CN108059651B - Separated flavor development peptide and preparation method and application thereof - Google Patents
Separated flavor development peptide and preparation method and application thereof Download PDFInfo
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- CN108059651B CN108059651B CN201810007698.8A CN201810007698A CN108059651B CN 108059651 B CN108059651 B CN 108059651B CN 201810007698 A CN201810007698 A CN 201810007698A CN 108059651 B CN108059651 B CN 108059651B
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- taste
- cordyceps militaris
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
-
- 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
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
- A23L2/56—Flavouring or bittering agents
-
- 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
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/20—Synthetic spices, flavouring agents or condiments
-
- 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
- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
-
- 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
-
- 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
-
- 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
Abstract
The invention provides an isolated taste peptide. According to an embodiment of the invention, the taste peptide has the amino acid sequence shown in SEQ ID NO 1. The flavor development peptide provided by the embodiment of the invention has an obvious flavor development effect, can simultaneously develop delicate flavor and sweet taste, has a low threshold value and high purity, can be applied to multiple branches in the field of food, and can improve the flavor and nutritive value of food.
Description
Technical Field
The invention belongs to the technical field of food biology, and particularly relates to a separated flavor-developing peptide, and a preparation method and application thereof.
Background
The flavor development system of food is very complex, and the flavor of the food is often the result of the combined action of different flavor development substances. The flavor development substances found at present mainly comprise two main types, including sulfur-containing compounds, volatile compounds such as acid, ketone, aldehyde and ester compounds, and non-volatile compounds such as nucleotide, amino acid, organic acid, saccharide and inorganic salt ions. Edible fungi are delicious food, and at present, research on flavor development substances of the edible fungi mainly focuses on volatile compounds and flavor development nucleotides and amino acids, while few reports are made on the flavor development characteristics of small molecular peptides in the edible fungi.
The flavor-presenting peptide is a small molecule peptide capable of producing different tastes such as acid, sweet, bitter, salty, fresh and the like, and is usually prepared by enzymolysis, microbial fermentation or chemical synthesis. The flavor development characteristics are often related to amino acid composition and amino acid sequence, and different flavor development peptides can generate different taste perception and flavor development intensity; the partial flavor peptide also has the function of improving the flavor, and can improve the original flavor and enhance the thick feeling of the food when being used together with the flavor enhancer and the sour agent. Besides the flavor development effect, as a small molecular peptide, the absorption and utilization efficiency of the small molecular peptide by a human body is higher than that of macromolecular protein and free amino acid, and the nutrition characteristic is excellent; meanwhile, certain antioxidant activity is also accompanied, which is beneficial to the processing and preservation of food. Therefore, the flavor development peptide is used as a flavor development base material or an auxiliary material to be applied to the food field, and has a great prospect.
Cordyceps militaris, also known as cordyceps militaris and cordyceps militaris, is an edible fungus with delicious taste and rich nutritive value. The cultivation technique is mature, usually the cordyceps militaris strain is inoculated to a culture medium for artificial cultivation, the fruiting body is harvested after being mature, and the cordyceps militaris strain is prepared by the steps of natural airing or drying and the like. From the aspect of nutritional active ingredients, the cordyceps militaris contains various substances with physiological activities such as cordycepin, cordycepic acid, cordyceps polysaccharide, superoxide dismutase and the like; in terms of flavor substances, the content of flavor nucleotides in cordyceps militaris is high, and the ratio of umami amino acid to sweet amino acid in cordyceps militaris protein is high, so that the cordyceps militaris protein is an excellent raw material for preparing flavor peptides.
In conclusion, the preparation of the flavor peptides with excellent flavor effect by using the cordyceps militaris as a raw material and combining the modern technologies of enzymolysis, separation, purification and identification has important significance for the discovery of novel flavor peptides and the application of the flavor peptides in the field of foods.
Disclosure of Invention
The present application is based on the discovery and recognition by the inventors of the following facts and problems:
the applicant takes cordyceps militaris as a raw material, prepares the flavor peptide with very obvious flavor development effect by improving enzymolysis, separation and purification technologies, can present delicate flavor and sweet taste at the same time, has low threshold value and high purity, can be applied to a plurality of branches in the field of food, such as seasonings, leisure food, beverages and the like, and can improve the flavor of the food and the nutritional value of the food.
