Method for ultrasonic-high pressure assisted three-phase extraction of polysaccharides from herba cistanche based on response surface prediction

Abstract

This application discloses a method for ultrasound-assisted high-pressure three-phase extraction of Cistanche deserticola polysaccharides based on response surface methodology. The method first uses single-factor experiments and response surface methodology to construct a quadratic regression model using ammonium sulfate mass fraction, tert-butanol to Cistanche deserticola slurry volume ratio, extraction temperature, and pH as factors to optimize the three-phase extraction process. Then, Cistanche deserticola powder is mixed with water at a specific material-to-liquid ratio and subjected to pulsed ultrasound and ultra-high pressure pretreatment sequentially. Ammonium sulfate and tert-butanol are added according to optimized parameters to construct a three-phase system. The resulting polysaccharides are obtained by centrifugation, dialysis, and freeze-drying. This application achieves precise prediction and optimization of the extraction process. The synergistic effect of ultrasound and high pressure significantly improves the extraction rate, and the simultaneous deproteinization of the three-phase system improves purity. The obtained polysaccharides possess excellent structural characteristics, antioxidant activity, and digestibility, making them suitable as prebiotics for use in functional foods and pharmaceuticals.

Description

Technical Field

[0001] This application relates to the field of plant active ingredient extraction technology, specifically to a method for ultrasonic-high pressure assisted three-phase extraction of Cistanche deserticola polysaccharides based on response surface methodology. Background Technology

[0002] Plant polysaccharides are large bioactive molecules composed of monosaccharide units linked by glycosidic bonds, playing a positive role in the growth, development, and metabolic regulation of organisms. Studies have shown that natural plant polysaccharides possess various beneficial effects, including antioxidant, anti-inflammatory, antibacterial, antiviral, antitumor, immune-enhancing, and gut microbiota-regulating properties. Among them, Cistanche deserticola (CD), a perennial parasitic plant of the Orobanchaceae family, is a traditional and precious Chinese medicinal herb, known as "desert ginseng," and has been officially listed as a food and medicine homology substance by the relevant Chinese authorities. Modern pharmacological studies have confirmed that Cistanche deserticola polysaccharides (CDPs) possess a variety of pharmacological activities, including immunomodulation, antioxidant, anti-inflammatory, hepatoprotective, neuroprotective, antitumor, anti-aging, anti-osteoporosis, antidepressant, and anti-Alzheimer's disease effects, showing particular potential in regulating the gut microbiota and protecting against inflammatory intestinal diseases (including colorectal cancer).

[0003] However, it is worth noting that the bioactivity of polysaccharides is largely determined by their fine structural properties. As a fundamental prerequisite for polysaccharide research and application, extraction techniques not only directly affect the yield of polysaccharides but also profoundly alter their chemical structure, molecular weight distribution, monosaccharide composition, uronic acid content, surface morphology, and thermal stability, thus determining their final bioactivity. For example, compared with traditional hot water extraction, ultrasonic-assisted extraction can reduce the molecular weight of polysaccharides and change their monosaccharide composition, thereby enhancing their antioxidant and enzyme-inhibiting activities; high-pressure assisted extraction can reduce the molecular weight and degree of esterification and increase the uronic acid content, thereby improving antioxidant, anti-glycation, and immunostimulatory activities; ultra-high pressure treatment can change the particle size and surface morphology of polysaccharides, endowing them with superior cell-protective effects. Based on the well-defined structure-activity relationship of polysaccharides, extraction methods have become a key factor determining the physicochemical properties and functional performance of polysaccharides.

[0004] Currently, research on extraction methods for Cistanche deserticola polysaccharides is relatively limited. Existing technologies mainly employ traditional methods such as hot water extraction and acid-base extraction, which suffer from problems such as long extraction times, low extraction efficiency, and easy damage to the natural structure of polysaccharides. The application of physical field-assisted technologies such as ultrasound-assisted extraction and high-pressure-assisted extraction in the extraction of Cistanche deserticola polysaccharides has not been fully developed. In particular, research on systematically comparing different extraction technologies to explore their effects on the differential regulation of the structure and activity of Cistanche deserticola polysaccharides remains a gap.

[0005] Therefore, developing a new, efficient, and mild extraction technology for Cistanche deserticola polysaccharides and establishing a correlation model between the extraction process and polysaccharide quality is of great significance for filling the aforementioned technological gaps and realizing the efficient extraction and high-value application of Cistanche deserticola polysaccharides. Summary of the Invention

[0006] In view of this, the purpose of this application is to provide a method for ultrasonic-high pressure-assisted three-phase extraction of Cistanche deserticola polysaccharides based on response surface methodology. This method first considers ammonium sulfate mass fraction, tert-butanol volume ratio, extraction temperature, and pH as factors, with the polysaccharide extraction rate as the response value. A quadratic regression prediction model is constructed using single-factor experiments and response surface methodology to determine the optimal process parameters for three-phase extraction. Based on this, Cistanche deserticola powder is mixed with water at a specific material-to-liquid ratio, and then subjected to pulsed ultrasonic and ultra-high pressure pretreatment to disrupt the cell wall structure. A three-phase extraction system is then constructed according to the optimal conditions obtained from response surface optimization. The resulting polysaccharides are obtained through centrifugation, dialysis, concentration, and freeze-drying. This application enables precise prediction and optimization of the extraction process. The synergistic cell-wall disruption effect of ultrasound and high pressure significantly improves the extraction rate. The three-phase extraction system simultaneously removes proteins to improve purity, resulting in polysaccharides with higher extraction rates, better structural integrity, and antioxidant activity.

[0007] To achieve the above objectives, this application provides the following technical solution:

[0008] In a first aspect, this application provides a method for ultrasonic-high pressure assisted three-phase extraction of Cistanche deserticola polysaccharides based on response surface methodology, comprising the following steps:

[0009] (1) Response surface optimization experiment, construct a quadratic regression prediction model, and determine the optimal process parameters for three-phase extraction;

[0010] (2) After drying, crushing and sieving the dried slices of Cistanche deserticola, Cistanche deserticola powder is obtained. The Cistanche deserticola powder is mixed with distilled water according to the material-liquid ratio to prepare Cistanche deserticola slurry.

[0011] (3) The Cistanche deserticola slurry is subjected to a combination of ultrasonic and high-pressure pretreatment;

[0012] (4) The pretreated material was combined with ammonium sulfate and tert-butanol to form a three-phase extraction system, and three-phase centrifugation was carried out under the optimized process conditions;

[0013] (5) After separation, the lower phase polysaccharide solution is collected, and after dialysis, concentration and freeze drying, Cistanche deserticola polysaccharide is obtained.

[0014] In some implementations, step (1) specifically includes:

[0015] Using ammonium sulfate mass fraction, the volume ratio of tert-butanol to Cistanche deserticola slurry, extraction temperature, and extraction pH as the factors investigated, and the extraction rate of Cistanche deserticola polysaccharides as the response value, a quadratic regression prediction model was constructed through single-factor experiments and response surface methodology. The equation of the quadratic regression prediction model is as follows:

[0016] y=-51.9873+0.2208A+12.7177B+0.8631C+12.0400D+0.0641AB+0.0046AC+0.0222AD-0.0586BC-0.3882BD-0.0479CD-0.0093A 2 -3.4227B 2 -0.0086C 2 -0.9025D 2 Wherein, y is the extraction rate of Cistanche deserticola polysaccharide, A is the mass fraction of ammonium sulfate, B is the volume ratio of tert-butanol to Cistanche deserticola slurry, C is the extraction temperature, and D is the extraction pH; the Cistanche deserticola slurry refers to the mixture obtained by mixing Cistanche deserticola powder and distilled water according to the material-liquid ratio.

[0017] In some embodiments, in the quadratic regression prediction model, the degree of influence of each factor on the extraction rate of Cistanche deserticola polysaccharides is as follows: ammonium sulfate mass fraction > extraction temperature = extraction pH > volume ratio of tert-butanol to Cistanche deserticola slurry.

[0018] In some preferred embodiments, the optimal process parameters obtained by solving the quadratic regression prediction model are: ammonium sulfate mass fraction of 30%, volume ratio of tert-butanol to Cistanche deserticola slurry of 1:1.5, extraction temperature of 40 ℃, and extraction pH of 6.

[0019] In some embodiments, in step (2), the ratio of Cistanche deserticola powder to distilled water is 1:20.

[0020] In some preferred embodiments, in step (3), the ultrasonic pretreatment uses pulsed ultrasound with the following parameters: frequency 20kHz, power 400 W, temperature 25℃, and a 2-second on / 2-second off cycle mode, with a total processing time of 30 min.

[0021] In some preferred embodiments, in step (3), the processing parameters for high-pressure pretreatment are: pressure 300 MPa, temperature 25 ℃, and processing time 10 min.

[0022] In some implementations, in step (3), the combined ultrasound and high pressure pretreatment is performed by first performing pulse ultrasound treatment and then performing ultra-high pressure treatment.

[0023] In some embodiments, the construction method of the three-phase extraction system in step (4) is as follows: the pretreated Cistanche deserticola slurry is magnetically stirred for 12 h under ambient temperature and light-proof conditions, the supernatant is collected by centrifugation and concentrated to 2 / 3 of the original volume; ammonium sulfate is added to the optimal ammonium sulfate mass fraction and the pH is adjusted to the optimal extraction pH; tert-butanol is added to the optimal tert-butanol to Cistanche deserticola slurry volume ratio; the system is allowed to stand for 1 h under the optimal extraction temperature conditions; and then centrifuged to separate the system into three phases.

[0024] In some implementations, in step (4), the centrifugation conditions are 6000 rpm and the centrifugation time is 10 min.

[0025] In some embodiments, in step (5), the lower phase polysaccharide solution containing ammonium sulfate is collected, dialyzed through a dialysis bag with a molecular weight cutoff of 3500 Da, concentrated, and freeze-dried to obtain purified Cistanche deserticola polysaccharide.

