CN107177653B - Method for preparing blood sugar reducing peptide by utilizing needle mushroom - Google Patents
Method for preparing blood sugar reducing peptide by utilizing needle mushroom Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention discloses a method for preparing hypoglycemic peptide by utilizing needle mushroom, which comprises the following steps: mixing needle mushroom dry powder with a sodium hydroxide aqueous solution with the mass concentration of 0.4-0.6%, carrying out water bath, adjusting the pH value of a supernatant to 4 +/-0.5, and carrying out refrigerated centrifugation to obtain a precipitate; adding water and neutral protease into the precipitate, then carrying out enzymolysis reaction under a shaking condition, preserving the temperature of the obtained enzymolysis reaction product at 95-100 ℃ for 10-15 min, then naturally cooling to room temperature, and centrifuging to obtain an enzymolysis liquid; performing ultrafiltration treatment on the enzymolysis liquid to obtain ultrafiltration enzymolysis liquid; and (4) spray drying the ultrafiltration enzymolysis liquid to obtain the flammulina velutipes blood sugar reduction peptide powder.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for preparing blood sugar reducing peptide by utilizing needle mushroom.
Background
At present, diabetes becomes a chronic disease which seriously threatens human health after tumors and cardiovascular diseases, and is one of three chronic diseases which threatens human health, postprandial hyperglycemia is a main risk factor of diabetes, and an alpha-glucosidase inhibitor can reduce the digestion of carbohydrates in food and delay the generation of glucose by inhibiting the activity of alpha-glucosidase on the brush border of chorionic membranes of small intestine epithelium, and has an important role in reducing postprandial blood sugar. At present, most of alpha-glucosidase inhibitors used for clinical treatment are chemically synthesized, and have certain side effects on human body after long-term use, such as damage to liver, flatulence, intestinal dysfunction and the like.
The bioactive peptide is a multifunctional compound derived from protein, plays an important regulation role in life activities, has the functions of promoting immunity, regulating hormone, resisting bacteria and viruses, resisting oxidation, reducing blood pressure, reducing blood fat, reducing blood sugar and the like, has the characteristics of easy digestion and high safety, and is a functional factor with great development prospect. The functional peptide with nutrition and physiological activity is extracted and prepared from natural protein, and the added value of protein-rich food and agricultural products can be effectively improved.
The needle mushroom is delicious in taste, has a certain health-care function, and can improve intelligence, resist fatigue, bacteria and inflammation and the like after being eaten frequently. The needle mushroom is rich in zinc and potassium, the potassium can control the increase of blood sugar, the zinc can increase the capability of proinsulin converted into insulin, the utilization of glucose by organisms is enhanced, and the increase of blood sugar is inhibited. The needle mushroom can also inhibit blood lipid increase, reduce cholesterol, prevent and treat cardiovascular and cerebrovascular diseases of diabetes, and reduce incidence of diabetic complications.
China is a big country for producing and consuming flammulina velutipes, the yield of the flammulina velutipes is increased along with the popularization of factory production of the flammulina velutipes, but the deep processing technology of the flammulina velutipes is backward, products with high added values are lacked, and the healthy and sustainable development of the flammulina velutipes industry is not facilitated. The protein content of the flammulina velutipes is high, the amino acid content is very rich, few researches on the preparation of bioactive peptides by enzymolysis of the flammulina velutipes protein are currently carried out, and the flammulina velutipes is adopted to prepare the antibacterial peptide in some researches.
201610381719.3 the invention "A Flammulina velutipes extract and its preparation method and application" teaches a preparation method of Flammulina velutipes extract, extracting Flammulina velutipes with water, precipitating with ethanol to obtain Flammulina velutipes extract; the needle mushroom extract mainly comprises polysaccharide, protein, vitamins and minerals. In-vitro experiments prove that the flammulina velutipes extract can inhibit the activity of in-vitro alpha glycosidase. In vivo experiments show that the needle mushroom extract can obviously reduce the blood sugar of rats with type 2 diabetes, regulate blood lipid metabolism disorder, improve insulin resistance, reduce the level of serum free fatty acid, increase the storage of liver glycogen and muscle glycogen, repair damaged pancreas and have obvious effect of treating type 2 diabetes.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for preparing hypoglycemic peptide by utilizing needle mushroom with low cost and good effect.