In a first aspect of the invention, the invention provides an isolated taste peptide. According to an embodiment of the invention, the taste peptide has the amino acid sequence shown in SEQ ID NO 1.
AYM(SEQ ID NO:1)。
The flavor development peptide provided by the embodiment of the invention has an obvious flavor development effect, can simultaneously develop delicate flavor and sweet taste, has a low threshold value and high purity, can be applied to multiple branches in the field of food, and can improve the flavor and nutritive value of food.
According to an embodiment of the invention, the above-mentioned taste peptide may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the taste peptide is derived from cordyceps militaris. Cordyceps militaris contains various physiologically active substances such as cordycepin, cordycepic acid, Cordyceps polysaccharide, superoxide dismutase, etc.; in terms of flavor substances, the content of flavor nucleotides in cordyceps militaris is high, and the ratio of umami amino acid to sweet amino acid in cordyceps militaris protein is high, so that the cordyceps militaris protein is an excellent raw material for preparing flavor peptides.
In a second aspect of the invention, the invention features an isolated nucleic acid. According to an embodiment of the invention, the nucleic acid encodes an gustducin as described above. The nucleic acid is introduced into the host cell to make the host cell express the taste peptide, so that the taste peptide has obvious taste effect, can present delicate flavor and sweet flavor at the same time, and has low threshold value.
According to an embodiment of the present invention, the above-mentioned nucleic acid may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the nucleic acid has a nucleotide sequence shown as SEQ ID NO 2-9.
GCTTATATG(SEQ ID NO：2)。
GCTTACATG(SEQ ID NO：3)。
GCCTATATG(SEQ ID NO：4)。
GCCTACATG(SEQ ID NO：5)。
GCATATATG(SEQ ID NO：6)。
GCATACATG(SEQ ID NO：7)。
GCGTACATG(SEQ ID NO：8)。
GCGTACATG(SEQ ID NO：9)。
The nucleic acid is introduced into a host cell, and the host cell efficiently expresses the taste peptide under the regulation and expression of a corresponding promoter, so that the taste peptide has obvious taste effect, can show both delicate flavor and sweet flavor, and has a low threshold value.
In a third aspect of the invention, the invention provides a construct. According to an embodiment of the invention, the construct carries a nucleic acid as described above. The nucleic acid is introduced into the host cell to make the host cell express the taste peptide, so that the taste peptide has obvious taste effect, can present delicate flavor and sweet flavor at the same time, and has low threshold value.
In a fourth aspect of the invention, a recombinant cell is provided. According to an embodiment of the invention, the recombinant cell is obtained by introducing a nucleic acid as described above or a construct as described above into a host cell. The recombinant cell according to the embodiment of the invention can express and/or secrete the taste peptide, can be used for preparing the taste peptide, has obvious taste effect, can show both umami taste and sweet taste, and has low threshold value.
It is to be noted that the term "construct" as used in the present invention refers to a genetic vector which comprises a specific nucleic acid sequence and is capable of transferring the nucleic acid sequence of interest into a host cell to obtain a recombinant cell. According to an embodiment of the present invention, the form of the construct is not particularly limited. According to an embodiment of the present invention, it may be at least one of a plasmid, a phage, an artificial chromosome, a Cosmid (Cosmid), and a virus, and is preferably a plasmid. The plasmid is used as a genetic carrier, has the characteristics of simple operation, capability of carrying larger fragments and convenience for operation and treatment. The form of the plasmid is not particularly limited, and may be a circular plasmid or a linear plasmid, and may be either single-stranded or double-stranded. The skilled person can select as desired. The term "nucleic acid" used in the present invention may be any polymer containing deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA, the length of which is not subject to any particular limitation. For constructs used to construct recombinant cells, it is preferred that the nucleic acid be DNA, as DNA is more stable and easier to manipulate than RNA.