[0026] Compared with existing technologies, it has at least the following advantages and beneficial effects:

[0027] 1. This application establishes a predictive model for polysaccharide extraction rate, considering the mass fraction of ammonium sulfate, the volume ratio of tert-butanol to Cistanche deserticola slurry, extraction temperature, and extraction pH as factors. Analysis of variance showed that the model was highly significant (P<0.0001), with no significant lack-of-fit terms, and the coefficient of determination R0 was [value missing]. 2 =0.9241, precision Adeq precision=11.1902>4, the relative deviation between the actual value and the predicted value in the verification experiment is <5%, indicating that the model fits well and has high prediction accuracy, and can be used for reliable optimization of the extraction process.

[0028] 2. This application introduces a combined ultrasonic and high-pressure pretreatment into a three-phase extraction system, significantly improving the extraction rate and purity of Cistanche deserticola polysaccharides. The cavitation effect of ultrasound and the mechanical disruption effect of ultra-high pressure synergistically break down the dense cell wall structure of Cistanche deserticola, promoting the efficient release of intracellular polysaccharides. The extraction rate is significantly higher than that of traditional water extraction and alcohol precipitation methods and single physical field-assisted methods. Simultaneously, the three-phase extraction system can remove proteins during the extraction process, avoiding the cumbersome additional protein removal steps required by traditional methods. Furthermore, the antioxidant activity of polysaccharides obtained from different extraction processes varies, with polysaccharides obtained through ultra-high pressure-assisted three-phase extraction exhibiting the strongest antioxidant capacity.

[0029] 3. The Cistanche deserticola polysaccharide obtained by the method of this application has good structural characteristics and bioactivity. The combined pretreatment reduces the molecular weight, particle size, and surface morphology of the polysaccharide, which is beneficial to improving the solubility and bioavailability of the polysaccharide. At the same time, the obtained polysaccharide has good digestive resistance and can resist digestion by α-amylase and simulated gastric juice, which provides a basis for it to play a prebiotic role in intestinal regulation. Attached Figure Description

[0030] Figure 1 This is a flowchart of the three-phase extraction process.

[0031] Figure 2 For response surface 3D plots and isoline plots.

[0032] Figure 3 The results are spectral and crystal structure analysis results; among which Figure 3 a represents the results of ultraviolet-visible spectroscopy analysis; 3b represents the results of Fourier transform infrared spectroscopy analysis; 3c represents the results of triple helix structure analysis; and 3d represents the X-ray diffraction curve.

[0033] Figure 4 The results include particle size, zeta potential, thermal stability measurements, and surface morphology analysis. Figure 4 a-4e represent particle size, Zeta potential, thermodynamic analysis of the four CDPs, TG curve, DTG curve, and scanning electron microscope images of the four CDPs, respectively. Figure 4 In e, AD represents CDPs-T, CDPs-U, CDPs-H, and CDPs-UH groups, respectively.

[0034] Figure 5 The results are for the determination of antioxidant activity, among which Figure 5 a is ABTS + Free radical scavenging ability, 5b is the DPPH free radical scavenging ability, and 5c is the hydroxyl free radical scavenging ability.

[0035] Figure 6 These are the results of in vitro digestion characteristic analysis; among which Figure 6 a-6e are the curves showing the change in the degree of hydrolysis of CDPs-T, CDPs-U, CDPs-H, CDPs-UH and inulin under different pH conditions after treatment with α-amylase over time. Figure 6 f-6j are curves showing the change in the degree of hydrolysis of CDPs-T, CDPs-U, CDPs-H, CDPs-UH, and inulin over time in artificial gastric fluid at different pH values.

[0036] Figure 7 The results are the analysis results of prebiotic activity and structure-activity relationship; among which Figure 7 a-7c represent the effects of different concentrations of CDPs on the proliferation of Bifidobacterium longum BB536, Lactobacillus plantarum L51-3, and Lactobacillus casei 129-2-1, respectively. Figure 7 d-7f represent the effects of different concentrations of CDPs on the pH of fermentation broths of Bifidobacterium longum BB536, Lactobacillus plantarum L51-3, and Lactobacillus casei 129-2-1, respectively. Figure 7 g is a heatmap showing the correlation between the structural features of CDPs and their antioxidant and prebiotic activities. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0038] The materials used in the following embodiments are not limited to those listed below, and other similar materials may be used instead. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0039] In this application, unless the context clearly indicates otherwise, the terms “including,” “comprising,” “containing,” “having,” etc., shall be understood as open-ended and mean “including but not limited to.”

[0040] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may be adjusted according to the desired performance. At a minimum, each numerical parameter should be interpreted based on the reported significant figures and by applying common rounding techniques.

[0041] 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 the subject matter of this application pertains. Before a detailed description of this application, the following explanations of terminology are provided to better understand this application.

[0042] 1. Three-phase separation method (TPP): This refers to a separation technique that, within the same reaction system, separates the system into three phases—an upper phase (organic solvent phase), a middle phase (denatured protein precipitate phase), and a lower phase (aqueous phase)—by adding a salting-out agent (such as ammonium sulfate) and an organic solvent (such as tert-butanol). This allows for the simultaneous extraction and preliminary purification of the target component. In this application, Cistanche deserticola polysaccharides are mainly enriched in the lower phase.

[0043] 2. Response surface methodology: This refers to a multiple regression analysis method that establishes a functional relationship between the response value (polysaccharide extraction rate in this application) and multiple influencing factors (ammonium sulfate mass fraction, tert-butanol to slurry volume ratio, extraction temperature, and extraction pH in this application) through reasonable experimental design. It is used for the optimization and prediction of process parameters. This application adopts a four-factor, three-level Box-Behnken design.

[0044] 3. Polysaccharide extraction rate: refers to the ratio of the mass of extracted polysaccharides to the total mass of Cistanche deserticola raw material powder, expressed as a percentage.

[0045] 4. Digestive resistance: This refers to the ability of polysaccharides to resist degradation in simulated digestive environments (such as α-amylase and simulated gastric juice). Polysaccharides with good digestive resistance can reach the colon intact and exert prebiotic effects. The Cistanche deserticola polysaccharides extracted in this application all exhibited resistance to digestion by α-amylase and simulated gastric juice.

[0046] The desert Cistanche deserticola used in this application was purchased from Alashan, Inner Mongolia, China. α-amylase (derived from Aspergillus oryzae, 30 U / mg) was purchased from Sigma-Aldrich. Dialysis bags (molecular weight cutoff 3500 Da) were purchased from Solarbio. Inulin (In), ammonium sulfate, and tert-butanol were all purchased from Aladdin. Lactic acid content kit (Addison, China).

[0047] Bifidobacterium longum BB536 (CICC24632), Lactobacillus plantarum L51-3 (CICC20999) and Lactobacillus casei 129-2-1 (CICC20990) were all purchased from the China Industrial Microbial Culture Collection Center.

[0048] All experimental results in the following examples are expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS 26.0 software. The significance level for all tests was set at P < 0.05. Data visualization and graphical representation were performed using Origin 2021 software.

[0049] The following are specific examples:

[0050] Example 1: Response surface methodology experiment to determine the optimal process parameters for three-phase extraction

[0051] 1.1 Basic Operation Procedure of Three-Phase Extraction

[0052] like Figure 1 As shown, the basic operation flow of three-phase extraction in this embodiment is as follows:

[0053] Dried Cistanche deserticola slices were baked at 50 ℃ for 12 h, pulverized, and passed through a 100-mesh sieve to obtain Cistanche deserticola powder. The powder was mixed with distilled water at a specific material-to-liquid ratio (the material-to-liquid ratio refers to the ratio of the mass of Cistanche deserticola powder to the volume of distilled water, in g / mL, i.e., w / v). The mixture was magnetically stirred for 12 h at room temperature and in the dark, and then centrifuged at 6000 rpm for 10 min. The supernatant was collected. First, a certain mass fraction of ammonium sulfate was added, and after it was fully dissolved, the pH was adjusted to a certain value using 1M HCl / NaOH. Then, a certain volume ratio (tert-butanol to Cistanche deserticola slurry volume ratio) of tert-butanol was added. The mixture was reacted at 40 ℃ for a certain time, and then centrifuged at 6000 rpm for 10 min. The system separated into three phases: the upper phase was the tert-butanol phase (which could be recovered by vacuum evaporation), the middle phase was the protein precipitate, and the lower phase was a polysaccharide solution containing ammonium sulfate. The lower phase polysaccharide solution was collected, dialyzed through a dialysis bag with a molecular weight cutoff of 3500 Da, concentrated, and freeze-dried to obtain Cistanche deserticola polysaccharide.

[0054] 1.2 Calculation of Cistanche deserticola polysaccharide extraction rate

[0055] The extraction rate of Cistanche deserticola polysaccharides was calculated according to the following formula:

[0056] ;

[0057] Where C is the concentration of Cistanche deserticola polysaccharide in the lower phase at equilibrium; V is the volume of the lower phase at equilibrium; and M is the total initial mass of Cistanche deserticola powder.

[0058] 1.3 Single-factor experiment

[0059] 1.3.1 Single-factor test of feed-liquid ratio

[0060] Following the basic operating procedure described above, and fixing other conditions (ammonium sulfate mass fraction 30%wt, tert-butanol volume ratio 1:5 (v / v), extraction temperature 40 ℃, extraction pH=6, extraction time 1 h), the effect of the solid-liquid ratio in the range of 1:10 to 1:30 on the extraction rate of Cistanche deserticola polysaccharides was investigated. The single-factor experimental scheme and results of the solid-liquid ratio are shown in Table 1 below:

[0061] Table 1. Single-factor experimental scheme and results for the feed-to-liquid ratio.

[0062]

[0063] The results show that the extraction rates at different material-to-liquid ratios ranged from 8.46% to 8.65%, with very small fluctuations. They did not show a significant upward or downward trend with increasing material-to-liquid ratio, indicating that the material-to-liquid ratio was not a key factor affecting the polysaccharide extraction rate under these experimental conditions.

