A dandelion compound peptide, its preparation method and uses

Abstract

The present invention relates to the field of traditional Chinese medicine, and relates to a dandelion compound peptide, a preparation method and uses thereof, comprising the following steps: (1) taking dry dandelion products, pulverizing, adding water, and performing steaming to obtain a liquid mixture; (2) then adding cellulase to the liquid mixture, maintaining the temperature, and stirring for enzymatic hydrolysis; then lowering the temperature, adding neutral protease, maintaining the temperature, and stirring for enzymatic hydrolysis; raising the temperature to inactivate the enzyme to obtain an enzymatic hydrolysate; (3) cooling the enzymatic hydrolysate, adding Bacillus licheniformis for fermentation, maintaining the temperature, and stirring for fermentation, and then raising the temperature for sterilization to obtain a fermentation broth; the present invention can extract active substances from metabolites of traditional Chinese medicine active substances, and the product is non-toxic.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and particularly relates to a dandelion compound peptide, a preparation method thereof and uses thereof. Background Art

[0002] Dandelion, with a sweet, slightly bitter and cold taste, belongs to the liver and stomach meridians, and has the effects of diuresis, laxation, jaundice reduction, choleresis, etc. Dandelion can treat heat toxins, carbuncles, sores, internal abscesses, red and swollen eyes, damp heat, jaundice, painful and difficult urination, furuncles and carbuncles, breast abscess, scrofula, toothache, red eyes, sore throat, lung abscess, intestinal abscess, damp heat jaundice, painful and difficult stranguria, acute mastitis, lymphadenitis, scrofula, furuncle and carbuncle, acute conjunctivitis, common cold and fever, acute tonsillitis, acute bronchitis, gastritis, hepatitis, cholecystitis, urinary tract infection, etc. Dandelion can be eaten raw, stir-fried or made into soup, and is a plant that can be used both as medicine and food. The dandelion plant contains vitamin A, taraxasterol, taraxacin, choline, organic acids, inulin, vitamin C and potassium, and also contains various healthy nutritional components such as iron, calcium, vitamin B2, vitamin B1, magnesium, vitamin B6, folic acid and copper. The specific element content of dandelion is mainly water. Every 60 grams of raw dandelion leaves contain 86% water, 1.6 grams of protein, 5.3 grams of carbohydrates, and the calories are about 108.8 kJ.

[0003] Conventional methods of consuming dandelion mainly include soaking in water, steaming and boiling for drinking or pulverizing for consumption. Modern extraction techniques have a history of thousands of years. The main method is to "separate and screen the effective components of traditional Chinese medicine", and solvent extraction is adopted, which is divided into water extraction, extraction with hydrophilic organic solvents (such as ethanol, methanol, etc.), and extraction with lipophilic organic solvents (chloroform, ether, etc.). Academician Wang Kui of the Chinese Academy of Sciences proposed: "Many conventional extracts of traditional Chinese medicine are actually prodrugs of the direct acting substances, and the metabolism and transformation of drugs in the body are everywhere; the activity and toxicity of traditional Chinese medicine are often manifested by the metabolites; the interaction between the components in traditional Chinese medicine compound is related to metabolism." Professor Yang Xiuwei of the School of Pharmacy of Peking University took the lead in carrying out the "research on the biotransformation of traditional Chinese medicine components by human intestinal bacteria suitable for the characteristics of traditional Chinese medicine" in China since 1995, established a systematic research platform for the absorption, distribution, metabolism, excretion, toxicity and efficacy (ADMET / Act.) of traditional Chinese medicine, and carried out the key basic scientific issues in the experimental medicine research of traditional Chinese medicine - determining the effective and toxic chemical components of traditional Chinese medicine from the biotransformation by intestinal bacteria.

