Preparation method and application of a cistanche polypeptide

Abstract

The application discloses a preparation method and application of a cistanche polypeptide, and belongs to the polypeptide preparation field. The preparation method is characterized by the following steps: after the cistanche is broken, the cistanche is subjected to two-stage specific enzymolysis by using, in sequence, trypsin, actinidin, ginger protease and flavor protease; after the enzyme is inactivated, the cistanche is inoculated with a compound bacterial liquid of bifidobacterium adolescentis and lactobacillus acidophilus for fermentation; and finally, the cistanche polypeptide is obtained through membrane separation and purification and drying. The cistanche polypeptide provided by the application has excellent antioxidant capacity and anti-fatigue effect, and the recovery rate of the target polypeptide is high, so that the cistanche polypeptide can be widely applied to antioxidant, anti-fatigue functional products and health foods.

Description

Technical Field

[0001] This application belongs to the field of polypeptide preparation, specifically relating to a method for preparing Cistanche deserticola polypeptide and its application. Background Technology

[0002] Cistanche deserticola, a traditional tonic herb, is rich in polysaccharides, phenylethanol glycosides, and proteins, which have been proven to have antioxidant and anti-fatigue effects. However, existing extraction technologies have significant limitations.

[0003] Conventional physical cell wall disruption or single enzymatic hydrolysis methods are insufficient to fully release active proteins within the cell wall, resulting in inadequate peptide yield. High-temperature or strong acid / alkali treatments can easily damage heat-sensitive functional peptides, causing a significant reduction in antioxidant activity and anti-fatigue effects. Some researchers have attempted combined enzymatic hydrolysis or microbial fermentation, but due to improper enzyme combinations or single-function microorganisms, they have been unable to synergistically release and modify active peptides, resulting in poor product performance.

[0004] In summary, how to achieve efficient preparation and efficacy enhancement of bioactive peptides through process innovation has become a bottleneck that the industry urgently needs to overcome. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a preparation process that combines stepwise enzymatic hydrolysis with multiple complex enzyme preparations and co-fermentation with complex bacteria, thereby achieving efficient preparation and enhanced efficacy of active substances in Cistanche deserticola polypeptides.

[0006] To achieve the above objectives, the present invention discloses the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing Cistanche deserticola polypeptide, the method comprising the following steps:

[0008] Step 1. Crush and sieve the dried Cistanche deserticola to obtain Cistanche deserticola powder. Mix the powder with drinking water and then perform cell wall breaking treatment to obtain Cistanche deserticola homogenate.

[0009] Step 2. Add trypsin to the Cistanche deserticola homogenate and hydrolyze for 5-7 hours to obtain hydrolysate 1. Inactivate the enzyme in hydrolysate 1. Add a compound enzyme preparation to the inactivated hydrolysate 1 and hydrolyze for 3-4 hours to obtain hydrolysate 2. Sterilize hydrolysate 2.

[0010] Step 3. Take the compound bacterial culture of Bifidobacterium adolescentis and Lactobacillus acidophilus and inoculate it into the sterilized enzymatic hydrolysate 2. The inoculation amount is 10-13 v / v% based on the volume of enzymatic hydrolysate 2. Ferment and culture at 35-37℃ and 100-150 r / min for 48-60 h to obtain the fermentation broth.

[0011] Step 4. Centrifuge the fermentation broth at 5000-8000 r / min to remove residue, take the supernatant and ultrafilter it with a 1000 Da ultrafiltration membrane, collect the filtrate and then nanofilter it with an 800 Da nanofiltration membrane to obtain the retentate and concentrate it. Spray dry the concentrate to obtain the Cistanche deserticola polypeptide.

[0012] Preferably, the compound enzyme preparation is composed of kiwifruit protease, ginger protease and flavor protease;

[0013] Preferably, the enzyme hydrolysate 1 is heated to 80-90℃ and kept at that temperature for 15-20 minutes to inactivate the enzyme;

[0014] Preferably, the sterilization conditions for the enzymatic hydrolysate 2 are 121°C for 20-30 minutes;

[0015] Preferably, the number of viable Bifidobacterium adolescentis in the compound bacterial solution is ≥1×10⁻⁶. 8 CFU / mL, viable Lactobacillus acidophilus count ≥1×10⁻⁶ 6 CFU / mL;

[0016] Preferably, in step 4, the retentate is concentrated in a rotary evaporator at 60-70°C and 50-60 r / min for 3-4 hours.

