CN111802507B - Method for removing bitter walnut peptide by using sugar alcohol liposome precursor - Google Patents
Method for removing bitter walnut peptide by using sugar alcohol liposome precursor Download PDFInfo
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- CN111802507B CN111802507B CN202010714515.3A CN202010714515A CN111802507B CN 111802507 B CN111802507 B CN 111802507B CN 202010714515 A CN202010714515 A CN 202010714515A CN 111802507 B CN111802507 B CN 111802507B
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- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
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Abstract
The invention discloses a method for removing bitter walnut peptide by using sugar alcohol liposome precursor, belonging to the technical field of food processing and safety, comprising the following steps: mixing materials, forming a film, mixing a protective agent and walnut peptide in Phosphate Buffered Saline (PBS), hydrating a lipid film, carrying out ultrasonic treatment, pre-freezing and freezing; the method utilizes an embedding method to carry out debitterizing walnut peptide by embedding a liposome precursor, and obtains a walnut peptide product with extremely low bitterness by adjusting the conditions of the mass ratio of the liposome precursor to the walnut peptide, the pH value, the carrier type and the like; because the sugar alcohol liposome precursor has a porous structure, bitter amino acid can be embedded well, so that a good debittering effect is achieved, the sugar alcohol liposome precursor does not interact with a peptide chain, and the loss of functional peptide is avoided; the method is simple and easy to implement, low in cost, excellent in debitterizing effect and high in application value.
Description
Technical Field
The invention relates to the technical field of food processing and safety, in particular to a method for removing bitter walnut peptide by using a sugar alcohol liposome precursor.
Background
The walnut peptide is a micromolecular substance extracted from walnut protein by utilizing a biological enzymolysis technology, and is rich in 18 amino acids. Because of its characteristics of scavenging free radicals and resisting oxidation, it is widely used in the fields of biomedicine, functional food, cosmetics, etc. However, in the application of the food field, the walnut peptide generated in the enzymolysis process of the walnut protein generally has bitter taste, which is probably caused by exposure of hydrophobic amino acid contained in the protein to molecules in the enzymolysis process of the walnut protein into polypeptide. As the degree of hydrolysis increases, the more hydrophobic amino acids are exposed, the more bitter taste increases. The bitter taste affects the quality and taste of the product, and the application of the walnut peptide in the field of food processing is severely limited. Therefore, the removal of the bitter taste generated by walnut protein hydrolysis is particularly important in food processing, so that the walnut protein hydrolysate needs to be subjected to debittering treatment.
For controlling the bitter taste of walnut peptide, there are 6 methods: selective separation, organic solvent extraction, protein modification, microbial fermentation, enzyme debittering and covering. The 6 methods for debitterizing the walnut peptides can reduce the bitter taste of the walnut peptides, but can greatly reduce the nutritional value of walnut hydrolysate and even influence the taste of the product. Therefore, it is very necessary to develop a new and efficient debittering method.
Disclosure of Invention
The invention aims to provide a method for utilizing sugar alcohol liposome precursor to remove bitter walnut peptide, so as to solve the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a method for utilizing sugar alcohol liposome precursor to remove bitter walnut peptide, which comprises the following steps:
(1) adding absolute ethyl alcohol into the mixture of the soybean lecithin, the cholesterol and the tween-80 to uniformly disperse the absolute ethyl alcohol to obtain a mixed solution;
(2) carrying out rotary evaporation on the mixed solution obtained in the step (1) until a transparent lipid film is formed;
(3) adding the freeze-drying protective agent and the walnut peptide into Phosphate Buffered Saline (PBS);
(4) pouring the Phosphate Buffer Solution (PBS) in the step (3) into the container in the step (2) to hydrate the lipid membrane;
(5) carrying out ultrasonic treatment on the hydrated lipid membrane in the step (4) to obtain liposome precursor suspension;
(6) pre-freezing the liposome precursor suspension in the step (5), and then freezing and drying to obtain the sugar alcohol liposome precursor debitterized walnut peptide freeze-dried powder.