In a fifth aspect of the invention, a method of preparing an odorant peptide is provided. According to an embodiment of the invention, the method comprises: (1) carrying out enzymolysis treatment on cordyceps militaris powder in flavourzyme and neutral protease liquid so as to obtain enzymolysis liquid; (2) carrying out ultrafiltration and first separation treatment on the enzymolysis liquid so as to obtain mixed flavor-developing peptide; and (3) subjecting the mixed taste-exhibiting peptide to a second separation treatment, which is a purification separation by reverse phase high performance liquid chromatography performed under a column of senkyo CAPCELL PAK ADME, so as to obtain the target taste-exhibiting peptide. By using the method provided by the embodiment of the invention to carry out combined enzymolysis digestion on the cordyceps militaris powder by the flavourzyme and the neutral proteinase, the obtained enzymolysis liquid has good taste; the objective flavor peptide is obtained by separating through a senkyo CAPCELL PAK ADME chromatographic column with stronger adsorption capacity to polar polypeptide, and the objective flavor peptide can be separated out efficiently. The target taste peptides obtained with the above method according to embodiments of the present invention are umami and sweet in taste, low threshold and high in purity.
According to an embodiment of the invention, the above-mentioned taste peptide may further comprise at least one of the following additional technical features:
according to the embodiment of the invention, the cordyceps militaris powder is obtained by processing cordyceps militaris as follows: drying, crushing and sieving the cordyceps militaris to obtain primary cordyceps militaris powder, mixing the primary cordyceps militaris powder with water, and boiling the mixed solution to obtain the cordyceps militaris powder. According to the embodiment of the invention, the cordyceps militaris can be subjected to primary crushing and refining treatment, so that further enzymolysis and digestion treatment are facilitated, and the primary cordyceps militaris powder is boiled with water, so that the phenomenon that the body is uncomfortable due to ingestion of raw cordyceps militaris can be avoided.
According to the embodiment of the invention, the drying is carried out at the temperature of 45-55 ℃. Further fully evaporating the water in the cordyceps militaris without losing the active ingredients in the cordyceps militaris.
According to the embodiment of the invention, the sieving is performed by sieving with a 60-100-mesh sieve. Thereby obtaining the cordyceps militaris powder with fine and uniform particles.
According to the embodiment of the invention, the ratio of the primary cordyceps militaris powder to the water is (10-20 g) to (100-200 g). Under the condition of the material-water ratio, the cordyceps militaris powder solution is not too thick or too thin, and is beneficial to subsequent enzymolysis reaction and treatment of enzymolysis liquid.
According to the embodiment of the invention, the cooking treatment time is 15-30 min. Further ensuring the cordyceps militaris to be fully cured.
According to the embodiment of the invention, the cooking treatment product is further cooled, and the temperature of the cooled product is 30-40 ℃. Thereby not destroying the activity of the flavor protease and the neutral protease which are added subsequently.
According to the embodiment of the invention, the flavourzyme and the neutral proteinase account for 0.5-2% of the mass of the cordyceps militaris powder. And the cordyceps militaris powder is completely digested by the combined enzymolysis of the flavourzyme and the neutral proteinase.
According to the embodiment of the invention, the enzymolysis treatment is carried out for 4-6 h at 45-55 ℃ and pH of 6.0-7.0. The flavor protease and the neutral protease can keep the optimal activity under the conditions of 45-55 ℃ and pH 6.0-7.0, and when the cordyceps militaris powder is subjected to enzymolysis for 4-6 hours, the thorough enzymolysis can be ensured, and excessive enzymolysis and the generation of unnecessary products can be avoided.
According to the embodiment of the invention, the method further comprises the step of carrying out enzyme inactivation and centrifugation on the enzymolysis treatment product. And then the enzymolysis supernatant is obtained, and the interference of the flavor protease and the neutral protease on the subsequent separation and purification is avoided.
According to the embodiment of the invention, the enzyme inactivation treatment is carried out in a water bath at 85-95 ℃ for 10-30 min. Further, the flavor protease and the neutral protease are completely denatured and inactivated under the above conditions.