In order to solve the technical problems, the invention provides a method for preparing hypoglycemic peptide by utilizing needle mushroom, which comprises the following steps:
1) mixing needle mushroom dry powder (capable of passing through a 60-mesh sieve) with the water content of less than or equal to 15% and sodium hydroxide aqueous solution with the mass concentration of 0.4% -0.6% according to the weight ratio of 1: mixing the materials according to the weight ratio of 11-13 (the needle mushroom dry powder is dissolved), carrying out water bath at 50-70 ℃ for 1.5-2.5 h, taking supernatant, and adjusting the pH value of the supernatant to 4 +/-0.5; freezing and centrifuging the supernatant after adjusting the pH value to obtain a precipitate (a needle mushroom protein precipitate);
2) mixing the precipitate obtained in the step 1) with water according to a weight ratio of 1: 3-5, adjusting the pH value to 7-7.5, then heating to 45-50 ℃, adding neutral protease accounting for 1% of the weight of the precipitate, and carrying out enzymolysis reaction under a shaking condition, wherein the enzymolysis temperature is 45-50 ℃, and the enzymolysis reaction time is 3-4 hours;
3) heating the enzymolysis reaction product obtained in the step 2) to 95-100 ℃ within 5-10 min, preserving the temperature for 10-15 min, then naturally cooling to room temperature, and centrifuging to obtain an enzymolysis liquid;
4) carrying out ultrafiltration treatment on the enzymolysis liquid obtained in the step 3) by adopting an ultrafiltration membrane under the working pressure of 0.1-0.2 Mpa and the working temperature of 20-45 ℃ to obtain ultrafiltration enzymolysis liquid, wherein the cutoff molecular weight of the ultrafiltration membrane is 3 KDa;
5) and spray drying the ultrafiltration enzymolysis liquid to obtain the flammulina velutipes blood sugar reduction peptide powder.
The improvement of the method for preparing the hypoglycemic peptide by utilizing the flammulina velutipes of the invention comprises the following steps: in step 2): the enzyme activity of the neutral protease is 100U/mg; the oscillation frequency is 150 to 200 r/min.
The method for preparing the hypoglycemic peptide by utilizing the flammulina velutipes is further improved as follows: the refrigerated centrifugation in the step 1) comprises the following steps: centrifuging the supernatant after the pH value is adjusted at the temperature of 3-5 ℃ at 7000-9000 r/min for 10-20 min.
The method for preparing the hypoglycemic peptide by utilizing the flammulina velutipes is further improved as follows: the centrifugation in the step 3) is as follows: centrifuging at 2500-3500 r/min for 15-25 min at 3-5 ℃, and obtaining supernatant which is the enzymolysis liquid.
The method for preparing the hypoglycemic peptide by utilizing the flammulina velutipes is further improved as follows: the spray drying of the step 5) comprises the following steps: the inlet air temperature is controlled at 170-190 deg.C (preferably 180 deg.C), and the outlet air temperature is controlled at 90-100 deg.C (preferably 95 deg.C).
The method for preparing the hypoglycemic peptide by utilizing the flammulina velutipes is further improved as follows:
in the step 1), mixing needle mushroom dry powder with a sodium hydroxide aqueous solution with the mass concentration of 0.5% according to a ratio of 1: 12, carrying out water bath at 60 ℃ for 2h, taking the supernatant, adjusting the pH value of the supernatant to 4, and centrifuging at 4 ℃ and 8000r/min for 15min to obtain a precipitate.
In the present invention, the room temperature is generally 10 to 30 ℃.
The method for preparing the functional peptide (blood sugar lowering peptide) by utilizing the needle mushroom has the following technical advantages:
1. the functional peptide of the needle mushroom prepared by the invention is obtained by biological enzyme hydrolysis, has strong effect of inhibiting the activity of alpha-glucosidase, has adjuvant therapy effect on patients with hyperglycemia, and is safe and free of toxic and side effects.
2. According to the invention, needle mushroom raw materials are converted by the technology, so that the utilization value of needle mushroom products can be effectively improved, and the method has important significance for continuously promoting agricultural quality improvement and efficiency improvement.
3. The method obtains the optimal preparation method through the selection of the optimal preparation process conditions, and selects the peptide segment with excellent activity to the maximum extent through the ultrafiltration membrane treatment technology, thereby effectively enhancing the content of the active peptide.
4. The product of the invention can be used as a medicine, a health food, a food additive, a drug synergist and the like, and has scientific and reasonable process, simple operation and stronger industrial implementation.
The flammulina velutipes hypoglycemic peptide is orally taken, and the dosage is about 4-5 g/day.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
The dried powder of needle mushroom in the following cases has a water content of less than or equal to 15% and can pass through a 60-mesh sieve.