Further, in the step (1), the mass ratio of the soybean lecithin to the cholesterol to the tween-80 is 4-6:1: 3-5.
Further, in the step (1), the mass ratio of the soybean lecithin to the cholesterol to the tween-80 is 5:1: 4.
Further, in the step (1), the soybean lecithin is 400mg, the cholesterol is 80mg and the Tween is 320 mg.
Further, in the step (3), the lyoprotectant is one of sucrose, trehalose and mannitol.
Further, in the step (3), the walnut peptide comprises phospholipid and walnut peptide in a mass ratio of 10: 1.
Further, in the step (3), the phosphate buffered saline PBS has a pH of 4-10.
Further, in step (3), the phosphate buffered saline PBS has a pH of 7.4.
Further, in the step (5), the ultrasonic treatment is ultrasonic for 1s, stopping for 1s, and circulating for 15 min.
Further, in the step (6), the pre-freezing is carried out for 12 hours at the temperature of minus 80 ℃, and the freezing dryer is carried out for 48 hours.
The invention adopts the sugar alcohol liposome precursor to embed the walnut peptide, and the liposome precursor is a bilayer microsphere similar to a biological membrane, so the invention has amphipathy, good biocompatibility and no toxicity. Therefore, the bitter amino acids of the walnut peptide can be well embedded, so that a good debittering effect is achieved, the cost is low, and the nutritional value of the walnut peptide is not influenced.
Compared with the prior art, the method has the beneficial effects that:
1. the walnut peptide used in the invention is a food source, and can be safely and effectively applied to the field of foods.
2. The method for preparing the liposome precursor has low cost, short time consumption and simple and easy operation.
3. The method utilizes a liposome precursor embedding method to debitterize the walnut peptide, has better effect compared with the conventional method, and has no influence on the structural texture and functionality of the walnut peptide.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described 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 without inventive exercise.
Fig. 1 is an atomic force image of the walnut peptide-embedded algal glycolipid plastid precursor prepared in example 1 observed under an atomic force microscope;
FIG. 2 is an IR spectrum of the algal glycolipid plastid precursor embedded with walnut peptide prepared in example 1.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but rather as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present disclosure, it is understood that each intervening value, to the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
A method for utilizing sugar alcohol liposome precursor debitterized walnut peptide comprises the following steps:
(1) accurately weighing 400mg of soybean lecithin, 80mg of cholesterol and 320mg of tween in a 100mL beaker, pouring 30mL of absolute ethanol solution, and putting the beaker into an ultrasonic cleaning instrument for 10 minutes to uniformly disperse the solution to obtain a mixed solution;
(2) pouring the mixed solution obtained in the step (1) into a rotary evaporator until a layer of transparent lipid film is formed;
(3) respectively weighing 40mg of walnut peptide and 1600mg of sucrose serving as a freeze-drying protective agent, and adding the walnut peptide and the sucrose into Phosphate Buffer Solution (PBS) at the pH value of 7.4;
(4) pouring the Phosphate Buffer Solution (PBS) in the step (3) into the rotary flask in the step (2) to hydrate the lipid membrane;
(5) carrying out ultrasonic treatment on the hydrated lipid membrane in the step (4), carrying out ultrasonic treatment for 1s, stopping for 1s, and circulating for 15min to obtain a liposome precursor suspension;
(6) pre-freezing the liposome suspension obtained in the step (5) for 12h in an environment of-80 ℃, and then freezing the liposome suspension in a freeze dryer for 48h to obtain the sugar alcohol liposome precursor debitterized walnut peptide freeze-dried powder.
