CN107779490B - Method for preparing bitter peptides by pepsin and application of bitter peptides - Google Patents
Method for preparing bitter peptides by pepsin and application of bitter peptides 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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- 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
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Abstract
The invention discloses a method for preparing bitter peptides by pepsin, which comprises the following steps: preparing a supernatant; enzyme hydrolysis to prepare bitter peptide crude liquid; preparing saturated hyaluronic acid; crude bitter peptide concentrates were prepared by non-newtonian fluid separation. The method has good effect of separating or removing bitter peptides, and is simple and easy to operate; the separated bitter peptides can be used for treating asthma and cardiovascular diseases.
Description
Technical Field
The invention belongs to the field of deep processing of aquatic products, and particularly relates to a method for preparing bitter peptides by pepsin and application of the bitter peptides.
Background
Because bitter taste is generally related to toxicity and drug property, uncomfortable mouthfeel of the bitter taste is avoided, but the bitter taste is also one of the most important elements for the long-term evolution and survival adaptation mode of human beings. The bitter taste of the food product is mainly derived from the hydrophobic amino acid groups exposed by the proteolytic peptides. Although bitter taste is not easily accepted in foods, it has been widely used in medicine, for example, many bitter peptides have angiotensin enzyme inhibitory effect. In addition, bitter receptors exist in tongue bud cells and lung trachea and lung bronchial epithelial cells, and recently, it is found that food-borne bitter molecules can become bitter receptor agonists of the lung trachea and bronchial epithelial cells, so that the bitter receptors can be possibly used for preparing anti-asthma medicines. The solubility, thermal stability and nutritional characteristics of fish protein are greatly improved after the fish protein is hydrolyzed by enzyme, however, bitter peptides are formed due to the exposure of hydrophobic amino acids and the change of spatial structure, so that the isolation of the bitter peptides or the removal of the bitter peptides is frequently required in the production of food or functional food or medicines. Although many methods for separating or removing bitter taste exist at present, many side effects are generally brought in, for example, the activated carbon is added into the enzymatic hydrolysate for selective separation, which is used at the earliest, and special odor of the activated carbon is brought in. The non-conventional method requires expensive instruments and cumbersome procedures, thereby limiting the commercialization of the food or functional food.
Hyaluronic acid is a food additive, and has been found to have many new biological functions in recent years. For example, hyaluronic acid has been found to be a ligand of the transmembrane receptor of CD44 in animal cells, regulates the activities of many normal cells, but is also involved in the infiltration and metastasis of cancer cells, and at the same time, is used for targeted and sustained delivery of drugs due to the high expression of hyaluronidase by cancer cells. Hyaluronic acid is a macromolecular polysaccharide substance widely existing in bacteria to mammalian cells, and the molecular weight of the hyaluronic acid varies from thousands to millions, but the hyaluronic acid has no antigenicity to human bodies. The physiological function and application characteristics of hyaluronic acid are closely related to molecular parameters such as molecular weight and molecular weight distribution. In addition, the high water locking property enables hyaluronic acid to show non-Newtonian fluid and viscoelastic behaviors, the in vivo and in vitro rheology is also being widely researched, a plurality of new applications are being developed, and the hyaluronic acid is widely used for drug slow-release delivery and degradation of hyaluronidase which is quite highly expressed by cancer cells. In recent years, the production by fermentation of hyaluronic acid has been widely used in the fields of foods, cosmetics, and the like. Early preparations of hyaluronic acid were from rooster combs, or some organs of animals, and hyaluronic acid is now entering the food field due to its fermentative production. Although the physical properties of hyaluronic acid are not well understood, many magical effects and functions of hyaluronic acid are being discovered in food research and manufacture. The non-newtonian fluid properties of hyaluronic acid are now widely used in the study of osteoarthritis and targeted drug delivery to cancer cells, however the physical properties of non-newtonian fluids of hyaluronic acid in vitro and the mechanism of physicochemical action with proteins/peptides are still unclear. In addition to the molecular weight of hyaluronic acid, its temperature, concentration and certain mediator molecules all affect its viscosity and rheological properties, and thus affect the enrichment or elimination of bitter peptides.
Therefore, there is a need to develop a more advantageous method for screening bitter peptides.
Disclosure of Invention
The invention aims to provide a method for preparing bitter peptides by pepsin, which solves the problems.
