CN114891062A - Method for improving functional characteristics of cannabis sativa protein through ultrasonic physical field assisted glycosylation - Google Patents
Method for improving functional characteristics of cannabis sativa protein through ultrasonic physical field assisted glycosylation Download PDFInfo
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Abstract
The invention discloses a method for improving the functional characteristics of hemp protein by ultrasonic physical field assisted glycosylation, which comprises the steps of firstly extracting hemp seed protein, adjusting the hemp seed protein to be neutral, carrying out ultrasonic treatment on the hemp protein, and controlling the ultrasonic power to be 100-200W/cm 2 The time is 20 to 80 minutes, and finally glycosylation is carried out for 3 to 24 hours at the temperature of between 40 and 90 ℃,and obtaining a final product. The modified hemp protein isolate prepared by the method has the advantages that the emulsibility is improved by 356.4%, and the surface hydrophobicity is improved by 231.4%; the solubility is improved by 47.6 percent, and the effect is obvious.
Description
Technical Field
The invention relates to a method for improving the functional characteristics of cannabis sativa protein by ultrasonic physical field assisted glycosylation, belonging to the technical field of food.
Background
Hemp is a plant with important industrial value widely planted in China, and the seeds of the hemp are valuable sources of traditional Chinese medicines and medical foods. Seeds typically contain about 20-25% protein, which is rich in essential amino acids and is more digestible than soy protein. However, in china, hemp seeds have long been used only for oil extraction, resulting in a large waste of protein. Recently, hemp seed protein isolate (HPI) has attracted increasing attention as a food ingredient, which has been included in many food products, such as dairy products and snacks. The functional properties of cannabis sativa protein are inferior compared to soy protein isolate, which greatly limits its industrial application. Therefore, it is necessary to improve the undesirable properties of the protein by modern techniques and to make better use of the protein.
Compared to many other food proteins, cannabis protein generally has limited functional properties (solubility, emulsifiability, etc.) due to its compact protein structure. Its use in food and industry will inevitably be greatly limited. Therefore, structural modifications of cannabis protein are necessary to improve its functional properties. Proteins have been subjected to physical, chemical or enzymatic treatments to convert them into stable forms with better functional properties and to be better utilized in the food industry. In recent years, many researchers have begun to combine proteins with sugars by the maillard reaction. This modification method is reported to be effective in improving the emulsifying property, solubility, antibacterial effect, antioxidant effect and reducing the allergy of proteins. In addition, the method utilizes naturally occurring reactions and no chemical reagents are used in the method. Therefore, this method is probably one of the most promising food application methods due to its safety.
The use of ultrasound in the food industry has increased in recent years due to its good effect on food and product modification. Ultrasonic waves are not only a fast, efficient, reliable alternative for improving food quality, but also have the potential to develop new products with unique functions. Currently, ultrasonic treatment technology is applied to aspects such as auxiliary extraction, physical modification, auxiliary modification and the like. The ultrasonic wave can loosen the structure of the protein molecules, increase the solubility of the protein and further increase the collision chance with sugar molecules, thereby improving the grafting degree of the protein. The influence of ultrasonic treatment on the functional properties of the ormosia protein is discussed by autumn fruits and the like, and the protein solubility is enhanced, the surface sparsity is increased and the emulsibility is obviously improved after the ultrasonic treatment; Martini-Velasco, A and the like use ultrasonic waves to treat broad bean protein, change the structure of the broad bean protein and improve the emulsifying capacity of the broad bean protein; li, S et al sonicate rice protein to improve its solubility.
Cannabis protein is mainly composed of edestin (edestin) and albumin (albumin). Edestin accounts for 60-80% of the total protein content, the remainder being albumin. Spherical edestin is located in aleurone particles in a large crystal substructure, and the crystallography technical research finds that the edestin has a structure similar to that of a glycinin hexamer and is composed of six identical subunits, each of which is formed by connecting an Acidic Subunit (AS) and a Basic Subunit (BS) through a disulfide bond, the compact structure of the lupulin enables the functional characteristics of the luplin to be poor and the traditional glycosylation process to be blocked, therefore, the ultrasonic wave energy is utilized to depend on the elastic mechanical wave propagated by the medium, the particles in the medium generate violent mechanical vibration in the transmission process of the ultrasonic wave, and the cavitation, the heat effect and the mechanical action among the media are caused, so that the structure of the hemp protein is further expanded, the structure of the protein molecules becomes loose, the solubility of the protein is increased, the collision chance with the polysaccharide molecules is increased, and the glycosylation degree of the protein is improved.