[0017] Preferably, in step 1, Cistanche deserticola and drinking water are mixed at a mass-to-volume ratio of 1:20-30 g / mL.

[0018] Preferably, the cell wall breaking in step 1 is carried out at 25,000-30,000 r / min for 10-15 min.

[0019] Preferably, the chymotrypsin activity is 1500-2000 U / g.

[0020] Preferably, the kiwi protease activity is 1000-1500 U / g, the ginger protease activity is 800-1000 U / g, and the flavor protease activity is 2000-2500 U / g.

[0021] More preferably, the amount of chymotrypsin added is 8-10% of the mass of the Cistanche deserticola homogenate, and the enzymatic hydrolysis is carried out at 35-37°C, with stirring every half hour during the enzymatic hydrolysis process.

[0022] More preferably, the amount of the compound enzyme preparation added is 5-7% of the mass of the enzymatic hydrolysate 1, and the enzymatic hydrolysis is carried out at 45-55℃ and a rotation speed of 10-20 r / min.

[0023] In a second aspect, the present invention provides a Cistanche deserticola polypeptide, which is prepared by the preparation method described in the first aspect.

[0024] Thirdly, the present invention provides the application of the Cistanche deserticola polypeptide described in the second aspect in the preparation of products with antioxidant and anti-fatigue effects.

[0025] Fourthly, the present invention provides the application of the Cistanche deserticola polypeptide described in the second aspect in the preparation of health food with antioxidant and anti-fatigue effects.

[0026] In this invention:

[0027] Based on the selectivity of different proteases for specific amino acid sequences or peptide bonds, the applicant combined numerous proteases in the prior art and screened out combinations suitable for producing Cistanche deserticola polypeptides. The preparation method of Cistanche deserticola polypeptides in this invention employs a stepwise enzymatic hydrolysis approach. The protease system composed of chymotrypsin, actinidin, ginger protease, and flavor protease in the two-step hydrolysis has different cleavage sites and specificities. Their combined use covers a wider range of cleavage sites and achieves synergistic effects, improving the efficiency of protein hydrolysis and ensuring complete protein digestion. Furthermore, this invention effectively controls the length distribution and types of polypeptides generated by adjusting the types, ratios, and hydrolysis conditions of different proteases, enhancing the bioactivity of the obtained Cistanche deserticola polypeptides, while simultaneously improving the recovery rate of the target polypeptides, reducing byproducts, and improving the unpleasant odor of the hydrolysate.

[0028] Bifidobacterium adolescentis is a strict anaerobic bacterium, while Lactobacillus acidophilus is a facultative anaerobic bacterium. They form a symbiotic system under anaerobic conditions. Bifidobacterium adolescentis metabolizes to produce short-chain fatty acids, which lower the pH value and activate the protease of Lactobacillus acidophilus. This cascade reaction produces glutathione analogue branched-chain amino acid peptides that can effectively scavenge free radicals, thereby improving the efficacy of the target polypeptides in Cistanche deserticola.

[0029] The beneficial effects of this invention are:

[0030] 1. This invention employs a specific combination of chymotrypsin, kiwifruit protease, ginger protease, and flavor protease for stepwise enzymatic hydrolysis, and further utilizes a dual-strain synergistic fermentation process of Bifidobacterium adolescentis and Lactobacillus acidophilus. This effectively overcomes the limitations of traditional single-enzyme methods or single-strain fermentation with low efficiency. This synergistic effect of enzymatic hydrolysis and fermentation can more fully extract the active peptides in Cistanche deserticola, thereby significantly enhancing the antioxidant capacity and anti-fatigue effect of the final product.

[0031] 2. The stepwise enzymatic hydrolysis method and the combined fermentation method provided by this invention effectively improve the yield of small molecule peptides of 800-1000 Da.

[0032] 3. This invention creatively integrates key steps such as high-speed cell disruption, specific compound enzymatic hydrolysis, and dual-strain synergistic fermentation, forming a highly efficient and stable preparation system. The process is simple and conducive to the development and application of subsequent functional products. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all of them. For clarity, not all features of the actual embodiments are described.

[0034] Based on the embodiments described in the implementation plan, other embodiments obtained by those skilled in the art without creative effort are all within the scope of protection of this invention.

[0035] Cistanche tubulosa: Cistanche tubulosa is also known as Cistanche tubulosa (Schenk) R.Wight.

