 days~11 days.
[0034] (2) Sterilize the fermented pine pollen at a temperature of 85°C for 30 minutes.
[0035] (3) Prepare a buffer solution with citric acid and disodium hydrogen phosphate. The pH of the buffer solution is 5.0. Add it to the sterilized pine pollen fermentation product at a material-to-liquid ratio of 1:20 to obtain a mixed solution A with a pH of 5.5.
[0036] (4) Add papain to mixture A. The amount of papain added is 2.5% of the mass of pine pollen. The enzymatic hydrolysis reaction is carried out at 50℃ for 2.5 h. After the enzymatic hydrolysis is completed, the enzyme is inactivated at 95℃ for 15 min to obtain mixture B.
[0037] (5) The mixture B was subjected to flash treatment with a flash voltage of 100 V and a flash time of 90 s to obtain mixture C.
[0038] (6) Centrifuge the mixture C at 12000 r / min for 10 min. The supernatant is the crude polysaccharide extract of pine pollen. Add an equal volume of trichloroacetic acid solution (volume percentage of 10%) to the supernatant to remove the protein in the crude polysaccharide extract of pine pollen. After rotary evaporation and concentration, dialyze the crude polysaccharide extract of pine pollen with protein removed using a 3500 Da dialysis bag for 8 h to obtain the dialysate. After vacuum freeze-drying, a relatively refined pine pollen polysaccharide is obtained.
[0039] This embodiment also identified three factors affecting the fermentation of pine pollen polysaccharides: inoculum quantity, fermentation days, and fermentation temperature. Single-factor experiments were conducted using the pine pollen polysaccharide extraction rate as a comparative indicator.
[0040] (1-1) Five different fermentation temperatures were selected for single-factor experiments, namely 35℃, 36℃, 37℃, 38℃ and 39℃.
[0041] The other two factors were set as follows: fermentation time 7 days and inoculum amount 20%.
[0042] The effect of fermentation temperature on the extraction rate of pine pollen polysaccharides is as follows: Figure 1 The extraction rate of pine pollen polysaccharides showed a trend of first increasing and then decreasing with the increase of fermentation temperature. The extraction rate of pine pollen polysaccharides was the highest when the fermentation temperature was 36℃, so the optimal fermentation temperature was 36℃.
[0043] (1-2) Five different fermentation days were selected for single-factor experiments, with fermentation days of 3d, 5d, 7d, 9d and 11d respectively.
[0044] The other two factors were set as follows: inoculum amount 20% and fermentation temperature 37℃.
[0045] The effect of fermentation days on the extraction rate of pine pollen polysaccharides is as follows: Figure 2 It can be seen that the extraction rate of pine pollen polysaccharides first increases and then slowly decreases with the increase of fermentation days. The extraction rate of pine pollen polysaccharides is the highest when the fermentation time is 5 days, so the optimal fermentation time is 5 days.
[0046] (1-3) Five different inoculation amounts were selected for single-factor experiments, with inoculation amounts of 5%, 10%, 15%, 20%, and 25%, respectively.
[0047] The other two factors were set as follows: fermentation time 7 days and fermentation temperature 37℃.
[0048] The effect of inoculum quantity on the extraction rate of pine pollen polysaccharides is as follows: Figure 3 It can be seen that the extraction rate of pine pollen polysaccharides first increases and then decreases with the increase of inoculum amount. When the inoculum amount is 20%, the extraction rate of pine pollen polysaccharides is relatively high, so the optimal inoculum amount is 20%.
[0049] Based on the results of the single-factor experiments above, an orthogonal experiment was conducted with fermentation temperature (A), fermentation time (B), and inoculum quantity (C) as independent variables. The design and results are as follows: Table 1 Experimental Design and Results In the table above, A. Fermentation temperature: 1, 2, 3 represent 35℃, 36℃, 37℃ respectively; B. Fermentation days: 1, 2, 3 represent 3d, 5d, 7d respectively; C. Inoculum quantity: 1, 2, 3 represent 15%, 20%, 25% respectively.