Disclosure of Invention
The technical problem is as follows:
the invention provides a method for improving the functional property of cannabis sativa protein, aiming at solving the problem of poor functional property of cannabis sativa protein.
The technical scheme is as follows:
the invention discloses a method for modifying hemp protein isolate, which is realized by the following technical scheme and comprises the following steps of improving the functional characteristics of hemp protein by ultrasonic physical field assisted glycosylation
(1) Protein extraction: extracting fructus Cannabis protein, adjusting to neutrality, and freeze drying;
(2) ultrasonic treatment: dissolving the hemp protein prepared in the step 1) in a phosphate buffer solution, and carrying out ultrasonic treatment at the temperature of 35-65 ℃ and the ultrasonic power of 100-200W/cm 2 Ultrasonic treatment is carried out for 20-80 minutes;
(3) and (3) glycosylation treatment: adding D-fructose into the ultrasonic-treated hemp protein solution, fully stirring, glycosylating for 3-24 hours at 40-90 ℃, and quickly putting the sample into an ice-water mixture to stop the reaction after glycosylation;
(4) and (3) freeze drying: dialyzing and freeze-drying the product obtained in the step 3) to obtain the final product.
One specific embodiment of the invention is as follows:
(1) extracting the hemp protein isolate: firstly, hemp seeds are fully crushed and degreased by 5 times of normal hexane. Dispersing the degreased powder in 5 times volume of ultrapure water, adjusting pH to 10.0 with 1mol/L NaOH, stirring at 25 deg.C for 2h, and centrifuging at 4 deg.C at 10000g for 30min to obtain supernatant. The pH of the supernatant was adjusted to 4.5 with 1mol/L HCl, and then centrifuged at 10000g for 30min at 4 deg.C, and the precipitate was collected and washed 3 times with water to remove salts. The resulting precipitate was redispersed in water, adjusted to pH 7.0 with 1mol/L NaOH and then freeze dried. The protein content in the dry powder was determined to be 82.25% (N.times.6.25) by Kjeldahl method.
(2) Ultrasonic treatment of the hemp protein isolate solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred with a magnetic stirrer at 4 ℃ to ensure adequate hydration of the solution. The protein solution was treated with an ultrasonic generator. The temperature is controlled to be 35-65 ℃, and the power is set to be 100-200W/cm 2 . The ultrasonic time is set to be 20-80 minutes.
(3) And (3) glycosylation treatment of the hemp protein isolate solution after ultrasonic treatment: adding D-fructose into the ultrasonic-treated hemp protein solution, fully stirring, and glycosylating for 3-12 hours at 40-90 ℃. After the glycosylation treatment, the sample was quickly placed in an ice-water mixture to stop the reaction.
(4) And (3) freeze drying: and dialyzing and freeze-drying the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain a cannabis sativa protein isolate powder product subjected to ultrasonic-assisted glycosylation.
In one embodiment of the present invention, the ultrasonic treatment process in step 2) is: the temperature is 35-65 ℃, and the ultrasonic power is 100-200W/cm 2 And carrying out ultrasonic treatment for 40-60 min.
Preferably, the ultrasonic treatment process in the step (2) is as follows: the temperature is 45 ℃, and the ultrasonic power is 150W/cm 2 And the ultrasonic treatment time is 40 min.
In one embodiment of the present invention, the glycosylation treatment process in the step 3) is: glycosylation is carried out for 3-24 hours at 50-80 ℃.
Preferably, the glycosylation treatment process in the step 3) is as follows: glycosylation is carried out for 6-12 hours at 60-70 ℃, and the optimal treatment process is as follows: glycosylation was carried out at 70 ℃ for 12 hours.