Example 2
(1) Accurately weighing 480mg of soybean lecithin, 80mg of cholesterol and 240mg of tween into a 100mL beaker, pouring 30mL of absolute ethanol solution, and putting into an ultrasonic cleaning instrument for 10 minutes to uniformly disperse the solution to obtain a mixed solution;
(2) pouring the mixed solution obtained in the step (1) into a rotary evaporator until a transparent lipid film is formed;
(3) respectively weighing 48mg of walnut peptide and 1600mg of trehalose serving as a freeze-drying protective agent, and adding the walnut peptide and the trehalose into Phosphate Buffer Solution (PBS), wherein the pH value is 7.4;
(4) pouring the Phosphate Buffer Solution (PBS) in the step (3) into the rotary flask in the step (2) to hydrate the lipid membrane;
(5) carrying out ultrasonic treatment on the hydrated lipid membrane in the step (4), and carrying out ultrasonic treatment for 1s and stopping for 1s, and circulating for 15min to obtain a liposome precursor suspension;
(6) pre-freezing the liposome suspension obtained in the step (5) for 12h in an environment of-80 ℃, and then freezing the liposome suspension in a freeze dryer for 48h to obtain the sugar alcohol liposome precursor debitterized walnut peptide freeze-dried powder.
Example 3
(1) Accurately weighing 320mg of soybean lecithin, 80mg of cholesterol and 400mg of tween into a 100mL beaker, pouring 30mL of absolute ethanol solution, and putting into an ultrasonic cleaning instrument for 10 minutes to uniformly disperse the solution to obtain a mixed solution;
(2) pouring the mixed solution obtained in the step (1) into a rotary evaporator until a transparent lipid film is formed;
(3) respectively weighing 32mg of walnut peptide and 800mg of mannitol serving as a freeze-drying protective agent, adding the walnut peptide and the mannitol into Phosphate Buffer Solution (PBS), and adjusting the pH value to 7.4;
(4) pouring the Phosphate Buffer Solution (PBS) in the step (3) into the rotary flask in the step (2) to hydrate the lipid membrane;
(5) carrying out ultrasonic treatment on the hydrated lipid membrane in the step (4), carrying out ultrasonic treatment for 1s, stopping for 1s, and circulating for 15min to obtain a liposome precursor suspension;
(6) pre-freezing the liposome suspension obtained in the step (5) for 12h in an environment of-80 ℃, and then freezing the liposome suspension in a freeze dryer for 48h to obtain the sugar alcohol liposome precursor debitterized walnut peptide freeze-dried powder.
Example 4
A method for utilizing sugar alcohol liposome precursor to remove bitter walnut peptide comprises the following steps:
(1) accurately weighing 400mg of soybean lecithin, 80mg of cholesterol and 320mg of tween in a 100mL beaker, pouring 30mL of absolute ethanol solution, and putting the beaker into an ultrasonic cleaning instrument for 10 minutes to uniformly disperse the solution to obtain a mixed solution;
(2) pouring the mixed solution obtained in the step (1) into a rotary evaporator until a layer of transparent lipid film is formed;
(3) respectively weighing 40mg of walnut peptide and 1600mg of trehalose serving as a freeze-drying protective agent, and adding the walnut peptide and the trehalose into Phosphate Buffer Solution (PBS) with the pH value of 9.5;
(4) pouring the Phosphate Buffer Solution (PBS) in the step (3) into the rotary flask in the step (2) to hydrate the lipid membrane;
(5) carrying out ultrasonic treatment on the hydrated lipid membrane in the step (4), carrying out ultrasonic treatment for 1s, stopping for 1s, and circulating for 15min to obtain a liposome precursor suspension;
(6) pre-freezing the liposome suspension obtained in the step (5) for 12h in an environment of-80 ℃, and then freezing the liposome suspension in a freeze dryer for 48h to obtain the sugar alcohol liposome precursor debitterized walnut peptide freeze-dried powder.