According to the embodiment of the invention, the centrifugal treatment is carried out for 10-20 min under the condition of 4000-6000 Xg. And then cell residues after enzymolysis treatment are precipitated to the bottom of the tube to obtain clear supernatant enzymolysis liquid.
According to an embodiment of the invention, the ultrafiltration treatment is performed by means of a 5000Da ultrafiltration membrane. And then the flavor-developing micromolecular substances are retained, the non-flavor-developing macromolecular substances are removed, the impurities of column chromatography samples are reduced, and the subsequent column chromatography separation is facilitated.
According to the embodiment of the invention, before the first separation treatment, the ultrafiltration permeate is subjected to reduced pressure concentration and freeze drying treatment. And after the freeze-drying is carried out to obtain powder, the control of the sample loading concentration during column chromatography is facilitated, and sensory evaluation is also facilitated.
According to the embodiment of the invention, the first separation treatment is column chromatography separation of a freeze-dried treatment product with the concentration of 5-15 mg/mL under Sephadex G-15. The separation range for Sephadex G-15 is < 1500Da, and the probability that the molecular weight of the tastant peptide is in this range is relatively large. The volume of the water flowing out of the substances with the molecular weight of more than 1500Da in the molecular sieve is fast; substances smaller than 1500Da can enter the molecular sieve, the peak emergence is slow, and the separation effect is achieved. When the concentration of the sample is 5-15 mg/mL, the separation effect on SephadexG-15 is better. If the concentration is too high, the peaks can be overlapped and cannot be separated; if the concentration is too low, the number of column chromatography is increased, and the efficiency is reduced. According to the embodiment of the invention, the first separation treatment can further separate the substances with or without flavor, and reduce the times of column chromatography and improve the efficiency while ensuring the separation effect.
According to an embodiment of the present invention, the conditions of the second separation process are: the concentration of the mixed flavor-presenting peptide is 1-5 mg/mL; the detection wavelength was 214 nm. Further, the flavor-developing substances and the non-flavor-developing substances are separated, so that the peaks can be completely separated, and collection and mass spectrometry are facilitated.
According to an embodiment of the invention, the mixed taste peptide has a taste threshold of 0.02% to 0.025% and a taste characteristic of umami, preferably a taste threshold of 0.023%; the flavor threshold value of the target flavor peptide is 0.029% -0.033%, the flavor characteristic is fresh and sweet, and preferably, the flavor threshold value is 0.031%. And performing sensory evaluation on the first separation treatment product to screen out mixed taste peptides with lower threshold values and fresh and sweet taste characteristics, further separating the mixed taste peptides to obtain a second separation treatment product, and performing sensory evaluation on the second separation treatment product again to screen out target taste peptides with lower threshold values and fresh and sweet taste characteristics. The target taste peptide provided by the embodiment of the invention has obvious taste development effect, can simultaneously present delicate flavor and sweet taste, and has low threshold value and high purity.
According to an embodiment of the invention, further comprising performing amino acid sequence analysis on the target taste peptide by electrospray quadrupole time-of-flight mass spectrometry. Further accurately determining the amino acid sequence of the target taste peptide and eliminating interfering substances.