The technical scheme of the invention is as follows:
a method for preparing bitter peptides by pepsin, which comprises the following steps:
(1) preparing a supernatant fluid: cleaning large yellow croaker, then descaling, cleaning with deionized water, cutting the large yellow croaker into fish blocks, pulping the fish blocks and buffer solution in a high-speed tissue triturator, centrifuging, and heating supernatant until endogenous enzyme is inactivated for later use;
(2) preparing bitter peptide crude liquid by enzyme hydrolysis: adding pepsin with the mass concentration of 10g/L into the supernatant, mixing and stirring uniformly, placing the mixture in a water bath kettle, adjusting the pH to 3.5, carrying out enzymolysis and cooling, adjusting the pH back to 7.0, fixing the volume, centrifuging, obtaining the supernatant which is the bitter peptide crude liquid, and taking the bitter peptide crude liquid for later use.
(3) Preparing saturated hyaluronic acid: adding hyaluronic acid sodium salt into a vessel filled with sterile water, fully stirring to form transparent viscous liquid, and covering a preservative film on the mouth of the vessel.
(4) Preparation of crude bitter peptide concentrate by non-newtonian fluid separation: adding the bitter peptide crude liquid into a container, then adding a transparent viscous liquid with the same volume as the bitter peptide crude liquid, wherein the upper layer of the solution is the transparent viscous liquid, the lower layer of the solution is the bitter peptide crude liquid, centrifuging or standing the solution in a centrifuge tube to collect components, determining the component positions of the bitter peptides through sensory evaluation, and collecting the bitter peptide components to obtain the bitter peptide crude concentrated solution.
Further, the fish blocks in the step (1) are 100 g.
Further, the mass ratio of the fish blocks to the buffer solution in the step (1) is 1: 5, and the buffer solution is 0.1 × PBS.
Further, the beating time in the step (1) is 5-10min, the rotation speed of the centrifugation is 6000r/min, the time is 5min, the heating time of the supernatant is 5min, and the temperature is 90 ℃.
Further, in the step (2), the temperature of the water bath in the water bath kettle is 50-60 ℃, the enzymolysis time is 4 hours, the temperature is raised to 95 ℃ after the enzymolysis, the enzyme is inactivated for 15min, and the centrifugal rotating speed is 12000r/min, and the time is 10 min.
Further, the pH in step (2) was adjusted by 1M HCl.
Further, after the step (2) is completed, sensory evaluation is also carried out on the bitter peptide crude liquid, and the sensory evaluation is carried out in the same manner as the sensory evaluation in the step (4): the standard curve of bitter peptides is established by 6-9 sensory evaluators, namely, the high, medium and low bitter concentrations are sequentially judged by a single blind method, and then the sensory bitter intensity of 5-100mg caffeine is sequentially tested to form the bitter standard curve.
Further, the transparent viscous liquid in the step (3) contains hyaluronic acid with a mass concentration of 20 g/L.
Further, the rotation speed of the centrifugation in the step (4) is 15000r/min, the temperature is 4 ℃, and the time is 30 min.
The bitter peptide prepared by the method can be used for preparing food for treating asthma.
The invention has the advantages that:
(1) the separation or removal effect of the bitter peptides is good, and the method is simple and easy to operate;
(2) the separated bitter peptides can be used for treating asthma and cardiovascular diseases.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein the content of the first and second substances,
fig. 1 is a graph of sensory evaluation criteria prepared from Mocca coffee;
FIG. 2 is a diagram showing how the bitter peptides generated by the hydrolysis of large yellow croaker protein by different enzymes are analyzed;
FIG. 3 is a Malvern rheometer analysis chart and an electron microscope chart;
FIG. 4 is a graph of bitter peptide diffusion from a non-Newtonian fluid vehicle of hyaluronic acid under shear and non-shear conditions as analyzed by sensory evaluation;
FIG. 5 is a graph of the Bradford method analyzing the spreading of bitter peptides in a 2% hyaluronic acid non-Newtonian fluid medium with and without shear;
FIG. 6 is an SDS-PAGE electrophoretic analysis chart of the tube protein collected under the action of non-Newtonian fluid shearing force and non-shearing force of hyaluronic acid.
Detailed Description
The invention provides a method for preparing bitter peptides by pepsin, which comprises the following steps:
(1) establishing a bitter taste standard;
(2) preparing a supernatant;
(3) enzyme hydrolysis to prepare bitter peptide crude liquid;
(4) preparing saturated hyaluronic acid;
(5) crude bitter peptide concentrates were prepared by non-newtonian fluid separation.