Example 1, a method for preparing hypoglycemic peptide using needle mushroom, sequentially comprising the following steps:
1) weighing 2kg of needle mushroom dry powder, adding 24kg of 0.5% sodium hydroxide aqueous solution, mixing, extracting at 60 ℃ for 2h, taking supernatant, adjusting the pH value of the supernatant to 4.0 by using 1moL/L HCl, freezing at 8000r/min (4 ℃), centrifuging for 15min to obtain 0.5kg of precipitate,
2) adding 1.5kg of water into the precipitate obtained in the step 1), uniformly stirring, adjusting the pH value to 7.0 by using 0.1moL/L sodium hydroxide, heating to 45 ℃, adding 5.0g of neutral protease (100U/mg), and carrying out enzymolysis reaction at 45 ℃ for 3 hours under a shaking condition (180 r/min);
3) heating the enzymolysis reaction product obtained in the step 2) to 95-100 ℃ within 10min, keeping the temperature for 15min, naturally cooling to room temperature, and centrifuging for 20min by adopting a J-6M type large-capacity refrigerated centrifuge (BECKMAN company) at the rotating speed of 3000r/min and at the temperature of 4 ℃ to obtain supernatant, namely the enzymolysis liquid.
4) And carrying out ultrafiltration treatment on the enzymolysis liquid by adopting an ultrafiltration membrane under the working pressure of 0.1-0.2 Mpa and the working temperature of 20-45 ℃ to obtain ultrafiltration enzymolysis liquid, wherein the cutoff molecular weight of the ultrafiltration membrane is 3 KDa.
5) And spray drying the ultrafiltration enzymolysis liquid (the technological parameters of spray drying are as follows: controlling the air inlet temperature: 180 ℃, air outlet temperature control: 95 ℃ to obtain the blood sugar reducing peptide of the flammulina velutipes.
The blood sugar lowering peptide powder of needle mushroom has yellowish color. The peptide powder was prepared into a solution of 3mg/mL, and the inhibitory activity against a-glucosidase was measured to be 60.2%.
Remarks explanation: the method for detecting the inhibitory activity of the alpha-glucosidase is as follows (the conventional technology):
50. mu.L of an enzymolysis peptide solution (prepared with 0.1mol/L phosphate buffer solution having a pH of 6.9) and 100. mu.L of a 10mg/mL α -glucosidase solution (prepared with 0.1mol/L phosphate buffer solution having a pH of 6.9) were added to an ELISA plate, mixed, and then stirred at 25 ℃ to prepare a mixtureThe mixture was left for 10min, 50. mu.L of a 5mmol/L PNPG (p-nitrophenyl- α -D-glucopyranoside) solution (prepared with 0.1mol/L phosphate buffer pH 6.9) was added, and after incubation at 37 ℃ for 30min, 50. mu.L of 0.67mol/L Na was added2CO3The reaction was stopped and absorbance at 405nm was measured. This system is called a sample.
The following 3 systems were set simultaneously: control, sample blank, and control blank.
Comparison: 50 μ L of the enzymatically hydrolyzed peptide solution was replaced with 50 μ L0.1mol/L phosphate buffer pH 6.9.
Blank sample: 100. mu.L of 0.1mol/L phosphate buffer pH 6.9 was used instead of 100. mu.L of 10mg/mL alpha-glucosidase solution;
control blank: the peptide solution and the α -glucosidase solution were replaced with 0.1mol/L of phosphate buffer at pH 6.9 corresponding to the respective volumes, and the inhibition ratios were calculated as follows.
Embodiment 2, a method for preparing hypoglycemic peptide by using needle mushroom, which comprises the following steps:
1) weighing 5kg of needle mushroom dry powder, adding 60kg of 0.5% sodium hydroxide aqueous solution, mixing, extracting at 60 ℃ for 2h, taking supernatant, adjusting the pH value of the supernatant to 4.0 by using 1moL/L HCl, freezing at 8000r/min (4 ℃) and centrifuging for 15min to obtain 1.3kg of precipitate;
2) adding 6.5kg of water into the precipitate obtained in the step 1), uniformly stirring, adjusting the pH value to 7.5 by using 0.1moL/L of sodium hydroxide, heating to 50 ℃, adding 13.0g of neutral protease (100U/mg), and carrying out enzymolysis reaction at 50 ℃ for 4 hours under a shaking condition (180 r/min);
the subsequent steps were the same as in example 1.
The obtained blood sugar lowering peptide powder of needle mushroom has yellowish color. The solution was prepared in a concentration of 3mg/mL, and the inhibitory activity against a-glucosidase was found to be 62.1%.
Comparative example 1, 5.0g of neutral protease (100U/mg) in step 2) of example 1 was changed to 5g of acidic protease (50U/mg), and pH was adjusted to 7.0, correspondingly to 4, and the same as in example 1 was repeated. The results obtained were:
the peptide powder was prepared into a 3mg/mL solution, and the inhibitory activity on a-glucosidase was measured to be 9.8%.
Comparative example 2, the step 1) of example 1 was omitted, namely, the supernatant was directly subjected to 8000r/min refrigerated centrifugation for 15min, while adjusting the pH of the supernatant to 4.0 with 1moL/L HCl;
the rest is equivalent to embodiment 1.