In a sixth aspect of the invention, a method of preparing an odorant peptide is provided. According to an embodiment of the invention, the method comprises: (1) pretreatment of raw materials: drying Cordyceps militaris at 45-55 ℃, crushing by using a traditional Chinese medicine crusher, and sieving by using a sieve of 60-100 meshes; uniformly mixing 10-20 g of cordyceps militaris powder and 100-200 g of purified water, boiling for 15-30 min, and cooling to 30-40 ℃; (2) enzymolysis: adjusting the pH value to 6.0-7.0, adding flavourzyme and neutral protease according to 0.5-2% of the mass of the cordyceps militaris powder, and stirring and carrying out enzymolysis for 4-6 h at the temperature of 45-55 ℃; after the enzymolysis is finished, inactivating the enzyme for 10-30 min in water bath at the temperature of 85-95 ℃; centrifuging at 4000-6000 Xg for 10-20 min, and taking supernatant to obtain cordyceps militaris enzymatic hydrolysate; (3) and (3) ultrafiltration separation: separating the Cordyceps militaris enzymatic hydrolysate with ultrafiltration membrane with cut-off molecular weight of 5000Da, collecting the permeate, concentrating under reduced pressure, and lyophilizing to obtain component P0; (4) and (3) column chromatography separation: redissolving the component P0 in purified water to ensure that the concentration of the component P0 is 5-15 mg/mL; using purified water as eluent, and carrying out column chromatography separation on the eluent by using Sephadex G-15; collecting the components, concentrating under reduced pressure, and freeze-drying to obtain components P1-P8; performing sensory evaluation, and screening P3 with the taste threshold value of 0.02-0.025% and the taste characteristic of fresh sweet taste; (5) and (3) reversed-phase high performance liquid chromatography purification: preparing the component P3 into a solution with the concentration of 1-5 mg/mL; eluting with 20mmol/L ammonium acetate/acetonitrile by using a Zishengtang CAPCELL PAKADME chromatographic column, detecting the wavelength at 214nm, collecting components P3-1-P3-4, concentrating under reduced pressure, and freeze-drying; carrying out sensory evaluation, and screening P3-3 with the taste threshold value of 0.029-0.033% and the fresh and sweet taste characteristic; (6) amino acid sequence analysis: the amino acid sequence of P3-3 was analyzed by electrospray quadrupole time-of-flight mass spectrometry. The target taste peptide obtained by the method provided by the embodiment of the invention has obvious taste development effect, fresh taste and sweet taste, low threshold value and high purity.
It should be noted that the preparation of the flavor peptide is not limited to the genetic engineering method or the method for purifying cordyceps militaris powder by enzymolysis, and those skilled in the art can select other processes such as chemical synthesis according to the experimental needs.
In a seventh aspect of the invention, the invention proposes the use of an aforementioned taste peptide or an aforementioned nucleic acid for the preparation of a food product. The taste-presenting peptide or nucleic acid according to the embodiments of the present invention is used in food products to improve the umami and sweet tastes of the food products and to improve the nutritional value of the food products.
In an eighth aspect of the invention, a food product is presented. According to an embodiment of the invention, the food product comprises the aforementioned taste peptide or the aforementioned nucleic acid or the taste peptide prepared according to the aforementioned method. The food provided by the embodiment of the invention has fresh and sweet flavor and high nutritional value.
According to an embodiment of the present invention, the food may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the food comprises at least one selected from the group consisting of a condiment, a snack food, a beverage.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a graph showing the results of column chromatography separation according to an embodiment of the present invention;
FIG. 2 is a graph showing the results of purification by reverse phase high performance liquid chromatography according to an embodiment of the present invention;
FIG. 3 is a graph showing the results of an electrospray quadrupole time-of-flight mass spectrometry analysis of the amino acid sequence of P3-3 according to an embodiment of the present invention;
FIG. 4 is a graph showing the results of an electrospray quadrupole time-of-flight mass spectrometry analysis of the amino acid sequence of P3-3 according to an embodiment of the present invention; and