In the present invention, preferably, the emulsification activity of the cannabis sativa protein isolate powder product subjected to the ultrasonic-assisted glycosylation treatment in the step (4) is increased by 356.4%.
In the present invention, preferably, the hydrophobicity of the surface of the cannabis sativa protein isolate powder product subjected to the ultrasonic-assisted glycosylation treatment in the step (4) is increased by 231.4%.
In the invention, preferably, the solubility of the cannabis sativa protein isolate powder product subjected to ultrasonic-assisted glycosylation treatment in the step (4) is improved by 47.6%.
Another object of the present invention is to provide a modified kenaf protein prepared by the above method.
Has the beneficial effects of
(1) The emulsibility of the cannabis sativa protein isolate subjected to ultrasonic-assisted glycosylation treatment is improved by 356.4%;
(2) the surface hydrophobicity of the hemp protein isolate subjected to ultrasonic-assisted glycosylation treatment is improved by 231.4%;
(3) the solubility of the cannabis sativa protein isolate subjected to ultrasonic-assisted glycosylation treatment is improved by 47.6%.
Drawings
Table 1: emulsibility, surface hydrophobicity and solubility of cannabis sativa protein isolate subjected to ultrasonic-assisted glycosylation treatment
FIG. 1: SDS-PAGE of ultrasonic-assisted glycosylation-treated cannabis sativa protein isolate
Note: 1: marker; 2, separating protein from the cannabis sativa L; 3: performing ultrasonic treatment for 20 min; 4: ultrasonic treatment is carried out for 40 min; 5: ultrasonic treatment is carried out for 60 min; 6: and (4) carrying out ultrasonic treatment for 80 min.
FIG. 2: emulsibility analysis of ultrasonic-assisted glycosylation-treated hemp protein isolate
FIG. 3: ultrasonic-assisted glycosylation-treated hemp protein isolate solubility analysis
FIG. 4: ultrasonic-assisted glycosylation-treated surface hydrophobicity analysis of hemp protein isolate
Detailed Description
The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.
Hemp seed: purchased from Guangxi Bama autonomous county.
Functional characteristic analysis of hemp protein isolate after ultrasonic physical field treatment
SDS-PAGE analysis
SDS-PAGE was performed using an electrophoresis system (Bio-Rad Laboratories, Inc., Hercules, USA) containing 12% separation gel and 5% concentration gel. The amount of the marker added was 5. mu.L, and the amount of the sample added was 10. mu.L/lane. The voltage of the concentrated gel and the voltage of the separating gel are respectively 80V and 120V. After electrophoresis, the gel was stained with Coomassie brilliant blue ultrafast staining solution for 1 hour, and then decolorized with ultrapure water. The gel was blotted by a fluorescence imaging System (ChemiDoc MP System, BIO-RAD, Shanghai, China).
2. Measurement of emulsification Activity
The samples were dissolved in phosphate buffer solution (10mM, pH 7.0) to obtain a final protein content of 2 mg/ml. For emulsion formation, the soybean oil and protein solutions (1:3, v/v) were homogenized at 24000rpm for 1 min. After homogenization for 0min and 10min, 50ll of the emulsion was immediately removed from the bottom of the beaker and diluted to 1:100 with 0.1% SDS solution. The absorbance of the diluted emulsion was recorded at 500 nm. The formula for calculation of EAI and ESI is as follows:
where EAI is the emulsified area per gram of protein (m) 2 (g)/g), ESI is emulsion stability index, DF is dilution factor (100), C is protein concentration (g/ml), u is optical path length (1cm), h is oil volume fraction (0.25), A0 and A10 are absorbance of the emulsion at 0min and 10min, respectively.
3. Determination of solubility
Protein solubility was determined by dispersing the samples in distilled water to obtain a final solution of 0.2% (w/w) in protein. The solution was centrifuged at 2400g for 30 minutes in a Kubota 6700 type centrifuge. The content of soluble protein before and after centrifugation was determined by Lowry method.