The present invention will be described in further detail with reference to specific embodiments in order to make the above objects, features and advantages more apparent and understandable.
A method for preparing bitter peptides by pepsin, which comprises the following steps:
the method comprises the following steps: establishing a bitter taste standard;
in one embodiment, this step may be specifically performed as follows: according to a national standard GB/T12315-2008 sensory analysis methodology sorting method, 6-9 sensory evaluators participate in the establishment of a standard curve of bitter peptides, namely, a single-blind method is used for sequentially judging the concentrations of high, medium and low bitter, and then the sensory bitter concentration (strength) of 5-100mg (caffeine) is sequentially tested to form a bitter standard curve of the self (stored in a brain without type);
step two: cleaning large yellow croaker, then descaling, cleaning with deionized water, cutting the large yellow croaker into fish blocks, pulping the fish blocks and buffer solution in a high-speed tissue triturator, centrifuging, and heating supernatant until endogenous enzyme is inactivated for later use;
in one embodiment, the method can be specifically implemented by washing large yellow croaker in Zhoushan, descaling, and carrying out enzymolysis under the conditions that deionized water is used for washing, cutting into blocks, weighing 100g, adding 0.1 × PBS buffer solution according to the mass ratio of the fish blocks to the buffer solution of 1: 5, pulping on a DS-1 high-speed tissue triturator for 5-10min, centrifuging at 6000r/min for 5min, taking supernatant, heating to 90 ℃ for 5min, and inactivating endogenous enzymes;
step three: preparing bitter peptide crude liquid by enzyme hydrolysis: adding pepsin with the mass concentration of 10g/L into the supernatant, mixing and stirring uniformly, placing the mixture in a water bath kettle, adjusting the pH to 3.5, carrying out enzymolysis and cooling, adjusting the pH back to 7.0, fixing the volume, centrifuging, obtaining the supernatant which is the bitter peptide crude liquid, and taking the bitter peptide crude liquid for later use.
In one embodiment, this step may be specifically performed as follows: adding pepsin 1% (W/V) into the supernatant, mixing, stirring, placing in 50-60 deg.C water bath (optimum temperature according to specific enzyme), adjusting pH to 3.5 with 1M HCl before enzymolysis, and performing enzymolysis for 4 hr; heating to 95 deg.C, inactivating enzyme for 15min, cooling, adjusting pH to 7.0, diluting to desired volume, centrifuging at 12000r/min for 10min, and collecting supernatant as bitter peptide crude solution. Sampling and sensory evaluation were performed at the designed time points during hydrolysis.
Step four: preparing saturated hyaluronic acid: adding hyaluronic acid sodium salt into a vessel filled with sterile water, fully stirring to form transparent viscous liquid, and covering a preservative film on the mouth of the vessel.
In one embodiment, this step may be specifically performed as follows: weighing required food-grade high-molecular (million-level) sodium hyaluronate to required sterile water, fully stirring until the transparent viscous liquid is 2% (W/V), wherein the pH value of natural sodium hyaluronate is 7.0, and in order to avoid influence on enzymatic hydrolysis peptide, covering a preservative film all the time, keeping the natural pH value and keeping a transparent bubble-free state.
Step five: preparation of crude bitter peptide concentrate by non-newtonian fluid separation: adding the bitter peptide crude liquid into a container, then adding a transparent viscous liquid with the same volume as the bitter peptide crude liquid, wherein the upper layer of the solution is the transparent viscous liquid, the lower layer of the solution is the bitter peptide crude liquid, centrifuging or standing the solution in a centrifuge tube to collect components, determining the component positions of the bitter peptides through sensory evaluation, and collecting the bitter peptide components to obtain the bitter peptide crude concentrated solution.