[0050] The K value represents an experimental index at a certain level under a certain factor. For example, the K1 value under column A in the table represents the sum of the yields of the three pine pollen polysaccharides at level 1 under factor A.
[0051] The k value is the average of the sums of the K values, such as k1 = K1 / 3; R is the range of k values, i.e., k max -k min .
[0052] The results of the orthogonal experiment are analyzed as follows: According to the experimental results of the nine groups in Table 1, the yield of polysaccharides is between 14.05% and 17.12%. According to the analysis in Table 2, the order of influencing factors is: inoculum quantity (C) > fermentation temperature (A) > fermentation time (B). The theoretical optimal fermentation process is: fermentation temperature of 37℃, fermentation time of 7 days, and inoculum quantity of 25%.
[0053] Table 2. Analysis of variance of orthogonal results Example 2: This embodiment provides an optimal method for enhancing the antioxidant activity of pine pollen polysaccharides through fermentation with Bifidobacterium longum, comprising the following steps: (1) After anaerobic activation of the Bifidobacterium longum strain, the bacterial concentration was adjusted to 1.24 × 10⁻⁶. 8 The bacterial solution was sprayed evenly onto pine pollen at a concentration of CFU / mL. The inoculum concentration was set at 25% (v:g). The mixture was stirred evenly and fermented at a temperature of 37℃ for 7 days.
[0054] (2) Sterilize the fermented pine pollen at a temperature of 85°C for 30 minutes.
[0055] (3) Prepare a buffer solution with citric acid and disodium hydrogen phosphate. The pH of the buffer solution is 5.0. Add it to the sterilized pine pollen fermentation product at a material-to-liquid ratio of 1:20 to obtain a mixed solution A with a pH of 5.5.
[0056] (4) Add papain to mixture A. The amount of papain added is 2.5% of the mass of pine pollen. The enzymatic hydrolysis reaction is carried out at 50℃ for 2.5 h. After the enzymatic hydrolysis is completed, the enzyme is inactivated at 95℃ for 15 min to obtain mixture B.
[0057] (5) The mixture B was subjected to flash treatment with a flash voltage of 100 V and a flash time of 90 s to obtain mixture C.
[0058] (6) Centrifuge the mixture C at 12000 r / min for 10 min. The supernatant is the crude polysaccharide extract of pine pollen. Add an equal volume of trichloroacetic acid solution (volume percentage of 10%) to the supernatant to remove the protein in the crude polysaccharide extract of pine pollen. After rotary evaporation and concentration, dialyze the crude polysaccharide extract of pine pollen with protein removed using a 3500 Da dialysis bag for 8 h to obtain the dialysate. After vacuum freeze-drying, a relatively refined pine pollen polysaccharide is obtained.
[0059] Comparative Example 1: Compared with Example 1, the pine pollen in this comparative example was not fermented with Bifidobacterium longum. The specific steps were as follows: (1) Prepare a buffer solution with citric acid and disodium hydrogen phosphate. The pH of the buffer solution is 5.0. Add it to pine pollen at a ratio of 1:20 to obtain a mixture A with a pH of 5.5.
[0060] (2) Papain was added to mixture A. The amount of papain added was 2.5% of the weight of pine pollen. The enzymatic hydrolysis reaction was carried out at 50℃ for 2.5 h. After the enzymatic hydrolysis was completed, the enzyme was inactivated at 95℃ for 15 min to obtain mixture B.
[0061] (3) The mixture B was subjected to flash treatment with a flash voltage of 100 V and a flash time of 90 s to obtain mixture C.
[0062] (4) Centrifuge the mixture C at 12000 r / min for 10 min. The supernatant is the crude polysaccharide extract of pine pollen. Add an equal volume of trichloroacetic acid solution (volume percentage of 10%) to the supernatant to remove the protein in the crude polysaccharide extract of pine pollen. After rotary evaporation and concentration, dialyze the crude polysaccharide extract of pine pollen with protein removed using a 3500 Da dialysis bag for 8 h to obtain the dialysate. After vacuum freeze-drying, a relatively refined pine pollen polysaccharide is obtained.