FIG. 5 is a graph showing the results of separation of mixed flavor peptides using an Agilent Zorbax SB-C18 chromatography column according to an embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Example 1
(1) Pretreatment of raw materials: oven drying Cordyceps militaris at 50 deg.C, pulverizing with a Chinese medicinal pulverizer, and sieving with 80 mesh sieve; mixing Cordyceps militaris powder 15g and purified water 200g, decocting for 20min, and cooling to 35 deg.C;
(2) enzymolysis: adjusting pH to 6.5 with edible soda ash, adding Flavourzyme (Neutrase, from Novoxil) and neutral protease (Flavourzyme, from Novoxil) according to 1.5% of the Cordyceps militaris powder, and stirring at 50 deg.C for enzymolysis for 6 hr; after the enzymolysis is finished, inactivating the enzyme for 10min in a water bath at 95 ℃; centrifuging at 6000 Xg for 15min, and collecting supernatant to obtain Cordyceps militaris enzymatic hydrolysate;
(3) and (3) ultrafiltration separation: separating the Cordyceps militaris enzymatic hydrolysate with ultrafiltration membrane with cut-off molecular weight of 5000Da, collecting the permeate, concentrating under reduced pressure, and lyophilizing to obtain component P0;
(4) and (3) column chromatography separation: redissolving the component P0 in purified water to a concentration of 10 mg/mL; using purified water as eluent, and carrying out column chromatography separation on the eluent by using Sephadex G-15; collecting the components, concentrating under reduced pressure, and lyophilizing to obtain components P1-P8, the results are shown in figure 1; performing sensory evaluation, and screening P3 with better flavor development effect;
(5) and (3) reversed-phase high performance liquid chromatography purification: preparing the component P3 into a solution with the concentration of 3 mg/mL; eluting with 20mmol/L ammonium acetate/acetonitrile by using Zishengtang CAPCELL PAK ADME chromatographic column, detecting wavelength at 214nm, collecting components P3-1-P3-4, concentrating under reduced pressure, and lyophilizing as shown in figure 2; performing sensory evaluation, and screening P3-3 with the best flavor development effect;
(6) amino acid sequence analysis: the amino acid sequence of P3-3 was analyzed by electrospray quadrupole time-of-flight mass spectrometry, and the results are shown in FIGS. 3 and 4, and the amino acid sequence of the taste peptide was identified as Ala-Tyr-Met (A-Y-M).
Example 2
(1) Pretreatment of raw materials: oven drying Cordyceps militaris at 45 deg.C, pulverizing with a Chinese medicinal pulverizer, and sieving with 100 mesh sieve; mixing 10g Cordyceps militaris powder and 150g purified water, decocting for 15min, and cooling to 40 deg.C;
(2) enzymolysis: adjusting pH to 6.0 with edible soda ash, adding Flavourzyme (Neutrase, from Novoxil) and neutral protease (Flavourzyme, from Novoxil) according to 1% of the weight of Cordyceps militaris powder, and stirring at 55 deg.C for enzymolysis for 5 hr; after the enzymolysis is finished, inactivating the enzyme for 15min in a water bath at 90 ℃; centrifuging at 5000 Xg for 20min, and collecting supernatant to obtain Cordyceps militaris enzymolysis solution;
(3) and (3) ultrafiltration separation: separating the Cordyceps militaris enzymatic hydrolysate with ultrafiltration membrane with cut-off molecular weight of 5000Da, collecting the permeate, concentrating under reduced pressure, and lyophilizing to obtain component P0;
(4) and (3) column chromatography separation: redissolving the component P0 in purified water to a concentration of 5 mg/mL; using purified water as eluent, and carrying out column chromatography separation on the eluent by using Sephadex G-15; collecting the components, concentrating under reduced pressure, and lyophilizing to obtain components P1-P8, the results are shown in figure 1; performing sensory evaluation, and screening P3 with better flavor development effect;
(5) and (3) reversed-phase high performance liquid chromatography purification: preparing the component P3 into a solution with the concentration of 2 mg/mL; eluting with 20mmol/L ammonium acetate/acetonitrile by using Zishengtang CAPCELL PAK ADME chromatographic column, detecting wavelength at 214nm, collecting components P3-1-P3-4, concentrating under reduced pressure, and lyophilizing as shown in figure 2; performing sensory evaluation, and screening P3-3 with the best flavor development effect;
(6) amino acid sequence analysis: the amino acid sequence of P3-3 was analyzed by electrospray quadrupole time-of-flight mass spectrometry, and the results are shown in FIGS. 3 and 4, and the amino acid sequence of the taste peptide was identified as Ala-Tyr-Met (A-Y-M).