[0036] The chymotrypsin, kiwi protease, ginger protease, and flavor protease are all commercially available. Before use, their enzyme activity was measured and they were appropriately diluted to ensure that the enzyme activity of chymotrypsin was 1500 U / g, kiwi protease was 1000 U / g, ginger protease was 1000 U / g, and flavor protease was 2000 U / g.

[0037] Bifidobacterium adolescentis: accession number GDMCC 1.1262, purchased from Guangdong Provincial Center for Microbial Culture Collection;

[0038] Lactobacillus acidophilus: accession number GDMCC 1.1807, purchased from Guangdong Provincial Center for Microbial Culture Collection.

[0039] Preparation of Cistanche deserticola polypeptide

[0040] Preparation of Example 1

[0041] Step 1. Place the dried Cistanche deserticola in a grinder and grind it through a 30-mesh sieve to obtain Cistanche deserticola powder. Mix the Cistanche deserticola powder with drinking water at a mass-volume ratio of 1:20 g / mL. The drinking water shall comply with GB 5749-2022. Then, perform cell wall breaking treatment by grinding at 30000 r / min for 10 min to obtain Cistanche deserticola homogenate.

[0042] Step 2. Add chymotrypsin to the Cistanche deserticola homogenate at a concentration of 8% of the homogenate mass. Hydrolyze at 35℃ and pH 7.5 for 7 hours, stirring every half hour during the hydrolysis process to obtain hydrolysate 1. Heat hydrolysate 1 to 80℃ and keep warm for 20 minutes to inactivate the enzyme. After inactivation, cool hydrolysate 1 to 45℃ and add a compound enzyme preparation at a concentration of 5% of the hydrolysate mass. Hydrolyze at 45℃, pH 5, and 10 r / min for 4 hours to obtain hydrolysate 2. Heat hydrolysate 2 to 121℃ for 20 minutes to sterilize.

[0043] The compound enzyme preparation consists of kiwifruit protease, ginger protease, and flavor protease in a mass ratio of 1:0.5:0.2.

[0044] Step 3. Take 1×10⁻⁶ live bacteria of Bifidobacterium adolescentis. 8 CFU / mL, Lactobacillus acidophilus viable count 1×10⁻⁶ 6 The CFU / mL compound bacterial culture was inoculated into the inactivated enzymatic hydrolysate 2 at an inoculation amount of 10 v / v% (volume ratio of bacterial culture to enzymatic hydrolysate 2). The culture was fermented at 35℃, 100 r / min, and under anaerobic conditions for 48 h to obtain the fermentation broth.

[0045] Step 4. Centrifuge the fermentation broth at 5000 r / min to remove residue, take the supernatant and ultrafilter it with a 1000 Da ultrafiltration membrane, collect the filtrate and then nanofilter it with an 800 Da nanofiltration membrane to obtain the retentate, place it in a rotary evaporator and concentrate it at 60℃ and 50 r / min for 4 h, and then spray dry the concentrated solution to obtain the Cistanche deserticola polypeptide.

[0046] Preparation of Example 2

[0047] Step 1. Place the dried Cistanche deserticola in a grinder and grind it through a 30-mesh sieve to obtain Cistanche deserticola powder. Mix the Cistanche deserticola powder with drinking water at a mass-volume ratio of 1:25 g / mL, wherein the drinking water conforms to GB 5749-2022. Then, perform cell wall breaking treatment by grinding at 25000 r / min for 10 min to obtain Cistanche deserticola homogenate.

[0048] Step 2. Add chymotrypsin to the Cistanche deserticola homogenate at a concentration of 9% of the homogenate's mass. Hydrolyze at 36℃ and pH 7.5 for 6 hours, stirring every half hour during the hydrolysis process to obtain hydrolysate 1. Heat hydrolysate 1 to 85℃ and incubate for 17 minutes to inactivate the enzyme. After inactivation, cool hydrolysate 1 to 50℃ and add a compound enzyme preparation at a concentration of 6% of its mass. Hydrolyze at 50℃, pH 5, and a rotation speed of 15 r / min for 3.5 hours to obtain hydrolysate 2. Sterilize hydrolysate 2 by heating it to 121℃ for 20 minutes.

[0049] The compound enzyme preparation consists of kiwifruit protease, ginger protease, and flavor protease in a mass ratio of 1:0.6:0.3.