[0064] 1.3.2 Single-factor experiment on extraction time

[0065] Following the basic operating procedure described above, and fixing other conditions (solid-to-liquid ratio 1:20, ammonium sulfate mass fraction 30%wt, tert-butanol volume ratio 1:1.5, extraction temperature 40 ℃, extraction pH=6), the effect of extraction time within the range of 0.5-1.5 h on the extraction rate of Cistanche deserticola polysaccharides was investigated. The single-factor experimental scheme and results of extraction time are shown in Table 2 below:

[0066] Table 2. Single-factor experimental design and results of extraction time.

[0067]

[0068] The results above indicate that as the extraction time increased from 0.5 h to 1.0 h, the polysaccharide extraction rate gradually increased from 8.07% to 8.53%. When the extraction time was further increased to 1.5 h, the extraction rate remained at around 8.54%, indicating that 1.0 h was close to the extraction equilibrium and further extending the time did not significantly improve the extraction rate.

[0069] 1.3.3 Single-factor experiment on the mass fraction of (NH4)2SO4

[0070] Following the basic operating procedure described above, and fixing other conditions (solid-to-liquid ratio 1:20, extraction time 1 h, tert-butanol volume ratio 1:1.5, extraction temperature 40 ℃, extraction pH=6), the effect of ammonium sulfate mass fraction in the range of 10% to 50% on the extraction rate of Cistanche deserticola polysaccharides was investigated. The single-factor experimental scheme and results of (NH4)2SO4 mass fraction are shown in Table 3 below:

[0071] Table 3. Single-factor experimental scheme and results for (NH4)2SO4 mass fraction

[0072]

[0073] As shown in Table 3 above, the polysaccharide extraction rate first increases and then decreases with the increase of ammonium sulfate mass fraction, reaching its highest level of 6.5% when the ammonium sulfate mass fraction is 30%. This is because as the ammonium sulfate mass fraction increases, the phase separation ability of the aqueous two-phase system is enhanced, making it easier for polysaccharides to accumulate in a certain phase, thereby increasing the polysaccharide extraction rate. However, when the ammonium sulfate mass fraction is too high, the excessive salting-out effect will damage the stability of the system and even cause co-precipitation, leading to a decrease in the polysaccharide extraction rate.

[0074] 1.3.4 Single-factor experiment on the volume ratio of tert-butanol

[0075] Following the basic operating procedure described above, and fixing other conditions (solid-to-liquid ratio 1:20, extraction time 1 h, ammonium sulfate mass fraction 30%wt, extraction temperature 40 ℃, extraction pH=6), the effect of the volume ratio of tert-butanol to Cistanche deserticola slurry within the range of 1:0.5 to 1:2.5 on the extraction rate of Cistanche deserticola polysaccharides was investigated. The single-factor experimental scheme and results of the tert-butanol volume ratio are shown in Table 4 below:

[0076] Table 4. Single-factor experimental scheme and results for tert-butanol volume ratio

[0077]

[0078] As shown in Table 4 above, the polysaccharide extraction rate first increased and then decreased with the increase of the tert-butanol volume ratio, reaching a maximum of 7.47% when the tert-butanol volume ratio was 1:1.5. This may be because when the amount of tert-butanol added is small, its interaction with ammonium sulfate is insufficient, failing to achieve an effective synergistic effect; while excessive tert-butanol addition will cause tert-butanol and sulfate to compete for water molecules, which is detrimental to the stability of the three-phase system, leading to a decrease in the extraction rate.

[0079] 1.3.5 Single-factor experiment on extraction temperature

[0080] Following the basic operating procedure described above, and fixing other conditions (solid-to-liquid ratio 1:20, extraction time 1 h, ammonium sulfate mass fraction 30% wt, tert-butanol volume ratio 1:1.5, extraction pH=6), the effect of extraction temperature in the range of 20 ℃ to 60 ℃ on the extraction rate of Cistanche deserticola polysaccharides was investigated. The single-factor experimental scheme and results of extraction temperature are shown in Table 5 below:

[0081] Table 5. Single-factor experimental scheme and results of extraction temperature

[0082]

[0083] As shown in Table 5 above, the polysaccharide extraction rate first increased and then decreased with increasing temperature, reaching its highest level of 8.93% at 40℃. This may be because higher temperatures intensify the thermal motion of molecules in the system, promoting polysaccharide dissolution. Simultaneously, the polysaccharides gradually expose a large number of hydroxyl groups, which facilitates the formation of more hydrogen bonds with water molecules, enhancing the extraction effect of the three-phase system. However, excessively high temperatures cause tert-butanol to volatilize, affecting its interaction with ammonium sulfate and thus decreasing the polysaccharide extraction rate.

[0084] 1.3.6 Extraction pH Single Factor Experiment

[0085] Following the basic operating procedure described above, and fixing other conditions (solid-to-liquid ratio 1:20, extraction time 1 h, ammonium sulfate mass fraction 30% wt, tert-butanol volume ratio 1:1.5, extraction temperature 40 ℃), the effect of extraction pH in the range of 4 to 8 on the extraction rate of Cistanche deserticola polysaccharides was investigated. The single-factor experimental scheme and results of extraction pH are shown in Table 6 below:

[0086] Table 6. Single-factor experimental scheme and results of extraction pH.

[0087]

[0088] As shown in Table 6 above, the polysaccharide extraction rate first increased and then decreased with increasing pH, reaching its highest level of 6.95% at pH 6. This may be because, under weakly acidic conditions, the synergistic effect of hydrogen bonding and ionic strength optimized the polysaccharide-solvent interaction, thus improving the extraction rate. However, as the pH of the system further increased, alkaline conditions led to decreased conformational stability of the polysaccharides or breakage of chemical bonds. Simultaneously, the presence of tert-butanol resulted in a dual effect, reducing the electrostatic interaction between salt ions and proteins, thus affecting the separation efficiency.

[0089] 1.4 Response Surface Design

[0090] Based on the results of the single-factor experiments, and in accordance with Table 7 below, the mass fraction of ammonium sulfate (A), the volume ratio of tert-butanol to Cistanche deserticola slurry (B), the extraction temperature (C) and the extraction pH (D) were used as the factors to be investigated, and the extraction rate of Cistanche deserticola polysaccharide (y) was used as the response value. A four-factor, three-level Box-Behnken design (BBD) was used to conduct response surface experiments, and a total of 27 experiments were conducted.

[0091] Table 7 Response Surface Design

[0092]

[0093] The response surface methodology and results of the 27 experiments are shown in Table 8.

[0094] Table 8 Response Surface Experiment - Extraction Rate of Cistanche deserticola Polysaccharides

[0095]

[0096] 1.5 Establishment of the Regression Model and Analysis of Variance

[0097] The experimental data in Table 8 were fitted using Design-Expert software to obtain the quadratic multiple regression equations for the extraction rate (y) of Cistanche deserticola polysaccharides on the mass fraction of ammonium sulfate (A), the volume ratio of tert-butanol to Cistanche deserticola slurry (B), the extraction temperature (C), and the extraction pH (D):

[0098] y=-51.9873+0.2208A+12.7177B+0.8631C+12.0400D+0.0641AB+0.0046AC+0.0222AD-0.0586BC-0.3882BD-0.0479CD-0.0093A 2 -3.4227B 2 -0.0086C 2 -0.9025D 2 .

[0099] Analysis of variance was performed on the quadratic regression model, and the results are shown in Table 9.

[0100] Table 9. Analysis of Variance Table

[0101]

[0102] As shown in the table above, the F-value of the model is 10.43, P < 0.0001, indicating that the model is highly significant; the F-value of the lack-of-fit term is 4.93, P = 0.1802 > 0.05, indicating that the lack-of-fit term is not significant. The model has a good fit and small experimental error, and can be used for the optimization and prediction of the three-phase extraction process of Cistanche deserticola polysaccharides. The coefficient of determination R of the model is... 2 =0.9241, indicating that the model can explain 92.41% of the response value variation; the corrected coefficient of determination R0 2 _Adj=0.8355; coefficient of variation CV%=4.14%<10%, indicating good experimental repeatability; precision=11.1902>4, indicating high prediction accuracy of the model.

[0103] The influence of each factor on the polysaccharide extraction rate was as follows: ammonium sulfate mass fraction (A) > extraction temperature (C) = extraction pH (D) > tert-butanol to Cistanche deserticola slurry volume ratio (B). Among them, A (P<0.0001), C (P=0.0003), and D (P=0.0003) had extremely significant effects on the polysaccharide extraction rate, while B (P=0.2465) had no significant effect; the quadratic terms of each factor were all extremely significant; the interaction between extraction temperature and extraction pH was significant (CD, P=0.0456), while the other interactions were not significant.

[0104] 1.6 Response Surface Analysis of Interactions Among Factors

[0105] Response surface 3D plots and contour plots can intuitively reflect the influence of the interaction between various factors on the response value. The closer the shape of the contour lines is to an ellipse, the more significant the interaction between the factors is, and vice versa. Figure 2 The response surface 3D plot and contour plot show that:

[0106] The interaction between extraction temperature (C) and extraction pH (D): The contour lines of both show a distinct elliptical shape, consistent with the analysis of variance results, indicating a significant interaction. Within a certain range, the polysaccharide extraction rate increases with increasing extraction temperature and pH approaching 6. However, when the temperature is too high or the pH deviates from 6, the extraction rate decreases significantly. This is because the synergistic effect of temperature and pH affects the solubility, hydrogen bonding ability, and stability of the three-phase system of polysaccharides.

[0107] The interactions among other factors—AB (ammonium sulfate mass fraction to tert-butanol volume ratio), AC (ammonium sulfate mass fraction to extraction temperature), AD (ammonium sulfate mass fraction to extraction pH), BC (tert-butanol volume ratio to extraction temperature), and BD (tert-butanol volume ratio to extraction pH)—are all nearly circular, indicating that the interactions are not significant, consistent with the results of the analysis of variance. This suggests that these factors mainly affect the polysaccharide extraction rate through single and secondary effects.