Example 3
(1) Pretreatment of raw materials: oven drying Cordyceps militaris at 55 deg.C, pulverizing with a Chinese medicinal pulverizer, and sieving with 60 mesh sieve; uniformly mixing 20g of cordyceps militaris powder and 200g of purified water, boiling for 30min, and cooling to 30 ℃;
(2) enzymolysis: adjusting pH to 7.0 with edible soda ash, adding Flavourzyme (Neutrase, from Novoxil) and neutral protease (Flavourzyme, from Novoxil) 2 wt% of Cordyceps militaris powder, stirring at 45 deg.C for enzymolysis for 6 hr; after the enzymolysis is finished, inactivating the enzyme in water bath at 85 ℃ for 30 min; centrifuging at 6000 Xg for 15min, and collecting supernatant to obtain Cordyceps militaris enzymatic hydrolysate;
(3) and (3) ultrafiltration separation: separating the Cordyceps militaris enzymatic hydrolysate with ultrafiltration membrane with cut-off molecular weight of 5000Da, collecting the permeate, concentrating under reduced pressure, and lyophilizing to obtain component P0;
(4) and (3) column chromatography separation: redissolving the component P0 in purified water to a concentration of 15 mg/mL; using purified water as eluent, and carrying out column chromatography separation on the eluent by using Sephadex G-15; collecting the components, concentrating under reduced pressure, and lyophilizing to obtain components P1-P8, the results are shown in figure 1; performing sensory evaluation, and screening P3 with better flavor development effect;
(5) and (3) reversed-phase high performance liquid chromatography purification: preparing the component P3 into a solution with the concentration of 5 mg/mL; eluting with 20mmol/L ammonium acetate/acetonitrile by using Zishengtang CAPCELL PAK ADME chromatographic column, detecting wavelength at 214nm, collecting components P3-1-P3-4, concentrating under reduced pressure, and lyophilizing as shown in figure 2; performing sensory evaluation, and screening P3-3 with the best flavor development effect;
(6) amino acid sequence analysis: the amino acid sequence of P3-3 was analyzed by electrospray quadrupole time-of-flight mass spectrometry, and the results are shown in FIGS. 3 and 4, and the amino acid sequence of the taste peptide was identified as Ala-Tyr-Met (A-Y-M).
Example 4
Sensory evaluation: the components after ultrafiltration, column chromatography and liquid chromatography were evaluated by Taste Dilution Analysis (TDA). Preparing a sample into an aqueous solution with the concentration of 5mg/mL, and mixing the aqueous solution according to the volume ratio of 1: 1, diluted by a factor of two with purified water. The sample solutions were presented to 8 sensory assessors in order of low to high concentration and the solutions at each dilution level were assessed by the triple point test. When the flavor difference between the sample solution at a certain dilution and the two blank samples (purified water) can be just identified, the dilution at that time is recorded as the dilution value (TD). TD values are the average of the results of each sensory panel, and the differences between the results should be less than or equal to a dilution level. And calculating the sample concentration (expressed by percentage content) corresponding to the TD value, namely the taste threshold value of the sample, and listing out the theoretical threshold values of monosodium glutamate, sucrose and sodium chloride for comparison. Meanwhile, each sensory assessor must describe the taste characteristics of each sample. The results are shown in Table 1:
table 1:
note: "-" indicates that the components were not measured or calculated because they had no noticeable taste.
Comparative example 1
The inventor screens the protease in the early stage, and carries out enzymolysis on cordyceps militaris powder by using different types of enzymes to obtain different cordyceps militaris enzymolysis products. Sensory evaluation sensory assessors described the taste characteristics of each sample. The results are shown in Table 2.
Table 2:
| protease type | Taste characteristics |
| Flavourzyme protease | Delicate, sweet and salty taste |
| Neutral protease | Delicate and sweet taste |
| Alkaline protease | Slightly weak sweet and bitter taste |
| Trypsin | Slightly sweet, sour and bitter |
| Papain | Weak sweet and sour taste |
| Flavourzyme + neutral protease | Has stronger delicate flavor, stronger sweet taste and salty taste |
The results of evaluation of taste characteristics in Table 2 show that: when the flavor protease and the neutral protease are used together, the taste development effect of the enzymolysis liquid is optimal. The flavor protease and the neutral protease are jointly used to obtain the enzymolysis liquid as the basic separation liquid, wherein the swelling class and the quantity of the taste peptide are rich, and the foundation is laid for the successful separation of the following taste peptide.
Comparative example 2
The inventors previously screened separation chromatography columns.