[0050] Step 3. Take 1×10⁻⁶ live bacteria of Bifidobacterium adolescentis. 8 CFU / mL, Lactobacillus acidophilus viable count 1×10⁻⁶ 6 The CFU / mL compound bacterial culture was inoculated into the inactivated enzymatic hydrolysate 2 at an inoculation amount of 12v / v% (volume ratio of bacterial culture to enzymatic hydrolysate 2). The culture was fermented at 36℃, 120r / min, and under anaerobic conditions for 54h to obtain the fermentation broth.

[0051] Step 4. Centrifuge the fermentation broth at 7000 r / min to remove residue, take the supernatant and ultrafilter it with a 1000 Da ultrafiltration membrane, collect the filtrate and then nanofilter it with an 800 Da nanofiltration membrane to obtain the retentate, place it in a rotary evaporator and concentrate it at 65℃ and 55 r / min for 3.5 h, and then spray dry the concentrate to obtain the Cistanche deserticola polypeptide.

[0052] Preparation of Example 3

[0053] Step 1. Place the dried Cistanche deserticola in a grinder and grind it through a 30-mesh sieve to obtain Cistanche deserticola powder. Mix the Cistanche deserticola powder with drinking water at a mass-volume ratio of 1:30 g / mL, wherein the drinking water conforms to GB 5749-2022. Then, perform cell wall breaking treatment by grinding at 25000 r / min for 10 min to obtain Cistanche deserticola homogenate.

[0054] Step 2. Add chymotrypsin to the Cistanche deserticola homogenate at a concentration of 10% of the homogenate's mass. Hydrolyze at 37℃ and pH 7.5 for 5 hours, stirring every half hour during the hydrolysis process to obtain hydrolysate 1. Heat hydrolysate 1 to 90℃ and incubate for 15 minutes to inactivate the enzyme. After inactivation, cool hydrolysate 1 to 55℃ and add a compound enzyme preparation at a concentration of 7% of its mass. Hydrolyze at 55℃, pH 5, and a rotation speed of 20 r / min for 3 hours to obtain hydrolysate 2. Sterilize hydrolysate 2 by heating it to 121℃ for 20 minutes.

[0055] The compound enzyme preparation consists of kiwifruit protease, ginger protease, and flavor protease in a mass ratio of 1:0.7:0.4.

[0056] Step 3. Take 1×10⁻⁶ live bacteria of Bifidobacterium adolescentis. 8 CFU / mL, Lactobacillus acidophilus viable count 1×10⁻⁶ 6 The CFU / mL compound bacterial culture was inoculated into the inactivated enzymatic hydrolysate 2 at an inoculation amount of 13v / v% (volume ratio of bacterial culture to enzymatic hydrolysate 2). The culture was fermented at 37℃, 150r / min, and under anaerobic conditions for 60h to obtain the fermentation broth.

[0057] Step 4. Centrifuge the fermentation broth at 8000 r / min to remove residue, take the supernatant and ultrafilter it with a 1000 Da ultrafiltration membrane, collect the filtrate and then nanofilter it with an 800 Da nanofiltration membrane to obtain the retentate, place it in a rotary evaporator and concentrate it at 70℃ and 60 r / min for 3 h, and then spray dry the concentrate to obtain the Cistanche deserticola polypeptide.

[0058] Preparation of Comparative Example 1

[0059] The preparation method of Example 2 was followed, except that in step 2, a compound enzyme preparation was first added to the Cistanche deserticola homogenate for enzymatic hydrolysis. The amount of compound enzyme preparation added was 6% of the Cistanche deserticola homogenate. Enzymatic hydrolysis was carried out at 50°C, pH=5, and a rotation speed of 15 r / min for 3.5 h to obtain hydrolysate 1. After enzyme inactivation, chymotrypsin was added to hydrolysate 1 at a mass of 9% of hydrolysate 1. Enzymatic hydrolysis was carried out at 36°C and pH=7.5 for 6 h to obtain hydrolysate 2.

[0060] The remaining steps and parameters are the same as in Example 2.

[0061] Preparation of Comparative Example 2

[0062] The preparation method was carried out according to Example 2, except that: the chymotrypsin in step 2 was replaced with trypsin, and the trypsin activity was 1500 U / g, and the amount added was 9% of the mass of Cistanche deserticola homogenate. The enzyme was hydrolyzed at 36°C and pH=7.5 for 6 hours to obtain enzyme hydrolysate 1.

[0063] The remaining steps and parameters are the same as in Example 2.