Example 5
A method for utilizing sugar alcohol liposome precursor to remove bitter walnut peptide comprises the following steps:
(1) accurately weighing 400mg of soybean lecithin, 80mg of cholesterol and 320mg of tween in a 100mL beaker, pouring 30mL of absolute ethanol solution, and putting the beaker into an ultrasonic cleaning instrument for 10 minutes to uniformly disperse the solution to obtain a mixed solution;
(2) pouring the mixed solution obtained in the step (1) into a rotary evaporator until a layer of transparent lipid film is formed;
(3) respectively weighing 40mg of walnut peptide and 800mg of mannitol serving as a freeze-drying protective agent, adding the walnut peptide and the mannitol into Phosphate Buffer Solution (PBS), and adjusting the pH to 4.5;
(4) pouring the Phosphate Buffer Solution (PBS) in the step (3) into the rotary flask in the step (2) to hydrate the lipid membrane;
(5) carrying out ultrasonic treatment on the hydrated lipid membrane in the step (4), carrying out ultrasonic treatment for 1s, stopping for 1s, and circulating for 15min to obtain a liposome precursor suspension;
(6) and (4) pre-freezing the liposome precursor suspension obtained in the step (5) for 12 hours in an environment of-80 ℃, and then freezing the liposome precursor suspension in a freeze dryer for 48 hours to obtain the sugar alcohol liposome precursor debitterized walnut peptide freeze-dried powder.
Comparative example 1
The difference from example 1 is that the walnut peptide in comparative example 1 is 50 mg.
Comparative example 2
The difference from example 1 is that cholesterol was 40mg in comparative example 2.
Comparative example 3
The difference from example 1 is that in comparative example 3, 160mg of tween was used.
Comparative example 4
The difference from example 1 is that in comparative example 4, 480mg of Tween was contained.
Comparative example 5
The difference from example 1 is that the pH of PBS in comparative example 5 is 11.0.
Comparative example 6
The difference from example 1 is that the pH of PBS in comparative example 6 is 3.0.
Comparative example 7
Except for example 1 that continuous sonication was performed for 15min in comparative example 7.
Examples 1 to 5 and comparative examples 1 to 7 were each subjected to three replicates.
Test example 1
10ml of the walnut peptide liposome precursor suspension in the examples 1-5 and the comparative examples 1-7 are respectively taken, centrifuged for 1min at 10000r/min, 2ml of upper layer solution containing free walnut peptide is absorbed, the absorbance is measured at 217nm by using an ultraviolet-visible spectrophotometer, and the upper layer solution is substituted into a walnut peptide standard curve. And calculating the encapsulation efficiency of the walnut peptide liposome precursor by using an equation of 'encapsulation efficiency (%) [ (total walnut peptide-free walnut peptide)/total walnut peptide ] × 100'. The measurement results of each example and comparative example are shown in table 1.
TABLE 1
The results show that the average encapsulation efficiency of the examples 1 to 5 is between 79.2 and 87.3 percent, which is higher than that of the comparative examples 1 to 7; the encapsulation efficiency of the examples 1 to 5 after standing for 7 days at 25 ℃ and 4 ℃ is not obviously reduced, and the reduction range is obviously smaller than that of the comparative examples 1 to 7; the bitter taste of examples 1 to 5 is significantly less than that of comparative examples 1 to 7.
Test example 2
Sensory evaluation test of the debittered walnut peptide: 10 professional chefs, 15 food processing professionals and 15 laymen formed a sensory evaluation panel, and sensory evaluation was performed on each group of walnut peptides according to the walnut peptide sensory evaluation table (table 2), and the average score (accurate to one place after the decimal point) was calculated, and the evaluation results are shown in table 3.
TABLE 2
| Bitter taste | Score value |
| Heavy bitter taste and unacceptable quality | 0-25 |
| Has strong bitter taste and is acceptable | 26-50 |
| Has little bitter taste and no influence on eating | 51-75 |
| Substantially free of bitter taste | 76-100 |
TABLE 3
As can be seen from Table 3, the average scores of the examples 1-5 and the comparative examples 1-7 are all more than 80, which shows that the bitterness of the walnut peptide freeze-dried powder obtained by the sugar alcohol liposome precursor debittering technology of the invention is thoroughly removed, the sensory effect is good and the product quality is high.
Test example 3
An atomic force image of the walnut peptide-embedded algal glycolipid plastid precursor prepared in example 1 was observed under an atomic force microscope, as shown in fig. 1.