In one embodiment, this step may be specifically performed as follows: the bitter peptides are collected by natural sedimentation or centrifugation of the components by utilizing the hydrophobic groups of the bitter peptides, the highly hydrophilic property of hyaluronic acid and the non-Newtonian fluid property. The bottom layer is bitter peptide crude liquid with good sensory evaluation, the upper layer is 2% hyaluronic acid with the same volume as the bitter peptide crude liquid, and viscous molecules slowly diffuse due to the non-Newtonian fluid property of the hyaluronic acid under the shearing conditions of centrifugation or gravity and the like. The diffusing molecular interactions hydrophilic molecules enter the fluid phase, while the non-hydrophilic bitter peptides remain in the aqueous phase. The difference in molecular diffusion was observed after centrifugation at 15000RPM, 4 ℃ for 30 min. Collecting components by centrifugal tube, determining component position of bitter peptide by sensory evaluation, and collecting bitter peptide component. The molecular weight was further confirmed by Bradford protein quantitation method and SDS-PAGE.
The bitter taste peptide primarily purified by the method can be used for asthma food therapy.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are further described below. The invention is not limited to the embodiments listed but also comprises any other known variations within the scope of the invention as claimed.
First, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The present invention is described in detail by using the schematic structural diagrams, etc., and for convenience of illustration, the schematic diagrams are not enlarged partially according to the general scale when describing the embodiments of the present invention, and the schematic diagrams are only examples, which should not limit the scope of the present invention. In addition, the actual fabrication process should include three-dimensional space of length, width and depth.
Please refer to fig. 1 to 6 for the performance test of the above steps one to five:
please refer to fig. 1 for a standard bitter curve obtained in step one, fig. 1 is a graph of sensory evaluation standard curve prepared from Mocca coffee, and an error bar indicates the standard error. The current methods of sensory evaluation remain the most common and effective methods due to the lack of a true instrument to measure bitterness. According to the method of Hyung Joo Suh and the like, the sensory evaluation of the coffee is that the concentrations of the coffee prepared according to the actual caffeine content are respectively as follows: 0, 0.025%, 0.05%, 0.1%, 0.2%, 03%, etc.; the corresponding score and bitterness were: 1-10 (0: no bitter taste). According to this scoring criterion, the taster tasted the bitter taste of the protein hydrolysate and scored in comparison with the criterion, and the final average value was used to represent the degree of bitter taste. As shown in FIG. 1, sensory concentration was lower than actual concentration (R) due to individual difference of participators on sensory standard curve20.9851), which may be errors due to solubility, and also systematic errors due to sensory insensitivity.
Although the debittering of the enzymatic hydrolysis peptide of soybean protein has been well established, the enzymatic hydrolysis and the debittering of fish protein, especially of large yellow croaker protein in Zhoushan have not been reported. For this purpose, we use 5 proteases to hydrolyze large yellow croaker protein, wherein the crude liquid of bitter taste peptide prepared by enzymatic hydrolysis in step three is obtained by hydrolyzing large yellow croaker protein with pepsin, please refer to fig. 2, and fig. 2 is an analysis diagram of bitter taste peptide generated by different enzymes hydrolyzing large yellow croaker protein. As shown in fig. 2, pepsin produced a very high bitter taste (peptides), essentially 5 times that of trypsin, which allows for the hyaluronic acid to separate the bitter taste peptides. Experiments also show that the addition of flavor enzymes produces substantially no bitter taste, while flavor enzymes produce a strong flavor, which results in a product with a much more moderate bitter taste, which is beneficial for animal feeding, or which improves the taste of the food, and therefore, in the method of the invention, pepsin is selected for the isolation of the highly bitter peptides.