[0063] Test Example 1: This test example uses the phenol-sulfuric acid method to determine the polysaccharide content in the crude pine pollen polysaccharide extract of Example 2 and Comparative Example 1, as follows: Prepare a glucose standard stock solution by serially diluting the glucose standard stock solution to a concentration range of 0~0.12 (mg / mL). Take 0.5 mL of the glucose standard dilution solution, add 0.25 mL of 6% phenol solution, mix well, and then quickly add 1.25 mL of concentrated sulfuric acid. Let it stand for 20 min, then boil it in a water bath for 15 min, and let it stand for another 1 h. Measure the absorbance at a wavelength of 490 nm with the reagent blank zeroed.
[0064] like Figure 7 As shown, the concentration range of the glucose standard is 0.02~0.14 (mg / mL), and the curve equation is y=5.6155x+0.0614, R 2 =0.9996, indicating a good linear relationship between glucose concentration and absorbance. This method for determining polysaccharide content has high accuracy and can be used to determine the polysaccharide content of pine pollen.
[0065] Take 0.5 mL of polysaccharide extract, add 0.25 mL of 6% phenol solution, mix well, and then quickly add 1.25 mL of concentrated sulfuric acid. Let stand for 20 min, then boil in a water bath for 15 min, and let stand for 1 h. Measure the absorbance at 490 nm wavelength with a reagent blank as the zero point. The polysaccharide content in Example 2 was 12.13 mg / mL, and the polysaccharide content in Comparative Example 1 was 15.94 mg / mL.
[0066] Test Example 2: This test example calculates the polysaccharide extraction rate of crude pine pollen extract from Example 2 and Comparative Example 1 based on the polysaccharide content obtained from Test Example 1, and the calculation is performed according to the following formula: Polysaccharide extraction rate = c * v / m Where c is the polysaccharide concentration obtained from the standard curve, in units of (mg / mL). v represents the volume of the extract, in mL. m represents the mass of pine pollen, in grams (g).
[0067] The polysaccharide extraction rates of Example 2 and Comparative Example 1 were calculated to be 16.81% (n=4, error ±0.3922) and 18.65% (n=4, error ±0.0518), respectively.
[0068] Test Example 3: This test example uses the Coomassie Brilliant Blue method to measure the protein content in the crude pine pollen polysaccharide extracts of Example 2 and Comparative Example 1, as detailed below: Prepare a stock solution of bovine serum albumin standard. Dilute the stock solution of bovine serum albumin standard stepwise to a concentration range of 0-100 (μg / mL). Mix the diluted bovine serum albumin standard solution with 2.5 mL of Coomassie brilliant blue solution, let stand for 5 min, and measure the absorbance at 595 nm.
[0069] like Figure 8 As shown, the concentration range of bovine serum albumin standard is 0~100 (μg / mL), and the curve equation is y=0.0234X+0.0034, R 2 =0.9994, indicating a good linear relationship between bovine serum albumin concentration and absorbance. This method for determining protein content has high accuracy and can be used to determine the protein content in crude polysaccharide extract of pine pollen.
[0070] 0.5 mL of crude pine pollen polysaccharide extract from Example 2 and Comparative Example 1 were mixed with 2.5 mL of Coomassie Brilliant Blue solution, and the absorbance was measured at 595 nm after standing for 5 min. The protein content in the crude pine pollen polysaccharide extract of Example 2 was 9.00%, and the protein content in the crude pine pollen polysaccharide extract of Comparative Example 1 was 10.75%, indicating that the crude pine pollen polysaccharide extract obtained by the method of the present invention has less impurity content, thus yielding pine pollen polysaccharide with higher purity.