4. Determination of surface hydrophobicity
Samples were dispersed in phosphate buffer (50mM, pH 7.4) to a final concentration of 0.1-0.5 mg/mL. mu.L of ANS (8.0mM) was added to 4mL of the protein buffer, and fluorescence intensity was measured at 390nm (excitation) and 470nm (emission) using Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) to calculate surface hydrophobicity.
Example 1
A process for modifying the protein separated from hemp by ultrasonic physical field includes such steps as treating the hemp by ultrasonic wave
(1) Extracting the hemp protein isolate: firstly, the hemp seeds are fully crushed and degreased by 5 times of normal hexane. Dispersing the degreased powder in 5 times volume of ultrapure water, adjusting pH to 10.0 with 1mol/L NaOH, stirring at 25 deg.C for 2h, and centrifuging at 4 deg.C at 10000g for 30min to obtain supernatant. The pH of the supernatant was adjusted to 4.5 with 1mol/L HCl, and then centrifuged at 10000g for 30min at 4 ℃ to collect the precipitate, which was then washed 3 times with water to remove the salts. The resulting precipitate was redispersed in water, adjusted to pH 7.0 with 1mol/L NaOH and then freeze dried.
(2) Ultrasonic treatment of the hemp protein isolate solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred with a magnetic stirrer at 4 ℃ to ensure adequate hydration of the solution. The protein solution was treated with an ultrasonic generator. The temperature is controlled at 45 ℃, and the power is set to be 150W/cm 2 . The ultrasound time was set at 20 minutes.
(3) And (3) glycosylation treatment of the hemp protein isolate solution after ultrasonic treatment: adding D-fructose into the ultrasonic-treated hemp protein solution, fully stirring, and glycosylating for 12 hours at 70 ℃. After the glycosylation treatment, the sample was quickly placed in an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and dialyzing and freeze-drying the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain a cannabis sativa protein isolate powder product subjected to ultrasonic-assisted glycosylation.
Example 2
A process for modifying the protein separated from hemp by ultrasonic physical field includes such steps as treating the hemp by ultrasonic wave
(1) Extracting the hemp protein isolate: firstly, the hemp seeds are fully crushed and degreased by 5 times of normal hexane. Dispersing the degreased powder in 5 times volume of ultrapure water, adjusting pH to 10.0 with 1mol/L NaOH, stirring at 25 deg.C for 2h, and centrifuging at 4 deg.C at 10000g for 30min to obtain supernatant. The pH of the supernatant was adjusted to 4.5 with 1mol/L HCl, and then centrifuged at 10000g for 30min at 4 ℃ to collect the precipitate, which was then washed 3 times with water to remove the salts. The resulting precipitate was redispersed in water, adjusted to pH 7.0 with 1mol/L NaOH and then freeze dried.
(2) Ultrasonic treatment of the hemp protein isolate solution: HPI was dissolved in 50mM phosphate buffer pH 7.4 and then stirred with a magnetic stirrer at 4 ℃ to ensure solubilizationThe liquid is fully hydrated. The protein solution was treated with an ultrasonic generator. The temperature is controlled at 45 ℃, and the power is set to be 150W/cm 2 . The ultrasound time was set at 40 minutes.
(3) And (3) glycosylation treatment of the hemp protein isolate solution after ultrasonic treatment: adding D-fructose into the ultrasonic-treated hemp protein solution, fully stirring, and glycosylating for 12 hours at 70 ℃. After the glycosylation treatment, the sample was quickly placed in an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and dialyzing and freeze-drying the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain a cannabis sativa protein isolate powder product subjected to ultrasonic-assisted glycosylation.
Example 3
A process for modifying the protein separated from hemp by ultrasonic physical field includes such steps as treating the hemp by ultrasonic wave
(1) Extracting the hemp protein isolate: firstly, the hemp seeds are fully crushed and degreased by 5 times of normal hexane. Dispersing the degreased powder in 5 times volume of ultrapure water, adjusting pH to 10.0 with 1mol/L NaOH, stirring at 25 deg.C for 2h, and centrifuging at 4 deg.C at 10000g for 30min to obtain supernatant. The pH of the supernatant was adjusted to 4.5 with 1mol/L HCl, and then centrifuged at 10000g for 30min at 4 ℃ to collect the precipitate, which was then washed 3 times with water to remove the salts. The resulting precipitate was redispersed in water, adjusted to pH 7.0 with 1mol/L NaOH and then freeze dried.