The results obtained were: after centrifugation, no precipitate is generated basically, and subsequent corresponding enzymolysis experiments cannot be carried out.
Comparative example 3-1, the weight of the aqueous sodium hydroxide solution in step 1) of example 1 was changed from 24kg to 30 kg; the rest is equivalent to embodiment 1.
The amount of precipitate obtained in step 1) was 0.46 kg.
Comparative example 3-2, the weight of the aqueous sodium hydroxide solution in step 1) of example 1 was changed from 24kg to 15 kg; the rest is equivalent to embodiment 1.
The amount of precipitate obtained in step 1) was 0.43 kg.
Comparative example 4, 5.0g of neutral protease (100U/mg) in step 2) of example 1 was changed to acid protease and papain (1: 1)10g, the pH value is correspondingly adjusted to 6 instead of adjusting the pH value to 7.0, and the enzymolysis temperature is changed to 58.62 ℃ at 45 ℃. The rest was equivalent to example 1.
The peptide powder was prepared into a 3mg/mL solution, and the inhibitory activity on a-glucosidase was measured to be 12.6%.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (5)
1. The method for preparing the hypoglycemic peptide by utilizing the flammulina velutipes is characterized by comprising the following steps of:
1) mixing needle mushroom dry powder with the water content less than or equal to 15% and sodium hydroxide aqueous solution with the mass concentration of 0.5% according to the weight ratio of 1: 12, carrying out water bath at 60 ℃ for 2h, taking supernatant, and adjusting the pH value of the supernatant to 4.0; freezing and centrifuging the supernatant after the pH value is adjusted to obtain a precipitate;
2) mixing the precipitate obtained in the step 1) with water according to a weight ratio of 1: 3-5, adjusting the pH value to 7-7.5, then heating to 45-50 ℃, adding neutral protease accounting for 1% of the weight of the precipitate, and carrying out enzymolysis reaction under a shaking condition, wherein the enzymolysis temperature is 45-50 ℃, and the enzymolysis reaction time is 3-4 hours;
the enzyme activity of the neutral protease is 100U/mg;
3) heating the enzymolysis reaction product obtained in the step 2) to 95-100 ℃ within 10min, preserving the temperature for 15min, then naturally cooling to room temperature, and centrifuging to obtain an enzymolysis liquid;
4) carrying out ultrafiltration treatment on the enzymolysis liquid obtained in the step 3) by adopting an ultrafiltration membrane under the working pressure of 0.1-0.2 Mpa and the working temperature of 20-45 ℃ to obtain ultrafiltration enzymolysis liquid, wherein the cutoff molecular weight of the ultrafiltration membrane is 3 KDa;
5) and spray drying the ultrafiltration enzymolysis liquid to obtain the flammulina velutipes blood sugar reduction peptide powder.
2. The method for preparing hypoglycemic peptide using enoki mushroom according to claim 1, wherein the step 2) comprises: the oscillation frequency is 180 r/min.
3. The method for preparing hypoglycemic peptide using enoki mushroom according to claim 2, wherein the step 1) of freeze-centrifugation is: the supernatant after pH adjustment was centrifuged at 8000r/min at 4 ℃ for 15 min.
4. The method for preparing hypoglycemic peptide using enoki mushroom according to claim 3, wherein the centrifugation of the step 3) is: centrifuging at 4 deg.C at 3000r/min for 20min to obtain supernatant as enzymolysis solution.
5. The method for preparing hypoglycemic peptide using enoki mushroom according to claim 4, wherein the spray drying of the step 5) is: the air inlet temperature is controlled at 180 ℃, and the air outlet temperature is controlled at 95 ℃.
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| CN105768112A (en) * | 2016-03-30 | 2016-07-20 | 福建农林大学 | Preparation method of edible and medicinal fungi protein peptide-zinc chelate |
| CN105779549A (en) * | 2016-05-20 | 2016-07-20 | 浙江省农业科学院 | Method for preparing functional peptide from royal jelly |
| CN105821104A (en) * | 2016-03-30 | 2016-08-03 | 福建农林大学 | Preparation method of edible fungus protein peptide and calcium chelate |
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| CN105768112A (en) * | 2016-03-30 | 2016-07-20 | 福建农林大学 | Preparation method of edible and medicinal fungi protein peptide-zinc chelate |
| CN105821104A (en) * | 2016-03-30 | 2016-08-03 | 福建农林大学 | Preparation method of edible fungus protein peptide and calcium chelate |
| CN105779549A (en) * | 2016-05-20 | 2016-07-20 | 浙江省农业科学院 | Method for preparing functional peptide from royal jelly |
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| "金针菇菇脚蛋白质的提取";朱丽萍等;《武汉工程大学学报》;20150615;第37卷(第6期);第21-26页 * |
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