[0004] The bilayer lipid structure of the biological membrane is the main obstacle for drugs to enter cells, while peptides are an ideal method to break through the biological membrane barrier and transport substances to cells. Small peptides can internalize into living cells, reach the cytoplasm and organelles, and have pharmacological activities. Swedish scientist Langere called small peptides 'cell-penetrating peptides'. Academician Sun Manji of the Chinese Academy of Military Medical Sciences and the Chinese Academy of Sciences proposed that when there are malfunctions in cells, macromolecular substances such as proteins and nucleic acids with repair capabilities are difficult to enter cells and reach effective concentrations, while cell-penetrating peptides can effectively transport other substances into cells and have biological functions themselves.

[0005] However, current conventional extraction methods of dandelion have ignored the extraction of small peptide active ingredients and the metabolism of toxic substances in dandelion. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to provide a dandelion compound peptide and its preparation method and uses. The preparation method of the dandelion compound peptide changes the traditional Chinese medicine solvent extraction method, and the obtained dandelion compound peptide product can achieve the perfect combination of the compound peptide and the active substances in dandelion, and reduce the antagonistic phenomenon of dandelion by metabolizing the allergens and toxic substances in dandelion, making the product safer.

[0007] To this end, the present invention provides the following technical solutions:

[0008] A preparation method of a dandelion compound peptide, comprising the following steps:

[0009] (1) Take dry dandelion, crush it, add water, and carry out steaming to obtain a liquid mixture.

[0010] (2) Cool the liquid mixture, then add cellulase, maintain the temperature, and stir for enzymatic hydrolysis; then cool down, add neutral protease, maintain the temperature, and stir for enzymatic hydrolysis; raise the temperature to inactivate the enzyme to obtain an enzymatic hydrolysate.

[0011] (3) Cool the enzymatic hydrolysate, add Bacillus licheniformis for fermentation, maintain the temperature, stir for fermentation, and then raise the temperature for sterilization to obtain a fermentation broth.

[0012] Optionally, in step (1), 10 - 12 times the weight of purified water of the dandelion raw material is added and stirred evenly, and steamed at 90°C - 95°C for 3 - 3.5 h to obtain a liquid mixture.

[0013] Optionally, in step (2), cool the feed liquid mixture to 56 - 58 °C, add the cellulase in an amount of 0.75 - 1.0% of the weight of the dandelion raw material, maintain the temperature at 56 - 58 °C, and stir for enzymatic hydrolysis for 30 - 45 min; the enzyme activity of the cellulase is 35 U / mg;

[0014] And / or, in step (2), then cool to 55 - 57 °C, add the neutral protease in an amount of 1 - 1.5% of the weight of the dandelion raw material, maintain the temperature at 55 - 57 °C, and stir for enzymatic hydrolysis for 3.0 - 4.0 h; the enzyme activity of the neutral protease is 30 U / mg;

[0015] And / or, in step (2), the stirring speed during the stirring enzymatic hydrolysis is 40 - 50 r / min.

[0016] Optionally, in step (3), the addition amount of Bacillus licheniformis is 0.05 - 0.07% of the weight of the dandelion raw material, and the viable count of Bacillus licheniformis is 3000×10 8 -4000×10 8 cfu / g;

[0017] And / or, the stirring speed of the stirring fermentation is 35 - 45 r / min.

[0018] Optionally, and / or, in step (3), cool the enzymatic hydrolysate to 35 °C - 37 °C;

[0019] And / or, in step (3), stir and ferment for 5 - 5.5 h;

[0020] And / or, in step (3), the conditions for heating and sterilization are heating to 90 °C - 95 °C for inactivation for 10 - 15 min.

[0021] Optionally, the following steps are further included:

[0022] (4), Cool the fermentation broth, adjust the pH to acidic, input purified air while stirring, carry out biosynthesis, and purify and concentrate the obtained synthesis broth.

[0023] Optionally, in step (4), cool the fermentation broth to 38 - 39 °C, adjust the pH to 3 - 3.5, convey purified air while stirring, and carry out biosynthesis for 1.5 - 2.0 h;

[0024] And / or, in the purification step, use a three - phase centrifuge for centrifugal purification, maintain the rotation speed at 16000 - 18000 r / min until the purification is completed.

[0025] Optionally, in step (4), use citric acid to adjust the pH;

[0026] And / or, the stirring speed in the step of conveying and purifying air while stirring is 22 - 24 r / min.