The 2% hyaluronic acid is loaded on the upper part of the protein peptide liquid hydrolyzed by the enzyme, moves downwards due to the small difference of specific gravity caused by the water locking capacity of the 2% hyaluronic acid, and due to the viscosity and the non-Newtonian fluid property, the 2% hyaluronic acid can move to the bottom after being centrifuged for 30 minutes or standing for 7-10 minutes, and meanwhile, the enzymolysis liquid at the bottom is pushed to float upwards, so the enzymolysis liquid moves upwards and also enters the non-Newtonian fluid, and the inertia of the Newtonian fluid and the repulsion of hyaluronic acid to non-hydrophilic molecules enable a certain amount of hydrophobin/peptide to move upwards. This was demonstrated on the one hand by sensory evaluation, followed by Bradford analysis. Experiments show that 2% of hyaluronic acid migrates as non-Newtonian fluid, translocation of hyaluronic acid from top to bottom is completed under 10min centrifugation (6000rpm, 4 ℃), and Newtonian fluid (bitter peptide) is subjected to position displacement with limited diffusion of fluid (2% hyaluronic acid). Whereas 0.5% of hyaluronic acid migrates as a newtonian fluid, translocation of hyaluronic acid from top to bottom is accomplished under 30min centrifugation (6000rpm, 4 ℃), with limited diffusion of the fluid, but the newtonian fluid (bitter peptides) is simply mixed with 2% hyaluronic acid. Since 2% hyaluronic acid is approximately heavier than water, both centrifugation and non-centrifugation result in translocation of hyaluronic acid from top to bottom. The process drives the hydrophilic peptide (pushing out the hydrophobic peptide) to drive the movement of the hydrophobic peptide in a lipid raft (lipid raft) like motion mode. Recent studies have shown that the degree of polymerization of hyaluronic acid molecules greatly affects the cell state, and hyaluronic acid in the outer layer of cells such as cancer cells, atherosclerotic plaque cells, and rheumatoid arthritis is often degraded by hyaluronidase or oxidized. Meanwhile, the receptor CD44 glycoprotein molecule of hyaluronic acid is highly expressed in lymphocytes and cancer cells. Lipid rafts on animal cell membranes are composed of hyaluronic acid and certain specific regions rich in cholesterol and glycoside lipids that carry G proteins, or other membrane factors, translocate, typically from inside to outside, in 15-30 minutes. Although there is no quantitative fluid tracking, the properties of hyaluronic acid as an in vivo, in vitro non-newtonian fluid should remain substantially the same, with integrity, and with the ability to diffuse small molecules of the non-newtonian fluid. Whereas 0.5% hyaluronic acid is mainly characterized by molecular diffusion and still exhibits substantially newtonian fluid characteristics. Although centrifugation slightly increased molecular aggregation, it was not very different from the resting tube. Therefore, 2% hyaluronic acid was selected in step four.
The results of the preparation of picopeptide semen by non-newtonian fluid separation in step five are shown in fig. 3, and fig. 3 is a marvin rheometer analysis diagram and an electron microscope diagram. As shown in fig. 3A, the bitter peptides change viscosity with different proportions of 2% hyaluronic acid under flow (shear) change by malvern rheometer analysis, as shown in fig. 3B, which is present predominantly as nanoscale particles under scanning electron microscopy.
The enzymatic hydrolysate was added to the bottom, but experimental results show that after centrifugation or standing the bitter peptides can move from the bottom through the hyaluronic acid layer to the top. The bitter peptide is mainly composed of hydrophobic amino acids exposed by the hydrolyzed oligopeptide or has a specific steric structure such as a proline residue is important to the bitter taste of the peptide, so that when the gravity of hyaluronic acid moves from the upper portion to the bottom portion, the negatively charged carboxyl group of hyaluronic acid and a part of basic amino acids or basic peptide form an ionic bond to move to the bottom portion, and the hydrophilic amino acid/peptide is adsorbed by the hyaluronic acid colloid, resulting in the hydrophobic peptide moving to the upper layer, i.e., the hydrophobic amino acid having the bitter taste, or the bitter peptide moving to the upper portion. Referring to FIG. 4, FIG. 4 is a graph of bitter peptide diffusion in a non-Newtonian fluid vehicle of hyaluronic acid under shear and non-shear conditions as analyzed by sensory evaluation. As can be seen from fig. 4, to observe the effect of newtonian or non-newtonian fluids on enzymatic hydrolysate protein/peptide, the protein content of the collected 10 fractions was displayed using coo horse brilliant blue (Bradford method). Referring to FIG. 5, FIG. 5 is a graph of the Bradford method analysis of bitter peptide diffusion in a 2% hyaluronic acid non-Newtonian fluid vehicle with and without shear forces, collected from top to bottom under tube numbers 1-10 for protein/peptide analysis and sensory evaluation.
Referring to FIG. 6, FIG. 6 is a SDS-PAGE electrophoretic analysis of the tube protein under the non-Newtonian shear force and non-shear force of the non-Newtonian fluid of hyaluronic acid. As shown in FIG. 6, since SDS-PAGE is a very sensitive analysis technique, SDS-PAGE shows that enzymatically hydrolyzed protein/peptide moves from the bottom to the upper layer by the action of non-Newtonian fluid, and part of it remains in the middle and lower part of the tube, so that molecular diffusion still plays a role. Also substantially consistent with Bradford analysis and sensory evaluation results. Of course, the diffusion effect of hyaluronic acid, the inertia and inertia of non-Newtonian fluids, and the shear and shear-free effects of many non-protein factors such as hyaluronic acid itself cannot be displayed by SDS-PAGE. Bands 1-5, 2% hyaluronic acid (centrifuged) were from collection tube 1 (top), 3, 5, 7, 9 (bottom), and bands 6-10, 2% hyaluronic acid (not centrifuged) were from collection tube 1, 3, 5, 7, 9.