[0108] 1.7 Prediction and Verification of Optimal Process Parameters

[0109] The optimal process parameters for the three-phase extraction of Cistanche deserticola polysaccharides were obtained by solving the quadratic regression equation using Design-Expert software: ammonium sulfate mass fraction 32.491%, tert-butanol to Cistanche deserticola slurry volume ratio 1:1.443 (v / v), extraction temperature 39.885 ℃, and extraction pH 5.576. Under these conditions, the model predicted an extraction rate of 12.162% for Cistanche deserticola polysaccharides.

[0110] To verify the reliability of the model, the process parameters were modified based on actual experimental operations: ammonium sulfate mass fraction 30%, tert-butanol to Cistanche deserticola slurry volume ratio 1:1.5, extraction temperature 40 ℃, and extraction pH 6. Three parallel verification experiments were conducted, and the actual extraction rate of Cistanche deserticola polysaccharides was measured to be 12.162%, with a relative deviation of <5% from the predicted value. This indicates that the established quadratic regression model has good predictive ability, and the optimized process parameters are reliable and feasible. The protein content of the Cistanche deserticola polysaccharides obtained under the optimal process was 4.58%, indicating that the three-phase extraction technology achieves efficient polysaccharide extraction while also having a good deproteinization effect, effectively improving the purity of the polysaccharides.

[0111] To further validate the optimal process conditions obtained from response surface methodology (ammonium sulfate mass fraction 30.25%, tert-butanol volume ratio 1:1.52, extraction temperature 40.18 ℃, pH 6.05), 12 additional validation experiments were conducted to investigate the effect of each single factor fluctuating near the optimal conditions on the polysaccharide extraction rate, as shown in Table 10.

[0112] Table 10 Results of 12 Groups of Verification Experiments

[0113]

[0114] When the process parameters are closest to the optimal conditions (i.e., 30% ammonium sulfate, 1:1.5 tert-butanol, 40 °C, and pH 6), the polysaccharide extraction rate reaches its highest level with good repeatability. For example, groups 5, 8, and 11 were all tested under optimal conditions, with extraction rates of 12.17%, 11.97%, and 12.02%, respectively. The average of these three groups was 12.05%, significantly higher than any other group. In contrast, when the mass fraction of ammonium sulfate deviated from the optimal value (e.g., from 30% to 20% or from 40%), the extraction rates decreased to 10.46% and 11.62%, respectively; when the volume ratio of tert-butanol changed to 1:1 or 1:2, the extraction rates were 11.08% and 11.35%; when the temperature increased to 50 °C or decreased to 30 °C, the extraction rates were 11.30% and 11.22%; and when the pH was adjusted to 5 or 7, the extraction rates were only 10.94% and 10.81%. The above comparison demonstrates that the optimal conditions predicted by the response surface methodology can indeed maximize the extraction rate of Cistanche deserticola polysaccharides, and these conditions exhibit good repeatability and stability.

[0115] Example 2: Preparation of Cistanche deserticola polysaccharides using different extraction processes

[0116] 2.1 Raw material pretreatment

[0117] Dried desert Cistanche slices were baked at 50 ℃ for 12 h, crushed, and passed through a 100-mesh sieve to obtain Cistanche powder. The Cistanche powder was mixed with distilled water at a material-to-liquid ratio of 1:20 (w / v) to prepare Cistanche slurry.

[0118] 2.2 Four Extraction Processes

[0119] The above-mentioned Cistanche deserticola slurry was subjected to the following four pretreatments:

[0120] (1) No pretreatment is required (for TPP method): Cistanche slurry is directly subjected to subsequent stirring extraction.

[0121] (2) Pulsed ultrasonic pretreatment (for U-TPP method): Cistanche slurry was subjected to pulsed ultrasonic pretreatment. The ultrasonic parameters were: frequency 20 kHz, power 400 W, temperature 25°C. A 2-second on / 2-second off cycle mode was adopted, and the total treatment time was 30 min.

[0122] (3) Ultra-high pressure pretreatment (for H-TPP method): Place the Cistanche deserticola slurry in an ultra-high pressure treatment device and treat it for 10 min at 300 MPa and 25 ℃.

[0123] (4) Ultrasonic-ultra-high pressure combined pretreatment (for UH-TPP method): First, the Cistanche slurry is subjected to pulse ultrasonic pretreatment (parameters as above), and then immediately subjected to ultra-high pressure treatment (parameters as above).

[0124] The Cistanche slurry after the above four pretreatments was magnetically stirred for 12 h at room temperature and in the dark, centrifuged at 6000 rpm for 10 min, and the supernatant was collected and concentrated to 2 / 3 of the original volume.

[0125] Ammonium sulfate was added to each of the four concentrated solutions to a mass fraction of 30%. After it was fully dissolved, the pH was adjusted to 6 with 1M HCl / NaOH. Then, tert-butanol was slowly added to the mixture to make the volume ratio of Cistanche deserticola slurry to tert-butanol 1:1.5 (v / v), and the mixture was allowed to stand at 40 °C for 1 h.

[0126] The system was then centrifuged at 6000 rpm for 10 min, and the system was divided into three phases: the upper phase was tert-butanol, which could be recovered by vacuum evaporation; the middle phase was protein precipitation; and the lower phase was a polysaccharide solution containing ammonium sulfate.

[0127] The lower phase polysaccharide solution was collected and dialyzed through a dialysis bag with a molecular weight cutoff of 3500 Da for 48 h (with deionized water replaced multiple times during the process). After concentration, it was freeze-dried to obtain purified Cistanche deserticola polysaccharides. The polysaccharides obtained by the above four processes were named as follows: CDPs-T (untreated), CDPs-U (ultrasound-assisted), CDPs-H (ultra-high pressure assisted), and CDPs-UH (ultrasound-ultra-high pressure combined assisted).

[0128] 2.3 Polysaccharide extraction rate and determination of basic chemical components

[0129] 2.3.1 Calculation of polysaccharide extraction rate

[0130] The polysaccharide extraction rate is calculated using the following formula:

[0131] ;

[0132] Where C is the concentration of Cistanche deserticola polysaccharide in the lower phase at equilibrium (mg / mL), V is the volume of the lower phase at equilibrium (mL), and M is the total initial mass of Cistanche deserticola powder (mg).

[0133] 2.3.2 Determination of total sugar content

[0134] Total neutral sugar content was determined using the phenol-sulfuric acid method, with D-glucose as the standard. Protein content was determined using the Coomassie brilliant blue method, with bovine serum albumin (BSA) as the standard.

[0135] 2.3.3 Determination of uronic acid content

[0136] The content of uronic acid was determined by the carbazole-sulfuric acid method, with D-galacturonic acid as the standard.

[0137] 2.3.4 Protein content determination

[0138] The protein content was determined by the Coomassie Brilliant Blue method, with bovine serum albumin (BSA) as the standard.

[0139] 2.3.5 Determination results

[0140] The extraction rates and main chemical components of the four CDPs are shown in Table 11.

[0141] Table 11 Physicochemical properties of CDPs-T, CDPs-U, CDPs-H and CDPs-UH

[0142]

[0143] Data are presented as the mean ± standard error (SD) of three replicates. Different lowercase superscript letters in the same row indicate significant differences (P < 0.05).

[0144] As can be seen from Table 11 above, the highest extraction rate of CDPs-UH is 14.11%, followed by CDPs-U and CDPs-H, while the lowest yield of CDPs-T is 10.03%. This may be because the cavitation effect of ultrasonic waves and the mechanical action of ultra-high pressure treatment can both effectively destroy the cell wall structure, promote the dissolution and dispersion of cistanche polysaccharides, and thus effectively improve the extraction rate of polysaccharides. The total sugar content is arranged in the order: CDPs-T (49.88%) < CDPs-H (52.83%) < CDPs-U (55.66%) < CDPs-UH (61.16%). This also shows that carbohydrates are the main components of CDPs. Glucuronic acid is a derivative formed by the oxidation of the hydroxyl group of monosaccharides into carboxyl groups. The order of the contents of the four glucuronic acids is: CDPs-UH (24.97%) < CDPs-U (26.3%) < CDPs-T (27.07%) < CDPs-H (31.11%). In addition, only a small amount of protein was detected in the four cistanche polysaccharides, ranging from 1.38 - 2.24% (Table 11). The results show that almost all free proteins have been removed from CDPs during the TPP separation process.

[0145] 2.4 Comparative experiment with the traditional water extraction and alcohol precipitation method

[0146] 2.4.1 Operating procedure of the traditional water extraction and alcohol precipitation method

[0147] (1) Traditional method without pretreatment: Dried Cistanche deserticola slices were baked at 50 °C for 12 h, pulverized, and passed through a 100-mesh sieve to obtain Cistanche deserticola powder. The powder was mixed with deionized water at a material-to-liquid ratio of 1:20 (w / v) to obtain a slurry. Without ultrasonic or high-pressure treatment, the slurry was directly stirred magnetically at room temperature in the dark for 12 h. The stirred slurry was centrifuged at 6000 rpm for 10 min, the supernatant was collected, and concentrated to two-thirds of the original volume. Anhydrous ethanol was slowly added to the concentrate to a final concentration of 80% (v / v), and the mixture was allowed to stand overnight at 4 °C to precipitate polysaccharides. The precipitate was then collected by centrifugation at 6000 rpm for 10 min, reconstituted with an appropriate amount of deionized water, and transferred to a dialysis bag with a molecular weight cutoff of 3500 Da for dialysis for 48 h, during which the deionized water was changed several times. The dialysis solution was concentrated and freeze-dried to obtain Cistanche deserticola polysaccharides extracted by the traditional method.

[0148] (2) Traditional method combined with ultrasound assistance: Cistanche deserticola powder and deionized water were mixed at a material-to-liquid ratio of 1:20, and then subjected to pulse ultrasound pretreatment. The ultrasound parameters were 20 kHz, 400 W, and 25 ℃, using a 2-second on / 2-second off cycle mode, with a total treatment time of 30 min. The slurry after ultrasound treatment was then magnetically stirred at room temperature in the dark for 12 h, followed by centrifugation (6000 rpm, 10 min), collection of supernatant, concentration to two-thirds of the original volume, addition of anhydrous ethanol to a final concentration of 80%, and standing overnight at 4 ℃ for alcohol precipitation. The precipitate was collected by centrifugation, redissolved, dialyzed (3500 Da, 48 h), concentrated, and freeze-dried to obtain the sample.