The appearance and the form of the trehalose-modified walnut peptide liposome precursor are observed by an atomic force microscope, and the liposome precursor can be observed to be spherical, smooth in surface, free of depression and good in form from the figure, so that the embedding of the walnut peptide by the liposome precursor is successful, and the hydration reconstruction effect is good. Meanwhile, the protective agent plays a role of a skeleton of the freeze-dried preparation, and the freeze-dried protective agent is reasonably selected, so that the redispersion of the liposome precursor in water is promoted, the system is uniformly dispersed, and the aggregation phenomenon is avoided.
Test example 4
The trehalose glycolipid plastid precursor embedded with the walnut peptide prepared in example 1 was examined by infrared spectroscopy, as shown in fig. 2.
As can be seen from the infrared spectrum, CH 2 Wave number of (2) at 2923.91cm -1 Here, it is shown that the lyoprotectant has no effect on the structure within the phospholipid bilayer; the graph shows that the frequency of stretching vibration of the polar head group C ═ O of phospholipid was 1737.02cm -1 It is shown that the addition of lyoprotectant has no effect on C ═ O.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (8)
1. A method for utilizing sugar alcohol liposome precursor to remove bitter walnut peptide is characterized by comprising the following steps:
(1) adding absolute ethyl alcohol into the mixture of the soybean lecithin, the cholesterol and the tween-80 to uniformly disperse the absolute ethyl alcohol to obtain a mixed solution;
(2) carrying out rotary evaporation on the mixed solution in the step (1) until a layer of transparent lipid film is formed;
(3) adding the freeze-drying protective agent and the walnut peptide into a phosphate buffer solution;
(4) mixing the phosphate buffer solution in the step (3) with the lipid membrane in the step (2) to hydrate the lipid membrane;
(5) carrying out ultrasonic treatment on the hydrated lipid membrane in the step (4) to obtain liposome precursor suspension;
(6) pre-freezing the liposome precursor suspension in the step (5), and then freeze-drying to obtain sugar alcohol liposome precursor debitterized walnut peptide freeze-dried powder;
the freeze-drying protective agent in the step (3) is one of sucrose, trehalose and mannitol.
2. The method of utilizing sugar alcohol liposome precursor to debitterized walnut peptide as claimed in claim 1, wherein: the mass ratio of the soybean lecithin, the cholesterol and the tween-80 in the step (1) is 4-6:1: 3-5.
3. The method of utilizing sugar alcohol liposome precursor to debitterized walnut peptide as claimed in claim 1, wherein: the pH value of the phosphate buffer PBS in the step (3) is 4-10.
4. The method of utilizing sugar alcohol liposome precursor to debitterized walnut peptide as claimed in claim 1, wherein: the mass ratio of the soybean lecithin to the walnut peptide powder is 10: 1.
5. The method of utilizing sugar alcohol liposome precursor to debitterized walnut peptide as claimed in claim 1, wherein: and (5) performing ultrasonic treatment for 1s, stopping for 1s, and circulating for 15 min.
6. The method of utilizing sugar alcohol liposome precursor to debitterized walnut peptide as claimed in claim 1, wherein: and (5) pre-freezing for 12 hours at the temperature of-80 ℃, and freeze-drying for 48 hours.
7. A freeze-dried powder of sugar alcohol liposome precursor debittered walnut peptide prepared according to the method of any one of claims 1 to 6.
8. The application of the sugar alcohol liposome precursor debitterized walnut peptide freeze-dried powder of claim 7 is characterized in that the application is the application of the sugar alcohol liposome precursor debitterized walnut peptide freeze-dried powder in preparation of food.
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| CN102406050A (en) * | 2011-11-17 | 2012-04-11 | 中国农业大学 | Walnut small-molecule polypeptide and preparation method thereof |
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| US5785984A (en) * | 1993-02-05 | 1998-07-28 | Kao Corporation | Taste-modifying method and bitterness-decreasing method |
| CN102406050A (en) * | 2011-11-17 | 2012-04-11 | 中国农业大学 | Walnut small-molecule polypeptide and preparation method thereof |
| CN107573400A (en) * | 2016-11-21 | 2018-01-12 | 广西大学 | A kind of method using beta-cyclodextrin Debittered soybean peptide |
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