[0064] Preparation of Comparative Example 3

[0065] The preparation method of Example 2 was followed, except that the kiwi protease in the compound enzyme preparation in step 2 was replaced with papain, and the enzyme activity of papain was 1000 U / g. The papain, ginger protease and flavor protease were compounded in a mass ratio of 1:0.6:0.3.

[0066] The remaining steps and parameters are the same as in Example 2.

[0067] Preparation of Comparative Example 4

[0068] The preparation method of Example 2 was followed, except that: ginger protease in step 2 was replaced with papain, and the enzyme activity of papain was 1000 U / g; kiwi protease, papain and flavor protease were compounded in a mass ratio of 1:0.6:0.3.

[0069] The remaining steps and parameters are the same as in Example 2.

[0070] Preparation of Comparative Example 5

[0071] The preparation method of Example 2 was followed, except that: the Bifidobacterium adolescentis in the compound bacterial solution in step 3 was replaced with Bifidobacterium breve with preservation number GDMCC 1.206, and fermented with Lactobacillus acidophilus to form a compound bacterial solution, and the viable number of Bifidobacterium breve was ensured to be the same as that of Bifidobacterium adolescentis.

[0072] The remaining steps and parameters are the same as in Example 2.

[0073] Preparation of Comparative Example 6

[0074] The preparation method of Example 2 was followed, except that Lactobacillus acidophilus in the compound bacterial solution in step 3 was replaced with Lactobacillus plantarum with accession number GDMCC 1.648, and fermented with Bifidobacterium adolescentis to form a compound bacterial solution, while ensuring that the number of viable Lactobacillus plantarum and Lactobacillus acidophilus were the same.

[0075] The remaining steps and parameters are the same as in Example 2.

[0076] Performance testing

[0077] 1. Antioxidant test

[0078] The Cistanche deserticola polypeptides prepared in Examples 1-3 and Comparative Examples 1-6 were diluted with deionized water to obtain test sample solutions 1-9 with a concentration of 0.01 wt%, and their antioxidant properties were tested.

[0079] 1.1 DPPH free radical scavenging

[0080] DPPH ethanol solution is violet in color and has a strong absorbance at 517 nm. If it combines with the sample solution to be tested, it will reduce the absorbance at 517 nm. This can be used to determine the ability of the sample solution to scavenge DPPH free radicals.

[0081] 1.1.1 Sample solution to be tested

[0082] The sample solutions to be tested, 1-9, are shown in Table 1 below:

[0083] Table 1. Experimental groups and corresponding Cistanche deserticola polypeptides

[0084] Positive control group (VC) 100 μg / mL VC aqueous solution Sample solution 1 A solution containing 0.01% of Cistanche deserticola polypeptide from Example 1 Sample solution 2 A solution containing 0.01% of Cistanche deserticola polypeptide from Example 2 Sample solution 3 A solution containing 0.01% of Cistanche deserticola polypeptide from Example 3 Sample solution 4 A solution containing 0.01% of Cistanche deserticola polypeptide (Comparative Example 1) 5 samples to be tested A solution containing 0.01% of Comparative Example 2 Cistanche deserticola polypeptide Sample solution 6 A solution containing 0.01% of Comparative Example 3 Cistanche deserticola polypeptide Sample solution 7 A solution containing 0.01% of Comparative Example 4 Cistanche deserticola polypeptide 8 samples to be tested A solution containing 0.01% Comparative Example 5 Cistanche deserticola polypeptide Sample solution to be tested 9 A solution containing 0.01% of Comparative Example 6 Cistanche deserticola polypeptide

[0085] 1.1.2 Experimental Design

[0086] DPPH was prepared with anhydrous ethanol to a concentration of 2×10⁻⁶. -4Take several portions of 2 mL each of the sample solution / VC, DPPH solution, and anhydrous ethanol. Mix 2 mL of the sample solution / VC and 2 mL of DPPH solution and let stand at room temperature for 30 min. Measure the absorbance at a wavelength of 517 nm to obtain Ai.

[0087] Mix 2 mL of the sample solution / VC and 2 mL of anhydrous ethanol, and then measure the absorbance as Aj according to the aforementioned method.

[0088] The absorbance was measured as Ac after mixing 2 mL of DPPH solution and 2 mL of deionized water according to the aforementioned method.

[0089] Each sample was measured in triplicate, and the average value was taken.

[0090] Calculate the scavenging rate of DPPH free radicals for each test sample according to the following formula (1) and record it in Table 2 below.