[0071] Test Example 4: This test case determined the antioxidant activity (DPPH radical scavenging, ABTS radical scavenging, and hydroxyl radical scavenging) of pine pollen polysaccharides in Example 2 and Comparative Example 1, as follows: 1. DPPH free radical scavenging experiment Mix 1 mL of sample solution with 1 mL of DPPH ethanol solution, incubate at room temperature in the dark for 30 min, and measure the absorbance at 517 nm. Calculate the DPPH free radical scavenging rate according to the following formula.
[0072] DPPH free radical scavenging rate (%) In the formula: A1: Absorbance of the sample solution + DPPH ethanol solution; A0: The control absorbance of the sample solution + ethanol solution (excluding free radicals); A: Absorbance of the DPPH ethanol solution.
[0073] The antioxidant activity of pine pollen polysaccharides obtained in Example 2 (0.5-5 mg / mL) and Comparative Example 1 (2-12 mg / mL) was determined by DPPH free radical scavenging assay. Figure 4As shown, the pine pollen polysaccharides obtained in Example 2 and Comparative Example 1 both exhibited high DPPH free radical scavenging rates. However, under the same DPPH free radical scavenging rate, the concentration of the pine pollen polysaccharide obtained by Bifidobacterium fermentation (Example 2) was lower than that of the unfermented pine pollen polysaccharide (Comparative Example 1), indicating that the pine pollen polysaccharide obtained by Bifidobacterium fermentation (Example 2) had a higher DPPH free radical scavenging rate than the unfermented pine pollen polysaccharide (Comparative Example 1).
[0074] 2. ABTS free radical scavenging experiment Mix 1 mL of sample solution with 1 mL of ABTS ethanol solution, incubate at room temperature in the dark for 30 min, and measure the absorbance at 734 nm. Calculate the ABTS free radical scavenging rate using the following formula.
[0075] ABTS free radical scavenging rate (%) In the formula: A 1： The absorbance is the sample solution + ABTS ethanol solution. A 0： The absorbance of the sample solution plus ethanol solution is the control absorbance (excluding free radicals); A: Absorbance of ABTS ethanol solution; The antioxidant activity of pine pollen polysaccharides obtained in Example 2 at concentrations of 1-5 mg / mL and those obtained in Comparative Example 1 at concentrations of 2-12 mg / mL was determined using an ABTS free radical scavenging assay. Figure 5 As shown, the pine pollen polysaccharides obtained in Example 2 and Comparative Example 1 both exhibited good scavenging activity against ABTS free radicals. However, at the same ABTS scavenging rate, the concentration of the pine pollen polysaccharide obtained by fermentation with Bifidobacterium (Example 2) was lower than that of the pine pollen polysaccharide obtained without fermentation (Comparative Example 1). This indicates that the pine pollen polysaccharide obtained by fermentation with Bifidobacterium (Example 2) had a higher scavenging rate against ABTS free radicals than the pine pollen polysaccharide obtained without fermentation (Comparative Example 1).
[0076] 3. Hydroxyl radical scavenging experiment First, add 1 mL of FeSO4 and 1 mL of salicylic acid-ethanol solution, then add 2 mL of sample solution, followed by 1 mL of H2O2 to start the reaction. Maintain the temperature at 37℃ in a water bath for 30 min, and measure the absorbance at 510 nm. Calculate the hydroxyl radical scavenging rate according to the following formula.
[0077] Hydroxyl radical scavenging rate (%) A1: Absorbance of the sample solution; A0: Absorbance of the sample solution without the colorimetric reagent H2O2 (excluding free radicals); A: Absorbance of hydroxyl radicals in the blank control; The antioxidant activity of pine pollen polysaccharides obtained in Example 2 (0.3-1.5 mg / mL) and Comparative Example 1 (0.2-1.5 mg / mL) was determined by a hydroxyl radical scavenging assay. Figure 6 As shown, the pine pollen polysaccharides obtained in Example 2 and Comparative Example 1 both exhibit good scavenging properties against hydroxyl radicals. Furthermore, under the same hydroxyl radical scavenging rate, the scavenging rates of pine pollen polysaccharides obtained by fermentation with Bifidobacterium (Example 2) and those obtained without fermentation (Comparative Example 1) are very similar.