[0149] (3) Traditional method combined with ultra-high pressure: Cistanche deserticola powder and deionized water were mixed at a material-to-liquid ratio of 1:20. The slurry was placed in an ultra-high pressure treatment device and treated at 300 MPa and 25℃ for 10 min. The slurry after ultra-high pressure treatment was then magnetically stirred at room temperature in the dark for 12 h. Subsequently, it was centrifuged (6000 rpm, 10 min) to obtain the supernatant, concentrated to two-thirds of the original volume, and anhydrous ethanol was added to a final concentration of 80%. The mixture was allowed to stand overnight at 4℃ for alcohol precipitation, centrifuged to collect the precipitate, redissolved, dialyzed (3500 Da, 48 h), concentrated, and freeze-dried to obtain the sample.

[0150] (4) Traditional method combined with ultrasonic and ultra-high pressure assisted treatment: Cistanche deserticola powder and deionized water were mixed at a material-liquid ratio of 1:20. First, pulse ultrasonic pretreatment was performed (20 kHz, 400 W, 25℃, 2 seconds on / 2 seconds off, 30 min in total), followed immediately by ultra-high pressure treatment (300 MPa, 25℃, 10 min). The slurry after combined pretreatment was then magnetically stirred at room temperature in the dark for 12 h. After that, it was centrifuged (6000 rpm, 10 min) to collect the supernatant, concentrated to two-thirds of the original volume, and anhydrous ethanol was added to a final concentration of 80%. The mixture was then allowed to stand overnight at 4℃ for alcohol precipitation, centrifuged to collect the precipitate, redissolved, dialyzed (3500 Da, 48 h), concentrated, and freeze-dried to obtain the sample.

[0151] 2.4.2 Comparison of experimental results

[0152] The extraction rates of the four samples obtained by the traditional water extraction and alcohol precipitation method were compared with those of the four samples (CDPs-T, CDPs-U, CDPs-H, and CDPs-UH) obtained by the three-phase extraction method. The results are shown in Table 12.

[0153] Table 12 Comparison of extraction rates between traditional water extraction and alcohol precipitation method and three-phase extraction method

[0154]

[0155] Based on the comparison of the above experimental data, the polysaccharide extraction rate of the traditional water extraction and alcohol precipitation method was significantly lower than that of the three-phase extraction method under the same pretreatment conditions. Specifically, without pretreatment, the extraction rate of the traditional method was only 7.87%, while that of the three-phase extraction method reached 10.03%; under ultrasound-assisted conditions, the traditional method was 10.14%, while that of the three-phase extraction method was 11.84%; under ultra-high pressure assisted conditions, the traditional method was 10.87%, while that of the three-phase extraction method was 12.16%; under combined ultrasound and ultra-high pressure assisted conditions, the traditional method was 11.56%, while that of the three-phase extraction method reached as high as 14.11%. It can be seen that, regardless of whether physical assistance methods are used, the extraction rate of the three-phase extraction method is 15%-30% higher than that of the traditional method. In addition, the protein content of the three-phase extraction method under the optimal process was only 4.58%, indicating that it can effectively remove protein while efficiently extracting polysaccharides, while the traditional water extraction and alcohol precipitation method lacks the ability to simultaneously remove protein, resulting in lower purity polysaccharides and requiring an additional protein removal step. Therefore, the three-phase extraction method not only has a higher extraction rate but also better process integration, enabling the extraction and preliminary purification of polysaccharides in one step. Its overall performance is significantly better than that of the traditional water extraction and alcohol precipitation method.

[0156] Example 3: Structural characterization of Cistanche deserticola polysaccharides

[0157] 3.1 Molecular weight determination

[0158] 3.1.1 Measurement Method

[0159] The molecular weight of CDPs was determined by high-performance liquid chromatography (HPLC). 50 mg of sample was dissolved in 10 mL of mobile phase (0.1 N NaNO3) and filtered through a 0.22 μm filter. Chromatographic analysis was performed using a Waters 2695 HPLC system equipped with a Waters 2410 differential refractive index detector and an Empower workstation. Chromatographic separation was performed on an Ultrahydrogel™ Linear column (300 mm × 7.8 mm inner diameter) at 40 °C, using 0.1 N NaNO3 solution as the mobile phase at a flow rate of 0.5 mL / min. Molecular weight calibration curves were established using a series of dextran standards T-2000 (Mw=2,000,000 Da), T-300 (Mw=300,600 Da), T-150 (Mw=135,030 Da), T-10 (Mw=9,750 Da), T-5 (Mw=2,700 Da) and glucose monomer (Mw=180 Da).

[0160] 3.1.2 Measurement Results

[0161] The molecular weight (Mw) determination results for the four CDPs are as follows:

[0162] The molecular weights of CDPs-T were 130.22 kDa, CDPs-U were 127.83 kDa, CDPs-H were 112.13 kDa, and CDPs-UH were 75.25 kDa. The molecular weights decreased after ultrasonic or ultra-high pressure treatment, possibly because these treatments may have caused varying degrees of damage to the molecular structure of the polysaccharides.

[0163] 3.2 Monosaccharide Composition Analysis

[0164] 3.2.1 Determination Method

[0165] The monosaccharide composition of CDPs was determined by pre-column derivatization of 1-phenyl-3-methyl-5-pyrazolone (PMP) combined with reversed-phase high-performance liquid chromatography (RP-HPLC). The specific steps were as follows: The polysaccharide sample was hydrolyzed with 4 mol / L trifluoroacetic acid at 110 °C for 2 h. After drying, the hydrolysate was derivatized with 0.5 mol / L PMP methanol solution and 0.3 mol / L NaOH at 70 °C for 100 min. After the reaction, the solution was neutralized with an equal volume of 0.3 mol / L HCl. Excess PMP was removed by chloroform extraction (3 times). The aqueous phase was filtered through a 0.45 μm filter before injection. Chromatographic analysis was performed on a ZORBAX Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm) at 30 °C and a detection wavelength of 250 nm. The mobile phase was 0.1 mol / L phosphate buffer (pH 6.7)-acetonitrile (83:17, v / v), with isocratic elution at a flow rate of 1.0 mL / min and an injection volume of 10 μL. PMP derivatization was performed using monosaccharide standards including mannose, ribose, rhamnose, glucose, galactose, xylose, arabinose, fucose, glucuronic acid, and galacturonic acid, and analysis was performed under the chromatographic conditions described above.

[0166] 3.2.2 Measurement Results

[0167] The monosaccharide composition and molar ratio of the four CDPs are shown in Table 13.

[0168] Table 13 Monosaccharide composition of Cistanche deserticola under different extraction techniques

[0169]

[0170] The results in the table above show that the four types of *Cistanche deserticola* polysaccharides are all composed of glucose, arabinose, galactose, rhamnose, galacturonic acid, glucuronic acid, mannose, xylose, glucosamine, fucose, and ribose, but their molar ratios differ, which is consistent with the NMR results. The results indicate that different extraction processes do not affect the types of monosaccharides in the polysaccharides, but they do alter their content, which is consistent with previous reports. The four types of *Cistanche deserticola* polysaccharides are mainly composed of glucose, arabinose, and galactose, with CDPs-T of 49.85%, 26.32%, and 12.37%, respectively. However, the proportions of glucose in CDPs-U, CDPs-H, and CDPs-UH all increased, while the proportions of galactose and arabinose decreased. These differences are likely closely related to the applied physical processes. These treatments may lead to different release effects of different monosaccharides during cell wall disruption, ultimately resulting in different proportions of them in the final extract.

[0171] 3.3 Spectroscopic and Crystal Structure Analysis

[0172] 3.3.1 Ultraviolet Spectroscopy Analysis

[0173] Ultraviolet (UV) scanning can identify homogeneous components of nucleic acids and proteins in polysaccharides. CDPs samples were dissolved in deionized water at a concentration of 1 mg / mL and then scanned in the 200–400 nm wavelength range at 25°C using a UV-2450 Shimadzu UV spectrophotometer.

[0174] 3.3.2 Infrared Spectroscopic Analysis

[0175] The lyophilized CDPs sample was mixed with KBr at a mass ratio of 1:100 (w / w) and pressed into a transparent slide. FT-IR spectrophotometry was performed on an FT-IR spectrophotometer (IR spirit-T type, Shimadzu Corporation, Japan), with a wavenumber range of 500–4000 cm⁻¹. -1 The resolution is 4.0 cm. -1 .

[0176] 3.3.3 Congo Red Test

[0177] The concentration of NaOH affects the maximum absorption wavelength of the complex formed by Congo red and a polysaccharide with a triple helix structure. A polysaccharide solution (3 mg / mL) and a Congo red solution (80 μmol / L) were mixed in a 1:1 ratio. Then, 1 mol / L NaOH solution was gradually added to achieve final NaOH concentrations of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L. These mixtures were incubated at room temperature for 5 min, and the maximum absorption wavelength (λmax) was scanned in the range of 200 to 800 nm using a UV-Vis spectrophotometer (UV-2450, Shimadzu Corporation, Japan). A Congo red solution without added polysaccharide served as a control.

[0178] 3.3.4 X-ray diffraction (XRD) analysis

[0179] The X-ray diffraction (XRD) patterns of the polysaccharides were analyzed using a benchtop X-ray diffractometer (Panaco Xpert powder model, Netherlands). Measurements were performed at room temperature, with an angle range of 2θ = 10° to 80° and a scan rate of 5° / min.

[0180] 3.3.5 Measurement Results

[0181] Figure 3 The results are spectral and crystal structure analysis results; among which Figure 3 a represents the results of ultraviolet-visible spectroscopy analysis; 3b represents the results of Fourier transform infrared spectroscopy analysis; 3c represents the results of triple helix structure analysis; and 3d represents the X-ray diffraction curve.