[0091] Clearance rate (%) = (1 - (Ai - Aj) / Ac) × 100% ... (1)

[0092] In the formula: Ai is the absorbance of 2 mL of the sample solution to be tested / VC + 2 mL of DPPH solution mixture; Aj is the absorbance of 2 mL of the sample solution to be tested / VC + 2 mL of anhydrous ethanol mixture; Ac is the absorbance of 2 mL of DPPH solution + 2 mL of deionized water.

[0093] Table 2 DPPH removal rate

[0094] Positive control group 95.16 Sample solution 1 93.25 Sample solution 2 98.34 Sample solution 3 96.62 Sample solution 4 76.18 5 samples to be tested 73.51 Sample solution 6 86.60 Sample solution 7 68.09 8 samples to be tested 80.42 Sample solution to be tested 9 75.85

[0095] Results analysis:

[0096] The experimental results show that Comparative Examples 1-4, by changing and replacing the stepwise enzymatic hydrolysis steps with chymotrypsin, actinidin, and ginger protease, respectively, resulted in a significant decrease in the antioxidant properties of Cistanche deserticola polypeptides compared to Example 2. This indicates that the stepwise enzymatic hydrolysis method and specific enzyme preparations in the hydrolysis process play a crucial synergistic role in the preparation of Cistanche deserticola polypeptides, and both are indispensable in the preparation process. The combination of chymotrypsin, actinidin, and ginger protease is specific and can better promote the decomposition of Cistanche deserticola proteins.

[0097] Compared to Example 2, Comparative Examples 5 and 6 showed a decrease in the antioxidant properties of Cistanche deserticola peptides. This indicates that the microbial fermentation steps of Bifidobacterium adolescentis and Lactobacillus acidophilus can enrich and modify small molecule peptides during the preparation of Cistanche deserticola peptides, thereby enhancing the antioxidant activity of Cistanche deserticola peptides. At the same time, the absence or replacement of either Bifidobacterium adolescentis or Lactobacillus acidophilus will lead to a decrease in antioxidant properties, indicating that the two strains enhance peptide activity through synergistic metabolism.

[0098] 2. Fatigue resistance test

[0099] Male BALB / c mice, weighing 20±2g, were used in the experiment. The experimental temperature was 20-24℃, and the relative humidity was 40-70%. The animals were acclimatized to the animal room environment for 4 days before the experiment. Different doses of the test solution were administered orally for 30 consecutive days. The negative control group was given the same volume of physiological saline, and the other treatments were the same as the dose groups. Each group consisted of 10 mice.

[0100] 2.1 Test material processing

[0101] The test samples were Cistanche deserticola polypeptides prepared in Example 2 and Comparative Examples 1-6. The recommended daily dose for human test samples based on Example 2 was 2g, and the recommended dose for samples based on an adult weight of 60kg was 0.033g / kg·d.

[0102] (1) In Example 2, three dosage groups were set up. The low, medium and high dosages were 0.033 g / kg, 0.167 g / kg and 0.333 g / kg of the test sample, respectively, which are equivalent to 1, 5 and 10 times the recommended dosage.

[0103] (2) Comparative Examples 1-6 were each set up with a dose group and compared with Example 2. The daily dosage of Comparative Examples 1-6 was the same as that of the medium dose group in Example 2, which was 0.167 g / kg.

[0104] (3) A negative control group was set up, and the mice were given the same volume of physiological saline.

[0105] Before the experiment, each of the above test samples was prepared into a test solution of each dose using physiological saline as a solvent. The test solution was administered by gavage, with a gavage volume of 0.8 mL per mouse.

[0106] 2.2 Mouse rotarod endurance time determination

[0107] Ten mice were used in each group. After three weeks of continuous drug administration, they underwent a one-week rotarod acclimatization training period (once daily, approximately 30 minutes). During the acclimatization training period, mice exhibiting significant behavioral abnormalities such as jumping, curling up on the rotarod, or poor climbing ability were excluded. After exclusion, the number of qualified mice remaining in each group was ≥6. On day 28 after the start of the experiment, 6 mice were randomly selected from each group of qualified mice for the formal rotarod endurance test. During the formal test, the mice were placed on the rotarod. The initial rotation speed of the rotarod was set to 5 r / min, and then linearly accelerated to a final rotation speed of 30 r / min within 50 seconds. The time from the start of the rotarod to the mouse falling due to fatigue was recorded, i.e., the rotarod endurance time. The results are shown in Table 3.