[0027] A dandelion compound peptide prepared by the preparation method of the dandelion compound peptide described above.

[0028] Use of the dandelion compound peptide described above in the preparation of feed additives, nutritional formula foods, health products, foods for special medical purposes or drugs.

[0029] The technical solution of the present invention has the following advantages:

[0030] 1. A preparation method of a dandelion compound peptide provided by the present invention includes the following steps: (1) Take dry dandelion, crush it, add water, and perform steaming to obtain a liquid mixture; (2) Cool down the liquid mixture, then add cellulase, maintain the temperature, and stir for enzymatic hydrolysis; then cool down, add neutral protease, maintain the temperature, and stir for enzymatic hydrolysis; raise the temperature to inactivate the enzyme to obtain an enzymatic hydrolysate; (3) Cool down the enzymatic hydrolysate, add Bacillus licheniformis for fermentation, maintain the temperature, and stir for fermentation, then raise the temperature for sterilization to obtain a fermentation broth; in the above method, first, cellulase is used to degrade cellulose in the liquid mixture to create conditions for improving the yield and the product; then neutral protease is added to control the molecular weight of the compound peptide and improve the product yield; then fermentation with Bacillus licheniformis is carried out, which can metabolize the allergens and toxic substances in dandelion, making the product safer in quality and easier to be absorbed and utilized by the animal body, fully meeting the requirements of food, health product, and drug raw materials. In summary, the present invention changes the way of separating and screening active substances from traditional Chinese medicine by the previous simple traditional Chinese medicine separation and screening technology, adopts a new Chinese medicine biological transformation method of enzyme-bacteria cooperation, can completely degrade and separate Chinese medicine active substances, and through fermentation, reshapes Chinese medicine compound peptides and other active substances, so that active substances can be extracted from the metabolites of Chinese medicine active substances. Not only small molecule peptides are obtained, but also active substances such as taraxasterol, taraxacin, and choline are retained. The animal body is easy to absorb and utilize, the yield is higher, and the toxic substances and allergens in Chinese medicine can be completely degraded and separated, and the product is non-toxic.

[0031] Furthermore, the above method has a short production cycle, low cost, does not produce any toxic and harmful substances, does not use organic solvents such as alcohol and acetone, is safe, has no toxic side effects, its product has a good taste and no peculiar smell, and can be widely applied in the fields of food, health products, drugs, etc.

[0032] Furthermore, the reaction conditions of the above method are mild, the operation is simple, further improving the safety and utilization value of the product.

[0033] Furthermore, the dandelion compound peptide obtained by the above method can be completely dissolved in water, which can be more conveniently added to foods, solid beverages, and liquid drinks, and is more suitable for use as a medicine.

[0034] 2. A method for preparing a dandelion compound peptide provided by the present invention further includes the following steps: (4) Cooling the fermentation broth, adjusting the pH to acidic, inputting purified air while stirring, and performing biosynthesis. The obtained synthesis solution is purified and concentrated; biosynthesis is carried out by utilizing the chelation effect of small peptides at the most suitable temperature and pH value for the gastrointestinal tract, so that this product can quickly enter cells after being eaten and participate in life activities faster. Detailed implementation manners

[0035] The following embodiments are provided to better further understand the present invention. They are not limited to the best implementation manner, and do not constitute a limitation to the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other existing technologies falls within the protection scope of the present invention.

[0036] For those not specifying specific experimental steps or conditions in the embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments not indicating the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0037] In the following embodiments:

[0038] Bacillus licheniformis is provided by Danone Company of France, and its activity is 3000×10 8 -4000×10 8 cfu / g.