[0182] like Figure 3As shown in a, the four CDPs did not have obvious absorption peaks in the 260-280 nm range, indicating that the samples contained virtually no protein or nucleic acid, which is consistent with the protein results in Table 11.

[0183] like Figure 3 As shown in b, the FT-IR spectra of the four CDPs all exhibit typical polysaccharide characteristic absorption peaks: 3600-3200 cm⁻¹. -1 The strong broadband region is attributed to OH stretching vibration; 2900-2800 cm -1 The weak band in the region is attributed to CH stretching vibration; 1618 cm -1 and 1420 cm -1 The absorption bands are attributed to the asymmetric and symmetric stretching vibrations of the carboxylate groups, respectively, indicating that all four polysaccharides contain uronic acid, which is consistent with the results in Table 11; 1155 cm -1 1107 cm -1 and 762 cm -1 Coc stretching vibration; 1074 cm -1 and 1028 cm -1 The positions at 918 cm⁻¹ represent the reducing C1-H group vibration and the CO stretching vibration, respectively, indicating that all four polysaccharides contain pyranoside bonds; -1 and 814 cm -1 The absorption peaks at these locations indicate the presence of α-glycosidic and β-glycosidic bonds, respectively. These data suggest that the extraction method has little effect on the chemical functional groups, and that CDPs contain all typical polysaccharide characteristic peaks.

[0184] Congo red can form complexes with polysaccharides having a triple helix conformation, causing a red shift in the maximum absorption wavelength (λmax) compared to Congo red in aqueous solution. This provides a convenient method for characterizing polysaccharide conformations. Figure 3 As shown in Figure c, the λmax of the four CDPs and Congo red decreased with increasing NaOH concentration, similar to the blank control, which is inconsistent with the triple-helix polysaccharide characteristics reported in the literature. Therefore, CDPs-T, CDPs-U, CDPs-H, and CDPs-UH do not possess a triple-helix structure. Furthermore, some studies have shown that monosaccharide composition may inhibit the formation of a triple-helix conformation in polysaccharides.

[0185] X-ray diffraction (XRD) is a technique for analyzing crystalline and amorphous materials of polysaccharides. Generally, crystalline materials exhibit sharp and narrow diffraction peaks, while amorphous components show broad peaks. For example... Figure 3As shown in Figure d, all four CDPs exhibit broad diffraction peaks in the 10–30° range, indicating that all samples possess an amorphous or semi-crystalline structure. Furthermore, the XRD spectra of the four CDPs show sharp and narrow peaks near 22.48°, 23.6°, 25.5°, 31.78°, and 34°, with highly similar diffraction curves, suggesting that the four polysaccharides share similar crystal structure characteristics. Compared to CDPs-T, the diffraction peaks of the other three groups of polysaccharides are broader, indicating that ultra-high pressure and / or ultrasonic pretreatment affects the crystal structure of the polysaccharides, and the crystal structure is closely related to physicochemical properties.

[0186] 3.4 Particle size, zeta potential, thermal stability and surface morphology analysis

[0187] 3.4.1 Particle size and zeta potential determination

[0188] CDPs samples were prepared into polysaccharide solutions of 1 mg / mL, and the zeta potential and particle size were measured at 25 °C using a potentiometer / particle size analyzer (Nano Brook 90Plus PALS, Brookhaven Corporation, Hartsville, NY, USA).

[0189] 3.4.2 Thermal stability determination

[0190] The thermal properties of four Cistanche deserticola polysaccharides were evaluated using thermogravimetric / differential thermogravimetric (TG / DTG) curves. Thermogravimetric analysis was performed using a Mettler-Toledo TGA / DSC 1 instrument. The thermal stability of the samples in the range of 50–500 °C was determined under argon purging at a heating rate of 10 °C / min and a gas flow rate of 50 mL / min.

[0191] 3.4.3 Surface morphology analysis

[0192] The surface microstructure of Cistanche deserticola polysaccharides was determined using a scanning electron microscope (SU8220, HITACHI, Japan). All samples were coated with a gold layer, and the accelerating voltage during the operation was 5 kV.

[0193] 3.4.4 Measurement Results

[0194] The stability of polysaccharide solutions or colloids is influenced by particle size and zeta potential, which greatly determines their potential applications. Generally, smaller particle size and higher absolute zeta potential enhance the dispersion and dissolution of polysaccharides, while larger particle size and lower zeta potential promote aggregation. This example evaluates the stability of CDPs by measuring particle size and potential.

[0195] Thermal stability is an important physicochemical property of polysaccharides in the food industry. Thermogravimetric analysis (TG) and differential thermogravimetric analysis (DTG) are commonly used to evaluate the thermal properties of polysaccharides.

[0196] Figure 4 The results include particle size, zeta potential, thermal stability measurements, and surface morphology analysis. Figure 4 a-4e represent particle size, Zeta potential, thermodynamic analysis of the four CDPs, TG curve, DTG curve, and scanning electron microscope images of the four CDPs, respectively. Figure 4 In e, AD represents the CDPs-T, CDPs-U, CDPs-H, and CDPs-UH groups, respectively. Results are expressed as mean ± SD; different letters indicate significant differences (P < 0.05). Figure 4 As shown in Figure a, the average particle size order of the four polysaccharides is: CDPs-T (933.05 nm) > CDPs-U (766.03 nm) > CDPs-H (747.67 nm) > CDPs-UH (707.51 nm). The results indicate that ultrasonic and / or ultra-high pressure treatment can effectively reduce the particle size of CDPs, making the particles more uniform and improving the stability of CDPs in aqueous solution. The polydispersity index (PDI) is an important indicator describing particle size distribution, ranging from 0 to 1. The smaller the PDI, the better the particle uniformity of the solution. Similarly, CDPs-UH has the lowest PDI, consistent with the average particle size distribution results.

[0197] like Figure 4 As shown in b, all four CDP polysaccharides carry a negative charge, indicating the presence of anionic groups in the polysaccharide chains. This is consistent with the conclusion in Table 11 that CDPs are acidic polysaccharides. Furthermore, particles with an absolute zeta potential below 30 mV exhibit insufficient repulsive force to maintain stable dispersion, indicating a general tendency for aggregation among the tested samples. In this study, the order of absolute zeta potential values ​​was: CDPs-UH > CDPs-H > CDPs-U > CDPs-T. While the observed aggregation trend may pose challenges in some applications, this moderate aggregation characteristic may actually contribute to enhancing the texture and mouthfeel of other foods (such as semi-solid foods like yogurt).

[0198] Thermal stability results are as follows Figure 4As shown in c and 4d, the TG curves exhibit relatively similar shapes, primarily showing two stages of mass loss: 50–200 °C (stage 1) and 200–500 °C (stage 2). In the TG curves of these CDPs, a slow mass loss occurs with increasing temperature, attributed to the evaporation of free and bound water within the CDPs. The mass loss rates for CDPs-T, CDPs-U, CDPs-H, and CDPs-UH are 10.08%, 7.35%, 8.23%, and 7.68%, respectively, with CDPs-H exhibiting the largest mass loss. The wide temperature range of CDPs-U in this stage indicates its strong water-binding capacity. The second mass loss is attributed to the thermal degradation of the polysaccharide chains, decarboxylation of acid groups, and the breakdown of the C-C bonds connecting the pyran rings. Furthermore, enhanced carbonization was observed in CDPs-T and CDPs-H, with coke yields of 37.06%, 31.10%, 37.30%, and 29.79% at 500 °C, respectively. This can be attributed to the retention of high molecular weight domains and more extensive intermolecular crosslinking, which promote coke formation through cyclization and condensation reactions during thermal decomposition.

[0199] The DTG curve shows the mass loss rate of the polysaccharide. For example... Figure 4 As shown in Figure d, the temperatures corresponding to the maximum mass loss rates of CDPs-T, CDPs-U, CDPs-H, and CDPs-UH occurred at 260.17 ℃, 297.67 ℃, 261.67 ℃, and 299.67 ℃, respectively. Differences in the monosaccharide composition, chemical composition, and structure of these polysaccharides may lead to some differences in their thermal stability and degradation characteristics. Although some studies suggest a positive correlation between uronic acid content and molecular weight and thermal stability, our data do not fully support these views, indicating that the factors affecting thermal stability may be more complex.

[0200] like Figure 4 As shown in e: the surface structure of CDPs-T is smooth; the surface of CDPs-U exhibits voids induced by ultrasound, becoming rough and loose; obvious irregularities in the layered structure are observed in CDPs-H; the surface of CDPs-UH becomes even more fragmented and rough.

[0201] Example 4: Determination of the antioxidant activity of Cistanche deserticola polysaccharides

[0202] Three different assay methods were used to evaluate the in vitro antioxidant activity of Cistanche deserticola polysaccharides (CDPs). CDP samples were dissolved in distilled water at different concentrations (0.25–1.0 mg / mL) prior to testing. Vitamin C (Vc) was used as a positive antioxidant reference.

[0203] 4.1 Determination of DPPH free radical scavenging ability

[0204] Take 100 μL of CDPs and Vc solutions of different concentrations and add 100 μL of 0.2 mmol / L DPPH anhydrous ethanol solution. Shake well and react in the dark at 37 ℃ for 30 min. After the reaction, take the solution and measure the absorbance of the sample at a wavelength of 517 nm.

[0205] 4.2 ABTS + Free radical scavenging capacity determination

[0206] Mix 7 mmol / L ABTS solution with 2.45 mmol / L potassium persulfate aqueous solution and incubate in the dark at room temperature for 12-16 h, mixing them at a 1:1 (v / v) ratio. Then dilute 20-40 times to stabilize the absorbance at 734 nm within the range of 0.700±0.02, obtaining an ABTS+ solution (7 mM). Take 50 μL of solutions with different concentrations of CDPs and Vc, and mix with 150 μL of ABTS+. + After the solution was shaken vigorously in a test tube, it was placed at 37 °C for 5 min and the absorbance was measured at 734 nm.