[0078] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.
Claims
1. A method for enhancing the antioxidant activity of pine pollen polysaccharides through Bifidobacterium fermentation, characterized in that, Includes the following steps: S1. Add Bifidobacterium bacterial solution to pine pollen for fermentation to obtain fermentation product; S2. Adjust the pH of the fermentation product from step S1 to acidic, and add protease for enzymatic hydrolysis to obtain the enzymatic hydrolysis product. S3. The enzymatic hydrolysis product obtained in step S2 is subjected to flash extraction, and the supernatant is collected by centrifugation to obtain crude polysaccharide extract of pine pollen. S4. Add trichloroacetic acid solution to the crude pine pollen polysaccharide extract obtained in step S3, and dialyze to obtain pine pollen polysaccharide with enhanced antioxidant activity.
2. The method for enhancing the antioxidant activity of pine pollen polysaccharides by fermentation with Bifidobacterium according to claim 1, characterized in that, The fermentation process conditions are as follows: the concentration of Bifidobacterium bacterial culture is (1~2)×10⁻⁶. 8 The inoculum concentration was CFU / mL, the inoculum size of Bifidobacterium was 5%–25%, the fermentation temperature was 35℃–39℃, and the fermentation time was 3 days–11 days.
3. The method for enhancing the antioxidant activity of pine pollen polysaccharides by fermentation with Bifidobacterium according to claim 2, characterized in that, In step S1, the fermentation conditions are as follows: the concentration of Bifidobacterium culture is 1.24 × 10⁻⁶. 8 The concentration of CFU / mL was 25% for Bifidobacterium inoculum, the fermentation temperature was 37℃, and the fermentation time was 7 days.
4. The method for enhancing the antioxidant activity of pine pollen polysaccharides by fermentation with Bifidobacterium according to claim 1, characterized in that, In step S2, adjust the pH of the fermentation product to 4.0~6.
0.
5. The method for enhancing the antioxidant activity of pine pollen polysaccharides by fermentation with Bifidobacterium according to claim 4, characterized in that, In step S2, the solution used to adjust the pH of the fermentation product is a mixture of citric acid and disodium hydrogen phosphate.
6. The method for enhancing the antioxidant activity of pine pollen polysaccharides by fermentation with Bifidobacterium according to claim 1, characterized in that, In step S2, before adjusting the pH of the fermentation product, the fermentation product is sterilized for 10-20 minutes at a temperature of 85°C.
7. The method for enhancing the antioxidant activity of pine pollen polysaccharides by fermentation with Bifidobacterium according to claim 1, characterized in that, In step S2, the protease is papain. The conditions for enzymatic hydrolysis are: the amount of enzyme added is (2~3)%, the hydrolysis time is (2~3) h, and the hydrolysis temperature is (45~50)℃.
8. The method for enhancing the antioxidant activity of pine pollen polysaccharides by fermentation with Bifidobacterium according to claim 1, characterized in that, In step S3, the flash extraction process conditions are: flash voltage of (90~110) V and flash time of (80~100) s; The centrifugation process conditions are as follows: centrifugation speed is (11000~13000) r / min, and centrifugation time is (5~15) min.
9. The method for enhancing the antioxidant activity of pine pollen polysaccharides by fermentation with Bifidobacterium according to claim 1, characterized in that, In step S4, the volume ratio of trichloroacetic acid solution to crude pine pollen polysaccharide extract is 1:1, wherein the trichloroacetic acid solution is selected as having a volume percentage of 5% to 15% trichloroacetic acid.
10. The method for enhancing the antioxidant activity of pine pollen polysaccharides by fermentation with Bifidobacterium according to claim 1, characterized in that, In step S4, the dialysis process conditions are as follows: the molecular weight cutoff of the dialysis bag used is ≥3500 Da, and the dialysis time is 8~10 h.