[0039] Cellulase is provided by Danisco Company, and its enzyme activity is 35 u / mg;

[0040] Neutral protease is provided by Danisco Company, and its enzyme activity is 30 U / mg;

[0041] Example 1

[0042] Weigh 1000 g of dry dandelion, crush it, add 10000 ml of purified water, stir evenly, cook at 90 °C for 3.0 h to obtain a liquid mixture; then cool the liquid mixture to 56 °C, add 7.5 g of cellulase, keep the temperature at 56 °C, the stirrer speed at 40 r / min, and stir and enzymolyze for 30 min; then cool to 55 °C, add 10 g of neutral protease respectively, keep the temperature at 55 °C, the stirrer speed at 40 r / min, and stir and enzymolyze for 3.0 h, and then filter with a plate and frame filter press; heat the filtrate to 90 °C to inactivate the enzyme for 10 min, then cool the temperature to 35 °C, add 0.5 g of Bacillus licheniformis for fermentation, keep the stirrer speed at 35 r / min and the temperature at 35 °C, and ferment for 5.0 h; then heat to 90 °C to sterilize for 10 min; then lower the temperature to 38 °C, adjust the pH value to 3.0 with citric acid, control the stirrer at 22 r / min, evenly convey purified air, and carry out biosynthesis for 1.5 h; centrifuge and purify the synthesis liquid with a three-phase centrifuge, with the speed maintained at 16000 r / min; further concentrate the purified liquid with a double-effect concentrator; spray-dry the concentrated liquid to obtain the dandelion compound peptide product.

[0043] Example 2

[0044] Weigh 100 kg of dry dandelion, crush it, add 1100 L of purified water, stir evenly, cook at 92 °C for 3.2 h to obtain a liquid mixture; then cool the liquid mixture to 57 °C, add 800 g of cellulase, keep the temperature at 57 °C, the stirrer speed at 45 r / min, and stir and enzymolyze for 40 min; then cool to 56 °C, add 1.2 kg of neutral protease respectively, keep the temperature at 56 °C, the stirrer speed at 45 r / min, and stir and enzymolyze for 3.5 h, and then filter with a plate and frame filter press; heat the filtrate to 92 °C to inactivate the enzyme for 12 min, then cool the temperature to 36 °C, add 60 g of Bacillus licheniformis for fermentation, keep the stirrer speed at 40 r / min and the temperature at 36 °C, and ferment for 5.3 h; then heat to 92 °C to inactivate for 12 min; lower the temperature to 38.5 °C, adjust the PH value to 3.2 with citric acid, control the stirrer at 23 r / min, evenly convey purified air, and carry out biosynthesis for 1.8 h; centrifuge and purify the synthesis liquid with a three-phase centrifuge, with the speed maintained at 17000 r / min; further concentrate the purified liquid with a double-effect concentrator; spray-dry the concentrated liquid to obtain the new traditional Chinese medicine dandelion compound peptide product.

[0045] Example 3

[0046] Weigh 500 kg of dry dandelion, crush it, add 6000 L of purified water and stir evenly. Under the condition of 95 °C, cook for 3.5 h to obtain a liquid mixture; then cool the liquid mixture to 58 °C, add 5 kg of cellulase, keep the temperature at 58 °C, keep the stirrer speed at 50 r / min, and stir and enzymolyze for 45 min; then cool to 57 °C, add 7.5 kg of neutral protease respectively, keep the temperature at 57 °C, keep the stirrer speed at 50 r / min, and stir and enzymolyze for 4.0 h, and then filter with a plate and frame filter press; heat the filtrate to 95 °C to inactivate the enzyme for 15 min, then cool the temperature to 37 °C, add 350 g of Bacillus licheniformis for fermentation, keep the stirrer speed at 45 r / min, keep the temperature at 37 °C, and ferment for 5.5 h; then heat to 95 °C to inactivate for 15 min; lower the temperature to 39 °C, adjust the pH value to 3.5 with citric acid, control the stirrer at 24 r / min, evenly transport purified air, and carry out biosynthesis for 2.0 h; centrifuge and purify the synthesis liquid with a three-phase centrifuge, and keep the speed at 18000 r / min; further concentrate the purified liquid with a double-effect concentrator; spray-dry the concentrated liquid to obtain a new traditional Chinese medicine dandelion compound peptide product.