When the Agilent Zorbax SB-C18 chromatographic column is used for separating the mixed flavor peptide, the P3-3 is found to have stronger polarity and can not be retained on C18, the separation result is shown in figure 5 (P3 is eluted by 0.1% formic acid-water/0.1% formic acid-acetonitrile, the detection wavelength is 214nm), the separation effect of P3 on the C18 column is not ideal, and the main component is almost eluted within 3 min;
when the chromatographic column of the senecio CAPCELL PAK ADME is adopted, the detection result is shown in figure 2, and P3-3 is successfully separated.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> Guangdong Dongyuang pharmaceutical Co., Ltd
RUYUAN NANLING HAOSHAN HAOSHUI FOOD Co.,Ltd.
<120> separated flavor development peptide, preparation method and application thereof
<130> PIDC4170358
<160> 9
<170> PatentIn version 3.3
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<223> amino acid sequence of taste-exhibiting peptide
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Ala Tyr Met
1
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Claims (4)
1. Use of a polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 1 or a nucleic acid consisting of a nucleotide sequence shown in SEQ ID NO. 2-9 in the preparation of food, characterized in that the polypeptide is used for taste development and the nucleic acid encodes the polypeptide.
2. The polypeptide of claim 1, wherein the polypeptide is derived from cordyceps militaris.
3. The use according to claim 1, wherein the method for preparing the polypeptide comprises:
(1) pretreatment of raw materials: drying Cordyceps militaris at 45-55 ℃, crushing by using a traditional Chinese medicine crusher, and sieving by using a sieve of 60-100 meshes; uniformly mixing 10-20 g of cordyceps militaris powder and 100-200 g of purified water, boiling for 15-30 min, and cooling to 30-40 ℃;
(2) enzymolysis: adjusting the pH value to 6.0-7.0, adding flavourzyme and neutral protease according to 0.5-2% of the mass of the cordyceps militaris powder, and stirring and carrying out enzymolysis for 4-6 h at the temperature of 45-55 ℃; after the enzymolysis is finished, inactivating the enzyme for 10-30 min in water bath at the temperature of 85-95 ℃; centrifuging at 4000-6000 Xg for 10-20 min, and taking supernatant to obtain cordyceps militaris enzymatic hydrolysate;
(3) and (3) ultrafiltration separation: separating the Cordyceps militaris enzymatic hydrolysate with ultrafiltration membrane with cut-off molecular weight of 5000Da, collecting the permeate, concentrating under reduced pressure, and lyophilizing to obtain component P0;
(4) and (3) column chromatography separation: redissolving the component P0 in purified water to ensure that the concentration of the component P0 is 5-15 mg/mL; using purified water as eluent, and carrying out column chromatography separation on the eluent by using Sephadex G-15; collecting the components, concentrating under reduced pressure, and freeze-drying to obtain components P1-P8; performing sensory evaluation, and screening P3 with the taste threshold value of 0.02-0.025% and the taste characteristic of fresh sweetness;
(5) and (3) reversed-phase high performance liquid chromatography purification: preparing the component P3 into a solution with the concentration of 1-5 mg/mL; eluting by using a Zishengtang CAPCELL PAK ADME chromatographic column and 20mmol/L ammonium acetate/acetonitrile, wherein the detection wavelength is 214nm, collecting the components P3-1-P3-4, and performing reduced pressure concentration and freeze-drying; performing sensory evaluation, and screening P3-3 with the taste threshold value of 0.029-0.033% and the taste characteristic of fresh and sweet taste;
(6) amino acid sequence analysis: the amino acid sequence of P3-3 was analyzed by electrospray quadrupole time-of-flight mass spectrometry.
4. The use according to claim 1, wherein the food comprises at least one selected from the group consisting of a condiment, a snack food, and a beverage.
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| CN102090606A (en) * | 2010-09-06 | 2011-06-15 | 山东好当家海洋发展股份有限公司 | Seafood seasoner and preparation method thereof |
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| CN105734099A (en) * | 2014-12-10 | 2016-07-06 | 黑龙江众生生物工程有限公司 | Preparation method of cordyceps militaris polypeptide |
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