(2) Ultrasonic treatment of the hemp protein isolate solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred with a magnetic stirrer at 4 ℃ to ensure adequate hydration of the solution. The protein solution was treated with an ultrasonic generator. The temperature was controlled at 45 ℃ and the power was set at 150W/cm 2. The ultrasound time was set at 60 minutes.
(3) And (3) glycosylation treatment of the hemp protein isolate solution after ultrasonic treatment: adding D-fructose into the ultrasonic-treated hemp protein solution, fully stirring, and glycosylating for 12 hours at 70 ℃. After the glycosylation treatment, the sample was quickly placed in an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and dialyzing and freeze-drying the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain a cannabis sativa protein isolate powder product subjected to ultrasonic-assisted glycosylation.
Example 4
A process for modifying the protein separated from hemp by ultrasonic physical field includes such steps as treating the hemp by ultrasonic wave
(1) Extracting the hemp protein isolate: firstly, the hemp seeds are fully crushed and degreased by 5 times of normal hexane. Dispersing the degreased powder in 5 times volume of ultrapure water, adjusting pH to 10.0 with 1mol/L NaOH, stirring at 25 deg.C for 2h, and centrifuging at 4 deg.C at 10000g for 30min to obtain supernatant. The pH of the supernatant was adjusted to 4.5 with 1mol/L HCl, and then centrifuged at 10000g for 30min at 4 deg.C, and the precipitate was collected and washed 3 times with water to remove salts. The precipitate obtained finally was redispersed in water, the pH adjusted back to 7.0 with 1mol/L NaOH and then freeze-dried.
(2) Ultrasonic treatment of the hemp protein isolate solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred with a magnetic stirrer at 4 ℃ to ensure adequate hydration of the solution. The protein solution was treated with an ultrasonic generator. The temperature was controlled at 45 ℃ and the power was set at 150W/cm 2. The ultrasound time was set at 80 minutes.
(3) And (3) glycosylation treatment of the hemp protein isolate solution after ultrasonic treatment: adding D-fructose into the ultrasonic-treated hemp protein solution, fully stirring, and glycosylating for 12 hours at 70 ℃. After the glycosylation treatment, the sample was quickly placed in an ice-water mixture to stop the reaction.
(4) And (3) freeze drying: and dialyzing and freeze-drying the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain a cannabis sativa protein isolate powder product subjected to ultrasonic-assisted glycosylation.
Ultrasonic-assisted glycosylation-treated cannabis sativa protein isolate SDS-PAGE analysis
SDS-PAGE analysis of the hemp protein isolate samples prepared in examples 1-4 was performed as shown in FIG. 1: the result shows that ultrasonic assistance accelerates the glycosylation reaction of the hemp protein, thereby increasing the molecular weight of the protein and gradually lightening the band.
Examples 1-4 were analyzed as follows:
emulsibility analysis of ultrasonic-assisted glycosylation-treated hemp protein isolate
The results of the analysis of the emulsifiability of the cannabis sativa protein isolate samples at 150W/cm2 and 45 ℃ for different ultrasonic treatment times (0, 20, 40, 60 and 80min, as shown in figure 2, show that the emulsifiability increases and then decreases with the increase of the ultrasonic treatment time, and is strongest at the ultrasonic treatment time of 40 min.
Ultrasonic-assisted glycosylation-treated hemp protein isolate solubility analysis
The solubility analysis of the hemp protein isolate samples prepared in examples 1-4 was performed as shown in FIG. 3: the results show that the solubility increased and then decreased with increasing sonication time, with the highest solubility at sonication time 40 min.
Ultrasonic-assisted glycosylation-treated surface hydrophobicity analysis of hemp protein isolate
The surface hydrophobicity of the protein isolate samples from cannabis prepared in examples 1-4 was analyzed as shown in figure 4: the results show that the surface hydrophobicity of the ambary separating protein is increased by ultrasonic treatment of the ambary separating protein at different times, the hydrophobicity of the ambary separating protein is gradually increased along with the increase of the time, and the surface hydrophobicity is strongest at the ultrasonic time of 40 min.