[0047] Example 4

[0048] The difference between this example and Example 1 is that the biosynthesis step is omitted, that is, the step of "then lower the temperature to 38 °C, adjust the pH value to 3.0 with citric acid, control the stirrer at 22 r / min, evenly transport purified air, and carry out biosynthesis for 1.5 h" is omitted, and the sterilized fermentation broth is directly centrifuged, purified, concentrated, and dried.

[0049] Comparative Example 1

[0050] The difference between this comparative example and Example 1 is that the step of adding Bacillus licheniformis for fermentation is omitted. The filtrate is directly cooled to 38 °C, the pH value is adjusted to 3.0 with citric acid, the stirrer is controlled at 22 r / min, evenly transport purified air, and carry out biosynthesis for 1.5 h; the synthesis liquid is centrifuged and purified with a three-phase centrifuge, and the speed is kept at 16000 r / min; the purified liquid is further concentrated with a double-effect concentrator; the concentrated liquid is spray-dried to obtain a dandelion compound peptide product.

[0051] Experimental Example 1

[0052] The molecular weight and the content of 2 - 5 peptides of the active peptides in the dandelion compound peptides obtained in the examples and comparative examples were investigated according to the national standard GB / T22492. The absorption rate detection method of the active peptides adopted the conventional animal intestinal absorption experiment method. The peptide content in the intestinal tract before and after culturing in the test sample was detected, and the absorption rate of the active peptides was calculated. The isolated small intestine (duodenum) of SD rats used for research was taken, and after conventional cleaning (Tyrode's solution) treatment, the small intestine was turned inside out so that the intestinal mucosa faced outward, and the intestinal contents were removed. Then, the dandelion compound peptides prepared in the examples and comparative examples were diluted with Tyrode's solution to a mass concentration of 0.5%. The small intestine was placed in a beaker containing the above - diluted solution (37°C), and air was filled into the intestine. After culturing at a constant temperature of 37°C for 1 hour, the polypeptide concentration in the diluted solution was detected. According to the polypeptide concentration before and after culturing, the absorption rate of the active peptides was calculated as follows: Absorption rate = (Polypeptide concentration before culture - Polypeptide concentration after culture) / Polypeptide concentration before culture × 100%.

[0053] The results are shown in the following table:

[0054] Table 1

[0055]

[0056] Experimental Example 2 Safety Detection

[0057] The allergic reaction probability was detected using the dandelion compound peptides obtained in the examples and comparative examples. The method was as follows:

[0058] Healthy guinea pigs (weighing 300 - 400 g), with an equal number of males and females, were randomly divided into 6 groups, with 20 in each group. The negative control group was given normal saline, the positive control group was given ovalbumin diluted with normal saline to 0.15 g / L, and the test groups were respectively given the dandelion compound peptides of Examples 1 - 3 and Comparative Example 1 (diluted with normal saline to a concentration of 0.2 g / L) by intraperitoneal injection, once every other day, for a total of 3 times, with a volume of 1 ml per animal. On the 14th and 21st days after the last injection, a volume of 2 ml per animal was given. Whether the animals showed allergic reactions was observed within 30 minutes after injection (the symptoms of allergic reactions were: 0 - normal, 1 - restlessness, 2 - erection, 3 - tremor, 4 - nose scratching, 5 - sneezing, 6 - coughing, 7 - rapid breathing, 8 - urination, 9 - defecation, 10 - lacrimation, 11 - dyspnea, 12 - snoring, 13 - purpura, 14 - unsteady gait, 15 - jumping, 16 - wheezing, 17 - spasm, 18 - rotation, 19 - Cheyne - Stokes respiration, 20 - death. The evaluation criteria were: 0 was negative for allergic reaction (-), 1 - 4 symptoms were weakly positive for allergic reaction (+), 5 - 10 symptoms were positive for allergic reaction (++), 11 - 19 symptoms were strongly positive for allergic reaction (+++), 20 was extremely strongly positive for allergic reaction (++++). The allergic reaction rate was calculated as: Number of animals with allergic reactions / Total number of animals.