[0108] 2.3 Determination of the exhaustive swimming time of mice

[0109] On day 30 after the start of the experiment, a swimming test to exhaustion was conducted in mice. The last gavage was administered on day 30. Thirty minutes after administration, six mice were randomly selected from each group of qualified mice and placed in swimming tanks. The water depth in the tanks was approximately 40 cm, and the water temperature was strictly maintained at 25 ± 1℃. A lead weight equivalent to 5% of the mouse's body weight was fixed to the mouse's tail. The time from entry into the water to exhaustion (i.e., swimming time to exhaustion) was recorded. Exhaustion was defined as the mouse's nose remaining submerged for 10 seconds without being able to return to the surface. The results are shown in Table 3.

[0110] 2.4 Determination of muscle and liver glycogen levels in mice

[0111] Six mice in each group were anesthetized with isoflurane after participating in the aforementioned exhaustive swimming experiment, and then euthanized by cervical dislocation. Liver and bilateral quadriceps muscle tissue were rapidly harvested. 150 mg of each tissue was accurately weighed and placed in pre-chilled homogenization tubes, with 1.5 ml of ice-cold physiological saline added. Under ice bath conditions, the tissues were homogenized at 20,000 rpm using a high-speed homogenizer until completely broken down and the solution homogeneous (10% w / v tissue homogenate). The homogenate was centrifuged at 7,000 rpm for 15 minutes at 4°C, and the supernatant was carefully collected. The glycogen content in the liver and muscle supernatants was measured strictly according to the instructions of the liver / muscle glycogen assay kit. The results are shown in Table 3.

[0112] 2.5 Statistical Methods

[0113] Statistical analysis was performed using SPSS 19.0. The rotarod endurance time, exhaustive swimming time, liver glycogen, and muscle glycogen levels of mice in each group were recorded. This indicates that if the test data is normally distributed, the independent t-test is used for statistical analysis; if the test data is not normally distributed, the rank-sum test is used. All statistical methods employ two-tailed tests with a significance level of α = 0.05.

[0114] 2.6 Experimental Results

[0115] Table 3 Results of anti-fatigue experiments for each group

[0116]

[0117] Note: “a” indicates that the difference is significant compared with the negative control group, P<0.05, “aa” indicates P<0.01; “b” indicates that the dose group in Example 2 is significantly different from that in Comparative Examples 1-6, P<0.05, “bb” indicates P<0.01.

[0118] 2.7 Results Analysis

[0119] According to the experimental results in Table 3, the Cistanche deserticola polypeptide prepared in Example 2 significantly prolonged the exercise time of mice and significantly increased the content of muscle glycogen and liver glycogen compared with the control group, effectively enhancing the anti-fatigue properties of mice. The results of Comparative Examples 5 and 6 show that the compound fermentation step of Bifidobacterium adolescentis and Lactobacillus acidophilus provided by the present invention can effectively enrich the active ingredients and improve the anti-fatigue effect of the product. The effects of Comparative Examples 1-4 also showed a significant decrease compared with Example 2, proving the importance of stepwise enzymatic hydrolysis and compound enzymatic hydrolysis of chymotrypsin, actinidin, ginger protease and flavor protease in the process preparation, which has a synergistic promoting effect on the increase of active peptide content in Cistanche deserticola polypeptide products.

[0120] 3. Yield determination of specific molecular weight peptides

[0121] In the preparation process of Examples 1-3 and Comparative Examples 1-6, 100 mL of the fermentation supernatant after centrifugation in step 4 was taken and thoroughly mixed with an equal volume of 10 g / 100 mL trichloroacetic acid. The mixture was allowed to stand at 4°C for 30 min, centrifuged at 8000 r / min for 5 min, and the peptide content in the supernatant was determined by the Coomassie brilliant blue method and denoted as M1.

[0122] Take 100 mL of the retentate after nanofiltration in step 4, mix it thoroughly with an equal volume of 10 g / 100 mL trichloroacetic acid, let it stand at 4 °C for 30 min, centrifuge at 8000 r / min for 5 min, and determine the peptide content in the retentate using the Coomassie brilliant blue method, denoted as M2.

[0123] The yield of 800-1000 Da molecular weight peptides was calculated according to the formula "K=M2 / M1×100%" (each sample was measured in triplicate and the average value was taken). The results are shown in Table 4.