Comparative example 1
A process for modifying the protein separated from hemp by ultrasonic physical field includes such steps as treating the hemp by ultrasonic wave
(1) Extracting the hemp protein isolate: firstly, the hemp seeds are fully crushed and degreased by 5 times of normal hexane. Dispersing the degreased powder in 5 times volume of ultrapure water, adjusting pH to 10.0 with 1mol/L NaOH, stirring at 25 deg.C for 2h, and centrifuging at 4 deg.C at 10000g for 30min to obtain supernatant. The pH of the supernatant was adjusted to 4.5 with 1mol/L HCl, and then centrifuged at 10000g for 30min at 4 ℃ to collect the precipitate, which was then washed 3 times with water to remove the salts. The resulting precipitate was redispersed in water, adjusted to pH 7.0 with 1mol/L NaOH and then freeze dried.
(2) Ultrasonic treatment of the hemp protein isolate solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred with a magnetic stirrer at 4 ℃ to ensure adequate hydration of the solution. The protein solution was treated with an ultrasonic generator. The temperature is controlled at 45 ℃, and the power is set to be 150W/cm 2 . The ultrasound time was set at 40 minutes.
(3) And (3) glycosylation treatment of the hemp protein isolate solution after ultrasonic treatment: adding fructo-oligosaccharide into the ultrasonic-treated hemp protein solution, fully stirring, and glycosylating at 70 ℃ for 12 hours. After the glycosylation treatment, the sample was quickly placed in an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and dialyzing and freeze-drying the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain a cannabis sativa protein isolate powder product subjected to ultrasonic-assisted glycosylation.
Comparative example 2
A process for modifying the protein separated from hemp by ultrasonic physical field includes such steps as treating the hemp by ultrasonic wave
(1) Extracting the hemp protein isolate: firstly, the hemp seeds are fully crushed and degreased by 5 times of normal hexane. Dispersing the degreased powder in 5 times volume of ultrapure water, adjusting pH to 10.0 with 1mol/L NaOH, stirring at 25 deg.C for 2h, and centrifuging at 4 deg.C at 10000g for 30min to obtain supernatant. The pH of the supernatant was adjusted to 4.5 with 1mol/L HCl, and then centrifuged at 10000g for 30min at 4 ℃ to collect the precipitate, which was then washed 3 times with water to remove the salts. The resulting precipitate was redispersed in water, adjusted to pH 7.0 with 1mol/L NaOH and then freeze dried.
(2) Ultrasonic treatment of the hemp protein isolate solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred with a magnetic stirrer at 4 ℃ to ensure adequate hydration of the solution. The protein solution was treated with an ultrasonic generator. The temperature is controlled at 45 ℃, and the power is set to be 150W/cm 2 . The ultrasound time was set at 40 minutes.
(3) And (3) glycosylation treatment of the hemp protein isolate solution after ultrasonic treatment: adding D sucrose into the ultrasonic-treated hemp protein solution, fully stirring, and glycosylating for 12 hours at 70 ℃. After the glycosylation treatment, the sample was quickly placed in an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and dialyzing and freeze-drying the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain a cannabis sativa protein isolate powder product subjected to ultrasonic-assisted glycosylation.
Comparative example 3
A process for modifying the protein separated from hemp by ultrasonic physical field includes such steps as treating the hemp by ultrasonic wave
(1) Extracting the hemp protein isolate: firstly, the hemp seeds are fully crushed and degreased by 5 times of normal hexane. Dispersing the degreased powder in 5 times volume of ultrapure water, adjusting pH to 10.0 with 1mol/L NaOH, stirring at 25 deg.C for 2h, and centrifuging at 4 deg.C at 10000g for 30min to obtain supernatant. The pH of the supernatant was adjusted to 4.5 with 1mol/L HCl, and then centrifuged at 10000g for 30min at 4 ℃ to collect the precipitate, which was then washed 3 times with water to remove the salts. The resulting precipitate was redispersed in water, adjusted to pH 7.0 with 1mol/L NaOH and then freeze dried.