[0059] The results are shown in the following table. It can be seen from the table that the probability of physical allergic reactions to the dandelion compound peptide in Examples 1-3 is significantly lower than that in Comparative Example 1, indicating that the dandelion compound peptide prepared by the present invention contains extremely low or no traditional Chinese medicine toxic substances and allergens, and the product is safe and non-toxic.

[0060] Table 2

[0061] Sample Constitutional allergic reaction rate Allergic reaction classification Negative control group 0％ Negative allergic reaction Positive control group 100％ Extremely strong positive allergic reaction Example 1 0％ Negative allergic reaction Example 2 0％ Negative allergic reaction Example 3 0％ Negative allergic reaction Comparative example 1 5％ Weak positive allergic reaction

[0062] Efficacy of Experimental Example 3

[0063] This experimental example examines the effects of the dandelion compound peptide obtained in Example 1, and the method of the 2001 edition of the "Chinese Pharmacopoeia" is used for the examination.

[0064] The results are as follows: The dandelion active peptide can improve cellular immunity, enhance the immune function of the body, and has obvious effects on acute mastitis, acute conjunctivitis, acute tonsillitis, and acute bronchitis; at the same time, it has good curative effects on gastritis, hepatitis, cholecystitis, and urinary tract infections. Compared with the dandelion extract, the effect is increased by more than five times, and the consumption amount is reduced by 80%.

[0065] Obviously, the above examples are only examples given for clear illustration and are not limitations on the implementation modes. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation modes here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for preparing a dandelion composite peptide, characterized in that: The steps include: (1) Take dried dandelion, crush it, add purified water 10-12 times the weight of the dandelion raw material, stir evenly, and cook at 90°C-95°C for 3-3.5 hours to obtain a feed-liquid mixture; (2) Then the feed-liquid mixture is cooled to 56-58°C, cellulase is added in an amount of 0.75-1.0% of the weight of the dandelion raw material, the temperature is maintained at 56-58°C, and the enzymolysis is stirred for 30-45 minutes; then the mixture is cooled to 55-57°C, neutral protease is added in an amount of 1-1.5% of the weight of the dandelion raw material, the temperature is maintained at 55-57°C, and the enzymolysis is stirred for 3.0-4.0 hours; the temperature is raised to inactivate the enzyme to obtain an enzymolysis solution; the enzymatic activity of the cellulase is 35U / mg; the enzymatic activity of the neutral protease is 30U / mg; the stirring speed during the stirring enzymolysis is 40-50r / min; (3) Cooling the enzymatic hydrolysate to 35°C-37°C, adding Bacillus licheniformis for fermentation, wherein the amount of Bacillus licheniformis added is 0.05-0.07% of the weight of the dandelion raw material, and the number of live bacteria of Bacillus licheniformis is 3000×10 8 -4000×10 8 cfu / g, maintain the temperature, stir and ferment for 5-5.5h, the stirring speed of the stirring fermentation is 35-45r / min, and then heat and sterilize, the condition of the temperature and sterilization is to heat to 90℃-95℃ for inactivation for 10-15min to obtain the fermentation liquid.

2. The method for preparing the dandelion composite peptide according to claim 1, characterized in that: The following steps are also included: (4) Cooling the fermentation liquid, adjusting the pH to acidic, introducing purified air while stirring, and performing biosynthesis. The resulting synthetic liquid is purified and concentrated.

3. The method for preparing the dandelion composite peptide according to claim 2, characterized in that: In step (4), the fermentation liquid is cooled to 38-39° C., the pH is adjusted to 3-3.5, and purified air is delivered while stirring to carry out biosynthesis for 1.5-2.0 hours; And / or, in the purification step, a three-phase centrifuge is used for centrifugal purification, and the rotation speed is maintained at 16000-18000r / min until the purification is completed.

4. The method for preparing the dandelion composite peptide according to claim 3, characterized in that: In step (4), citric acid is used to adjust the pH; And / or, the stirring speed in the step of conveying purified air while stirring is 22-24 r / min.

5. Use of the dandelion composite peptide prepared by the preparation method of the dandelion composite peptide according to any one of claims 1 to 4 in the preparation of feed additives or medicines.

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