[0207] 4.3 Determination of OH radical scavenging ability

[0208] 1.00 mL of polysaccharide solutions and vitamin C of different concentrations were sequentially added to 1.00 mL of 6 mmol / L FeSO4 solution and 1.00 mL of 6 mmol / L salicylic acid-ethanol solution. After thorough mixing, 1.00 mL of 6 mmol / L H2O2 was added. The mixture was placed in a water bath at 37℃ for 60 min, and the absorbance was measured at a wavelength of 510 nm. The scavenging rate was calculated using the following formula:

[0209] Clearance rate (%) = [1 - (A1 - A2) / A0] × 100%;

[0210] Where A0 is the absorbance of the mixture without the sample (replaced with water), A1 is the absorbance of the reaction solution, and A2 is only the absorbance of the sample.

[0211] 4.4 Statistical Analysis

[0212] All experimental results are expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS 26.0 software. The significance level for all tests was set at P < 0.05. Data visualization and graphical representation were performed using Origin 2021.

[0213] 4.5 Measurement Results

[0214] Figure 5 The results are for the determination of antioxidant activity, among which Figure 5 a is ABTS +Free radical scavenging capacity; 5b represents DPPH free radical scavenging capacity, and 5c represents hydroxyl free radical scavenging capacity. Uppercase letters indicate significant differences between treatment groups (P<0.05); lowercase letters indicate significant differences within each treatment group (P<0.05). Figure 5 Analysis:

[0215] DPPH is a stable lipophilic and nitrogen-centered free radical that can be scavenged by proton donation from antioxidants, forming a stable DPPH-H molecule. DPPH free radical scavenging activity is widely used to evaluate the antioxidant capacity of polysaccharides. For example... Figure 5 As shown in Figure a, the four Cistanche deserticola polysaccharides exhibited significant free radical scavenging effects within a concentration range of 0.2–1.0 mg / mL, with activity gradually increasing with increasing concentration. At the same concentration, their scavenging abilities followed the order: CDPs-H > CDPs-T > CDPs-U > CDPs-UH. At a concentration of 1.0 mg / mL, CDPs-H achieved a DPPH scavenging rate of 86.10%, second only to vitamin C at the same concentration. The differences in DPPH free radical scavenging abilities among the different polysaccharides may stem from differences in monosaccharide composition and surface morphology.

[0216] ABTS + The free radical scavenging ability indicates the antioxidant's ability to scavenge oxidative free radicals. Compared with the positive control, all four desert Cistanche polysaccharides showed the ability to scavenge ABTS. + The ability of free radicals, and exhibits a clear dose-response relationship ( Figure 5 b). At a concentration of 1.0 mg / mL, the ABTS of CDPs-H + The strongest free radical scavenging ability was 96.20%, followed by CDPs-T (92.53%), CDPs-U (93.48%), and finally CDPs-UH (92.46%) (P<0.05). This may be because the ultra-high pressure treatment led to partial degradation of the polysaccharides, exposing more active groups and enhancing their antioxidant properties.

[0217] Hydroxyl radicals are highly reactive and are considered harmful oxidants to organisms. Polysaccharides may scavenge hydroxyl radicals by donating electrons or hydrogen. Figure 5 As shown in c, the scavenging capacity of the four CDPs is closely related to the increase of concentration. The hydroxyl radical scavenging capacity of CDPs-H is the highest at 1.0 mg / mL (36.49%), but it is still significantly lower than that of Vc (P<0.05), consistent with the highest content of uronic acid. At the same concentration, the order of scavenging capacity is: CDPs-H > CDPs-T > CDPs-U > CDPs-UH. In summary, CDPs-H has the strongest antioxidant capacity.

[0218] Example 5: In vitro digestion characteristics analysis

[0219] 5.1 α-Amylase digestion tolerance

[0220] The tolerance of CDPs samples to α-amylase was determined using the method reported by Al-Sheraji et al. (2012). α-amylase was dissolved in 20 mM sodium phosphate buffer containing 6.7 mM NaCl, and the pH of the solution was adjusted to 4, 5, 6, 7, and 8 with 5 M NaOH, respectively, to achieve a final enzyme activity of 2 U / mL. 100 mg of the sample was dissolved in buffers at different pH values ​​and incubated at 37°C for 6 h. Samples were taken at 0, 1, 2, 4, and 6 h to determine the reducing sugar and total sugar content, and the degree of hydrolysis was calculated using the aforementioned formula. Inulin (In) was used as a positive control.

[0221] 5.2 Determination of digestive tolerance to artificial gastric juice

[0222] Formula for artificial gastric fluid: Weigh out 8.25 g of Na₂HPO₄·H₂O, 14.35 g of NaH₂PO₄, 8 g of NaCl, 0.2 g of KCl, 0.0755 g of anhydrous CaCl₂, and 0.18 g of MgCl₂·6H₂O, dissolve them, and bring the volume to 1000 mL with distilled water. Adjust the pH of the solution to 1, 2, 3, 4, and 5 with 5 M HCl, respectively. Then, dissolve 100 mg of CDPs and inulin (In, positive control) in buffer solutions of different pH values ​​to a total concentration of 1.0% (w / v), and incubate at 37 °C for 6 h. Samples were taken at 0, 1, 2, 4, and 6 h to determine the reducing sugar and total sugar content of the mixture. The reducing sugar content was determined using the 3,5-dinitrosalicylic acid (DNS) method, and the total sugar content was determined using the phenol-sulfuric acid method. The degree of hydrolysis of the sample was calculated using the following formula:

[0223] Degree of hydrolysis (%) = (Reducing sugar content released / (Total sugar - Initial reducing sugar content)) × 100%;

[0224] The released reducing sugar refers to the difference between the reducing sugar content at a specified time and the initial reducing sugar content.

[0225] 5.3 Measurement Results

[0226] Figure 6 These are the results of in vitro digestion characteristic analysis; among which Figure 6 a-6e are the curves showing the change in the degree of hydrolysis of CDPs-T, CDPs-U, CDPs-H, CDPs-UH and inulin under different pH conditions after treatment with α-amylase over time. Figure 6f-6j represent the hydrolysis degree of CDPs-T, CDPs-U, CDPs-H, CDPs-UH, and inulin in artificial gastric fluid at different pH values ​​over time. Now, combined with... Figure 6 The analysis is as follows:

[0227] Within a pH range of 4-8, the degree of hydrolysis of CDPs with α-amylase was measured over time. Figure 6 (a-6e). According to prebiotic standards, tolerance to α-amylase is a prerequisite for prebiotic candidates so that they can reach the cecum-colon and be utilized by probiotics. The degree of hydrolysis increased significantly with prolonged incubation time, reaching its maximum at 4 h. Further extension of incubation time did not significantly change the degree of hydrolysis. On the other hand, pH significantly affected the degree of hydrolysis of the CDPs samples. The degradation degree of the four CDPs samples increased in the following order of pH: 4 < 5 < 6 < 8 < 7.

[0228] This pH sensitivity may be attributed to amylase in saliva, which plays a crucial role in polysaccharide hydrolysis. Amylase activity is pH-dependent, with an optimal pH range of 6.7–7.0, within which it is most stable and active. At pH 7, enzyme activity peaks, resulting in the highest degree of hydrolysis. Conversely, at pH 4, far below the optimal pH, the enzyme's structure and function are more impaired, leading to a significant decrease in activity and the lowest degree of hydrolysis. After 6 h of incubation in artificial oral solution at pH 7, the maximum degrees of hydrolysis for CDPs-T, CDPs-U, CDPs-H, and CDPs-UH were 9.89%, 9.35%, 8.89%, and 9.54%, respectively, close to the degree of hydrolysis of In (9.51%), revealing that CDPs exhibit high tolerance to α-amylase. Literature reports that polysaccharide hydrolysis is closely related to its sugar structure, composition, and conformation. This strong enzyme tolerance of CDPs-UH may be related to its complex monosaccharide composition and α- / β-mixed glycosidic bonds.

[0229] Figure 6f-6j shows the digestion results of four CDPs in human gastric juice at different pH values ​​over time. As the pH value increased, the percentage of hydrolysis of the four CDPs gradually decreased, reaching its maximum after 6 h of incubation at pH 1. This may be because the glycosidic bonds of polysaccharides are more easily broken down at low pH, and the extended incubation time also facilitates hydrolysis into monosaccharides and disaccharides under acidic conditions. The maximum degree of hydrolysis of CDPs-T, CDPs-U, CDPs-H, and CDPs-UH after 6 h of incubation in gastric juice at pH 1 were 2.95%, 2.45%, 1.80%, and 1.93%, respectively, close to the degree of hydrolysis of In (2.82%), indicating that these four polysaccharides maintain good stability in acidic environments. Food typically stays in the human stomach for 2-4 hours. The four CDPs all showed resistance to gastric juice exceeding 95%, suggesting that most CDPs can safely reach the intestines without severe hydrolysis by human gastric juice, making them available for utilization by beneficial intestinal bacteria. Therefore, CDPs may be a promising type of prebiotic.

[0230] Example 6 Prebiotic Activity Assay

[0231] 6.1 Determination Method

[0232] To determine whether the polysaccharides exhibited prebiotic activity, *Bifidobacterium longum* BB536, *Lactobacillus plantarum* L51-3, and *Lactobacillus casei* 129-2-1 were used, referencing previous reports. MRS medium supplemented with 0.05% (w / v) L-cysteine ​​and free of carbohydrates was used as the basal medium. The basal MRS medium served as a blank control (control group), and the recognized prebiotic inulin (In) served as a positive control. Carbohydrates (CDPs-T, CDPs-U, CDPs-H, CDPs-UH) were added to the basal MRS medium after filtration sterilization, with final concentrations of 0.25%, 0.5%, 1.0%, and 2.0% (w / v), respectively. The activated bacterial suspension (OD) was then... 600 =0.60±0.01) was inoculated into MRS medium to achieve a final probiotic concentration of 2% (v / v). These cultures were incubated at 37°C for 48 h, and the absorbance was measured at a wavelength of 600 nm. The pH of the fermentation broth was also measured using a pH meter after 48 h of bacterial fermentation.