Example one
The embodiment shows that the preparation method of the bitter peptide by pepsin comprises the following steps:
washing large yellow croaker in Zhoushan, descaling, wherein the enzymolysis conditions comprise that after being washed by deionized water, the large yellow croaker is cut into pieces, weighed to be 100g, 0.1 × PBS buffer solution is added according to the protein mass ratio of 1: 5, the mixture is beaten on a DS-1 high-speed tissue triturator for 5min, and centrifuged at 6000r/min for 5min, supernatant is taken and heated to 90 ℃ and 5min to inactivate endogenous enzymes, 1 percent (W/V) of required pepsin is respectively added into the supernatant, the mixture is uniformly stirred, the mixture is placed in a 50-60 ℃ water bath kettle (according to the optimal temperature of the specific enzymes), before the enzymolysis process, the pH is adjusted to 3.5 by 1M of HCl, enzymolysis is carried out for 4h, the temperature is increased to 95 ℃, enzyme is inactivated for 15min, the cooling is carried out, the pH value is adjusted to 7.0, the volume is fixed, the supernatant is centrifuged at 12000r/min, the supernatant is taken to carry out enzymatic hydrolysis process of bitter peptide.
Weighing required food-grade high-molecular (million-level) sodium hyaluronate into sterile water, fully stirring until transparent viscous liquid (2%) is obtained, wherein the pH value of natural sodium hyaluronate is 7.0, and in order to avoid influence on enzymatic hydrolysis peptide, the natural pH value is kept by plastic film covering, and the transparent non-bubble state is kept.
The bitter peptide is collected by utilizing the hydrophobic groups of the bitter peptide, the high hydrophilic property and the non-Newtonian fluid property of hyaluronic acid, natural sedimentation or centrifugation and components. The bottom layer plus the organoleptically evaluated enzymatically hydrolyzed bitter peptides and the top layer plus an equal volume of 2% hyaluronic acid, due to its non-newtonian fluid properties, centrifugation, or gravity all contribute to slow diffusion under the action of viscous molecules under shear conditions. The diffusing molecular interactions hydrophilic molecules enter the fluid phase, while the non-hydrophilic bitter peptides remain in the aqueous phase. The molecular diffusion difference is observed after centrifugation at 15000r/min, 4 ℃ and 30 min. Collecting components in the centrifugal tube after centrifugation or standing, then determining the component position of the bitter peptide by sensory evaluation, and collecting the bitter peptide component.
In conclusion, the invention discloses a method for preparing bitter peptides by pepsin, and the research shows that the pepsin hydrolyzes large yellow croaker protein to generate the most obvious bitter taste. 2% of hyaluronic acid itself is found in the bitter taste peptide (enzymolysis liquid) enriched by hyaluronic acid, and the characteristic of non-Newtonian fluid is kept in the enzymolysis liquid with the same volume, namely, shear force inertia and viscosity enable the protein/peptide to diffuse slowly, but hyaluronic acid also passes through the enzymolysis liquid under the action of centrifugation and gravity, the apparent viscosity of the liquid is increased due to the increase of resistance caused by the viscous stress of the non-Newtonian fluid, the molecular diffusion is avoided when the enzymolysis liquid moves to the upper part, certain protein enrichment is formed, as shown in figure 5, but at the same time, hydrophobic bitter taste peptide is enriched on the upper part of the tube due to the hydrophilic action and the repulsion of hydrophobic groups, as shown in figure 4. In contrast, 0.5% hyaluronic acid exhibits mainly rapid diffusion in admixture with the enzymatic hydrolysate, i.e. newtonian fluid characteristics with stress proportional to strain rate. Since the complex diffusion coefficient of protein/peptide in non-Newtonian fluid is beyond the scope of the present study, it can not be analyzed formulaically, but the movement of protein and bitter peptide shows the possibility of simple and rapid separation of bitter peptide, which provides the initial condition for the application of hyaluronic acid in food industry. In addition, although hyaluronic acid translocates in vitro quite differently from cell membrane lipid rafts, its non-newtonian fluid properties are similar to those of hyaluronic acid in vivo, i.e. the motile character of the molecule promotes the movement of both self and interacting molecules. Compared with a standing test tube, experiments show that the separation method of the bitter peptides which are hydrophilic and repel hydrophobic of hyaluronic acid does not need centrifugation, and the bitter peptides can be eliminated by a gravity (shear force) moving path according to the hydrophilic and hydrophobic characteristics, so that the aim of enriching the bitter peptides is effectively fulfilled. From the above experimental results, further tests, such as analysis of molecular weight to change the water locking effect, further change the fluid characteristics, the column diameter ratio of hyaluronic acid and enzymatic hydrolysate, etc., may increase the migration and enrichment of bitter peptides. Therefore, the method may become the basis of the optimal process conditions for isolating, or removing, the bitter peptides.