[0124] Table 4 Yields of peptides with specific molecular weights

[0125] Example 1 73.65 Example 2 77.53 Example 3 78.10 Comparative Example 1 55.08 Comparative Example 2 63.47 Comparative Example 3 58.15 Comparative Example 4 50.34 Comparative Example 5 64.72 Comparative Example 6 59.19

[0126] As shown in Table 4, specific enzymatic hydrolysis and fermentation steps in the preparation of Cistanche deserticola polypeptides are key factors in improving the yield of target molecular weight polypeptides. The absence or replacement of these key factors will lead to a decrease in yield. The stepwise enzymatic hydrolysis method and the compound fermentation method provided by this invention work together to effectively improve the yield of small molecule peptides of 800-1000 Da in Cistanche deserticola polypeptide products and can effectively reduce by-products.

[0127] In summary, high-speed cell wall disruption promotes the release of protein components in Cistanche deserticola, while the enzymatic hydrolysis of specific compound enzyme preparations promotes efficient protein hydrolysis. Furthermore, the combined fermentation of Bifidobacterium adolescentis and Lactobacillus acidophilus achieves the production and enrichment of active peptides, thus realizing a dual improvement in the yield and efficacy of active peptides, providing a new path for the high-value utilization of Cistanche deserticola.

[0128] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but all such changes and modifications fall within the scope of protection of the present invention.

Claims

1. A method for preparing Cistanche deserticola polypeptide, characterized in that, The preparation method includes the following steps: Step 1. Crush and sieve the dried Cistanche deserticola to obtain Cistanche deserticola powder. Mix the powder with drinking water and then perform cell wall breaking treatment to obtain Cistanche deserticola homogenate. Step 2. Add trypsin to the Cistanche deserticola homogenate and hydrolyze for 5-7 hours to obtain hydrolysate 1. Inactivate the enzyme in hydrolysate 1. Add a compound enzyme preparation to the inactivated hydrolysate 1 and hydrolyze for 3-4 hours to obtain hydrolysate 2. Sterilize hydrolysate 2. Step 3. Take the compound bacterial culture of Bifidobacterium adolescentis and Lactobacillus acidophilus and inoculate it into the sterilized enzymatic hydrolysate 2. The inoculation amount is 10-13 v / v% based on the volume of enzymatic hydrolysate 2. Ferment and culture at 35-37℃ and 100-150 r / min for 48-60 h to obtain the fermentation broth. Step 4. Centrifuge the fermentation broth at 5000-8000 r / min to remove residue, take the supernatant and ultrafilter it with a 1000 Da ultrafiltration membrane, collect the filtrate and then nanofilter it with an 800 Da nanofiltration membrane to obtain the retentate and concentrate it. Spray dry the concentrate to obtain the Cistanche deserticola polypeptide. The compound enzyme preparation consists of kiwifruit protease, ginger protease, and flavor protease.

2. The preparation method according to claim 1, characterized in that, In step 1, Cistanche deserticola and drinking water are mixed at a mass-to-volume ratio of 1:20-30 g / mL.

3. The preparation method according to claim 1, characterized in that, The cell wall breaking process in step 1 is carried out at 25,000-30,000 r / min for 10-15 min.

4. The preparation method according to claim 1, characterized in that, The amount of chymotrypsin added is 8-10% of the mass of Cistanche deserticola homogenate, and enzymatic hydrolysis is carried out at 35-37℃, with stirring every half hour during the enzymatic hydrolysis process.

5. The preparation method according to claim 1, characterized in that, The amount of the compound enzyme preparation added is 5-7% of the mass of the enzymatic hydrolysate 1, and the enzymatic hydrolysis is carried out at 45-55℃ and a stirring speed of 10-20 r / min.

6. The preparation method according to claim 1, characterized in that, The enzyme inactivation treatment described in step 2 involves heating the enzyme hydrolysate 1 to 80-90℃ and holding it at that temperature for 15-20 minutes.

7. The preparation method according to claim 1, characterized in that, In step 4, the retentate is placed in a rotary evaporator and concentrated for 3-4 hours at 60-70℃ and 50-60r / min.

8. A Cistanche deserticola polypeptide, characterized in that, The Cistanche deserticola polypeptide is prepared by the preparation method according to any one of claims 1-7.

9. The application of the Cistanche deserticola polypeptide according to claim 8 in the preparation of products with antioxidant and anti-fatigue effects.

10. The application of the Cistanche deserticola polypeptide according to claim 8 in the preparation of health food with antioxidant and anti-fatigue effects.

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