[0233] 6.2 Measurement Results

[0234] Figure 7 The results are the analysis results of prebiotic activity and structure-activity relationship; among which Figure 7 a-7c represent the effects of different concentrations of CDPs on the proliferation of Bifidobacterium longum BB536, Lactobacillus plantarum L51-3, and Lactobacillus casei 129-2-1, respectively. Figure 7d-7f represent the effects of different concentrations of CDPs on the pH of fermentation broths of Bifidobacterium longum BB536, Lactobacillus plantarum L51-3, and Lactobacillus casei 129-2-1, respectively. Figure 7 g is a heatmap showing the correlation between CDP structural features and antioxidant and prebiotic activities. Now, combined with... Figure 7 The analysis is as follows:

[0235] The bioactivity of polysaccharides is closely related to their physicochemical properties and structure. This study selected three widely used and recognized probiotic bacteria in the food industry (Bifidobacterium longum, Lactobacillus plantarum, and Lactobacillus casei) to evaluate the prebiotic activity of CDPs. Figure 7 As shown in a-7c, the OD values ​​of CDPs and inulin group after 48 h of culture were... 600 All concentrations were significantly higher than the carbon-free group (P<0.05). Within the experimental concentration range, CDPs promoted the proliferation and growth of the three probiotics in a dose-dependent manner, which is related to the characteristics of polysaccharides and probiotics. On the one hand, probiotics have different abilities to utilize effective polysaccharides by secreting different enzymes; on the other hand, the chemical structure of plant polysaccharides is complex and diverse, requiring various carbohydrate-activating enzymes to hydrolyze polysaccharide segments.

[0236] All polysaccharide groups showed higher prebiotic activity than the In group, with CDPs-UH exhibiting significantly higher activity than other polysaccharide groups (P<0.05). The utilization of polysaccharides by probiotics depends on various factors, including physicochemical properties, molecular weight, monosaccharide composition, and structure. Firstly, total sugar content plays a crucial role. The highest prebiotic activity observed in CDPs-UH is likely due to its highest total sugar content among the CDPs samples, consistent with literature findings that higher total sugar content correlates with better proliferation. Furthermore, molecular weight (Mw) is a significant factor influencing carbon source decomposition and probiotic utilization. Generally, low molecular weight polysaccharides effectively promote probiotic proliferation, consistent with the lowest molecular weight found in CDPs-UH in this study. Secondly, differences in monosaccharide composition also affect probiotic growth. Previous studies have shown that glucose, arabinose, and galactose exhibit significant prebiotic activity. CDPs-UH contains higher levels of glucose, arabinose, and galactose. Finally, polysaccharide structure also influences probiotic proliferation. Compared to the other three polysaccharides, CDPs-UH has a rough and porous surface, which greatly increases the likelihood of it being transported into the probiotic cells for metabolism and absorption. Therefore, a single factor is insufficient to determine the prebiotic activity of CDPs. Thus, CDPs-UH possesses optimal prebiotic activity, which is closely related to its highest total sugar content, lowest molecular weight, high content of glucose, arabinose, and galactose, and its microstructure.

[0237] The changes in pH value were also examined in different groups after 48 h of incubation. Figure 7(d-7f). pH value not only reflects the acidification activity of the tested strains but also indicates the utilization of carbohydrates by that specific strain. Polysaccharides are metabolized by bacteria to produce lactic acid and short-chain fatty acids, leading to a decrease in the pH value of the culture medium. The results showed that, compared with the group without a carbon source, the pH value of probiotics cultured in media containing CDPs or inulin decreased significantly after 48 h of fermentation (P<0.05). The results also showed that the proliferation of the above probiotics varied depending on the type and concentration of prebiotics used in the basal medium. The acidification activity of the probiotic strains increased with increasing prebiotic concentration. At a polysaccharide concentration of 2%, the pH value of the CDPs-UH group rapidly decreased to 4.66–4.79 after 48 h; while the pH value of the In group was 5.19–5.30 during the same period (P<0.05). Under the same polysaccharide concentration, the pH value of the CDPs-UH group decreased the most after 48 h.

[0238] Correlation analysis of the structural characteristics (including monosaccharide composition, chemical composition, molecular weight, particle size, and potential) of CDPs extracted from four different processes with their antioxidant and prebiotic properties is as follows: Figure 7 As shown in g. The results showed that uronic acid content, xylose and galacturonic acid, arabinose, galactose, rhamnose, glucosamine, and ribose all had significant positive effects on DPPH and hydroxyl radical scavenging capacity (P<0.05). Conversely, the free radical scavenging activity of CDPs increased with decreasing total sugar content and glucose content. ABTS + Regarding free radical scavenging ability, only uronic acid content, xylose and galacturonic acid showed a significant positive correlation (P<0.05), which means that as the uronic acid content or the ratio of xylose and galacturonic acid increases, the ABTS+ free radical scavenging activity also increases, while other structural features have no significant effect.

[0239] Regarding the degree of α-amylase hydrolysis, uronic acid content and galacturonic acid showed a significant negative correlation, as did particle size (P<0.05). In terms of the degree of hydrolysis in artificial gastric juice, lower molecular weight, smaller particle size, and higher absolute potential value (i.e., better dispersibility) indicated better tolerance of CDPs to artificial gastric juice.

[0240] Regarding the probiotic proliferation effect, total sugar content and glucose content showed a significant positive correlation (P<0.05), meaning that as the total sugar content and glucose content increased, the OD of the three probiotics increased. 600 The values ​​also increased accordingly. However, glucosamine, ribose, molecular weight, particle size, and potential showed a significant negative correlation (P<0.05).

[0241] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A method for ultrasonic-high pressure assisted three-phase extraction of Cistanche deserticola polysaccharides based on response surface methodology, comprising the following steps: (1) Response surface optimization experiment, construct a quadratic regression prediction model, and determine the optimal process parameters for three-phase extraction; (2) After drying, crushing and sieving the dried slices of Cistanche deserticola, Cistanche deserticola powder is obtained. The Cistanche deserticola powder is mixed with distilled water according to the material-liquid ratio to prepare Cistanche deserticola slurry. (3) The Cistanche deserticola slurry is subjected to a combination of ultrasonic and high-pressure pretreatment; (4) The pretreated material was combined with ammonium sulfate and tert-butanol to form a three-phase extraction system, and three-phase centrifugation was carried out under the optimized process conditions; (5) After separation, the lower phase polysaccharide solution is collected, and after dialysis, concentration and freeze drying, Cistanche deserticola polysaccharide is obtained.

2. The method according to claim 1, wherein step (1) specifically includes: Using ammonium sulfate mass fraction, the volume ratio of tert-butanol to Cistanche deserticola slurry, extraction temperature, and extraction pH as the factors investigated, and the extraction rate of Cistanche deserticola polysaccharides as the response value, a quadratic regression prediction model was constructed through single-factor experiments and response surface methodology. The equation of the quadratic regression prediction model is as follows: y=-51.9873+0.2208A+12.7177B+0.8631C+12.0400D+0.0641AB+0.0046AC+0.0222AD-0.0586BC-0.3882BD-0.0479CD-0.0093A 2 -3.4227B 2 -0.0086C 2 -0.9025D 2 Wherein, y is the extraction rate of Cistanche deserticola polysaccharide, A is the mass fraction of ammonium sulfate, B is the volume ratio of tert-butanol to Cistanche deserticola slurry, C is the extraction temperature, and D is the extraction pH; the Cistanche deserticola slurry refers to the mixture obtained by mixing Cistanche deserticola powder and distilled water according to the material-liquid ratio.

3. According to the method described in claim 2, in the quadratic regression prediction model, the degree of influence of each factor on the extraction rate of Cistanche deserticola polysaccharide is as follows: ammonium sulfate mass fraction > extraction temperature = extraction pH > volume ratio of tert-butanol to Cistanche deserticola slurry.

4. The method according to any one of claims 1-3, wherein the optimal process parameters obtained by solving the quadratic regression prediction model are: ammonium sulfate mass fraction of 30%, volume ratio of tert-butanol to Cistanche deserticola slurry of 1:1.5, extraction temperature of 40 ℃, and extraction pH of 6.

5. According to the method of claim 1, in step (2), the ratio of Cistanche deserticola powder to distilled water is 1:

20.

6. According to the method of claim 1, in step (3), the ultrasonic pretreatment adopts pulsed ultrasound with the following parameters: frequency 20 kHz, power 400 W, temperature 25℃, adopting a 2-second on / 2-second off cycle mode, and the total processing time is 30 min.

7. According to the method of claim 1, in step (3), the processing parameters of the high pressure pretreatment are: pressure 300MPa, temperature 25℃, and processing time 10 min.

8. According to the method of claim 1, in step (3), the combined ultrasound and high pressure pretreatment is to first perform pulse ultrasound treatment and then perform ultra-high pressure treatment.

9. According to the method of claim 1, in step (4), the method for constructing the three-phase extraction system is as follows: the pretreated Cistanche deserticola slurry is magnetically stirred for 12 h under ambient temperature and light-proof conditions, the supernatant is collected by centrifugation and concentrated to 2 / 3 of the original volume; ammonium sulfate is added to the optimal ammonium sulfate mass fraction and the pH is adjusted to the optimal extraction pH; tert-butanol is added to the optimal tert-butanol to Cistanche deserticola slurry volume ratio; the system is allowed to stand for 1 h under the optimal extraction temperature conditions; then the system is centrifuged to separate into three phases under the centrifugation conditions of 6000 rpm and centrifugation time of 10 min.

10. According to the method of claim 1, in step (5), the lower phase polysaccharide solution containing ammonium sulfate is collected, dialyzed through a dialysis bag with a molecular weight cutoff of 3500 Da, concentrated, and freeze-dried to obtain purified Cistanche deserticola polysaccharide.

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