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (8)
1. A method for preparing bitter peptides by pepsin, which is characterized by comprising the following steps:
(1) preparing a supernatant fluid: cleaning large yellow croaker, then descaling, cleaning with deionized water, cutting the large yellow croaker into fish blocks, pulping the fish blocks and buffer solution in a high-speed tissue triturator, centrifuging, and heating supernatant until endogenous enzyme is inactivated for later use;
(2) preparing bitter peptide crude liquid by enzyme hydrolysis: adding pepsin with the mass concentration of 10g/L into the supernatant, mixing and stirring uniformly, placing the mixture in a water bath kettle, adjusting the pH to 3.5, carrying out enzymolysis and cooling, adjusting the pH back to 7.0, fixing the volume, and centrifuging to obtain a supernatant which is the bitter peptide crude liquid, and taking the bitter peptide crude liquid for later use;
(3) preparing saturated hyaluronic acid: adding hyaluronic acid sodium salt into a vessel filled with sterile water, fully stirring to form transparent viscous liquid, and covering a preservative film on the mouth of the vessel, wherein the transparent viscous liquid contains hyaluronic acid with the mass concentration of 20 g/L;
(4) preparation of crude bitter peptide concentrate by non-newtonian fluid separation: adding the bitter peptide crude liquid into a container, adding the transparent viscous liquid in the step (3) with the same volume as the bitter peptide crude liquid, wherein the upper layer of the solution is the transparent viscous liquid, the lower layer of the solution is the bitter peptide crude liquid, centrifuging or standing the solution in a centrifuge tube to collect components, determining the component positions of the bitter peptides through sensory evaluation, and collecting the bitter peptide components to obtain the bitter peptide crude concentrated solution.
2. The process for the preparation of bitter peptides with pepsin according to claim 1, characterized in that: the fish blocks in the step (1) are 100 g.
3. The method for preparing bitter taste peptide by pepsin according to claim 1, wherein the mass ratio of the fish pieces to the buffer solution in step (1) is 1: 5, and the buffer solution is 0.1 × PBS.
4. The process for the preparation of bitter peptides with pepsin according to claim 1, characterized in that: beating time in the step (1) is 5-10min, the rotation speed of centrifugation is 6000r/min, the time is 5min, the heating time of the supernatant is 5min, and the temperature is 90 ℃.
5. The process for the preparation of bitter peptides with pepsin according to claim 1, characterized in that: and (3) in the step (2), the temperature of the water bath in the water bath kettle is 50-60 ℃, the enzymolysis time is 4 hours, the temperature is raised to 95 ℃ after the enzymolysis, the enzyme is deactivated for 15min, and the centrifugal rotating speed is 12000r/min and the time is 10 min.
6. The process for the preparation of bitter peptides with pepsin according to claim 1, characterized in that: the pH in step (2) was adjusted by 1M HCl.
7. The process for the preparation of bitter peptides with pepsin according to claim 1, characterized in that: after the step (2) is finished, sensory evaluation is also carried out on the bitter peptide crude liquid, and the sensory evaluation is carried out in the same way as the sensory evaluation in the step (4): the standard curve of bitter peptides is established by 6-9 sensory evaluators, namely, the high, medium and low bitter concentrations are sequentially judged by a single blind method, and then the sensory bitter intensity of 5-100mg caffeine is sequentially tested to form the bitter standard curve.
8. The process for the preparation of bitter peptides with pepsin according to claim 1, characterized in that: and (4) centrifuging at the rotating speed of 15000r/min at the temperature of 4 ℃ for 30 min.
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