(2) Ultrasonic treatment of the hemp protein isolate solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred with a magnetic stirrer at 4 ℃ to ensure adequate hydration of the solution. The protein solution was treated with an ultrasonic generator. The temperature is controlled at 45 ℃, and the power is set to be 150W/cm 2 . The ultrasound time was set at 40 minutes.
(3) And (3) glycosylation treatment of the hemp protein isolate solution after ultrasonic treatment: adding D stachyose into the ultrasonic-treated hemp protein solution, fully stirring, and glycosylating for 12 hours at 70 ℃. After the glycosylation treatment, the sample was quickly placed in an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and (3) dialyzing and freeze-drying the HPI sample subjected to ultrasonic-assisted glycosylation to obtain a cannabis sativa protein isolate powder product subjected to ultrasonic-assisted glycosylation.
Comparative example 4
A process for modifying the protein separated from hemp by ultrasonic physical field includes such steps as treating the hemp by ultrasonic wave
(1) Extracting the hemp protein isolate: firstly, the hemp seeds are fully crushed and degreased by 5 times of normal hexane. Dispersing the degreased powder in 5 times of ultrapure water, adjusting pH to 10.0 with 1mol/L NaOH, stirring at 25 deg.C for 2h, and centrifuging at 4 deg.C at 10000g for 30min to obtain supernatant. The pH of the supernatant was adjusted to 4.5 with 1mol/L HCl, and then centrifuged at 10000g for 30min at 4 ℃ to collect the precipitate, which was then washed 3 times with water to remove the salts. The resulting precipitate was redispersed in water, adjusted to pH 7.0 with 1mol/L NaOH and then freeze dried.
(2) Ultrasonic treatment of the hemp protein isolate solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred with a magnetic stirrer at 4 ℃ to ensure adequate hydration of the solution. The protein solution was treated with an ultrasonic generator. The temperature is controlled at 45 ℃, and the power is set to be 150W/cm 2 . The ultrasound time was set at 40 minutes.
(3) And (3) glycosylation treatment of the hemp protein isolate solution after ultrasonic treatment: adding D-fructose into the ultrasonic-treated hemp protein solution, fully stirring, and glycosylating for 3 hours at 70 ℃. After the glycosylation treatment, the sample was quickly placed in an ice-water mixture to stop the reaction.
(4) And (3) freeze drying: and dialyzing and freeze-drying the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain a cannabis sativa protein isolate powder product subjected to ultrasonic-assisted glycosylation.
Comparative example 5
A process for modifying the protein separated from hemp by ultrasonic physical field includes such steps as treating the hemp by ultrasonic wave
(1) Extracting the hemp protein isolate: firstly, the hemp seeds are fully crushed and degreased by 5 times of normal hexane. Dispersing the degreased powder in 5 times volume of ultrapure water, adjusting pH to 10.0 with 1mol/L NaOH, stirring at 25 deg.C for 2h, and centrifuging at 4 deg.C at 10000g for 30min to obtain supernatant. The pH of the supernatant was adjusted to 4.5 with 1mol/L HCl, and then centrifuged at 10000g for 30min at 4 ℃ to collect the precipitate, which was then washed 3 times with water to remove the salts. The resulting precipitate was redispersed in water, adjusted to pH 7.0 with 1mol/L NaOH and then freeze dried.
(2) Ultrasonic treatment of the hemp protein isolate solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred with a magnetic stirrer at 4 ℃ to ensure adequate hydration of the solution. The protein solution was treated with an ultrasonic generator. The temperature is controlled at 45 ℃, and the power is set to be 150W/cm 2 . The ultrasound time was set at 40 minutes.
(3) And (3) glycosylation treatment of the hemp protein isolate solution after ultrasonic treatment: adding D-fructose into the ultrasonic-treated hemp protein solution, fully stirring, and glycosylating for 6 hours at 70 ℃. After the glycosylation treatment, the sample was quickly placed in an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and dialyzing and freeze-drying the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain a cannabis sativa protein isolate powder product subjected to ultrasonic-assisted glycosylation.
Comparative example 6
A process for modifying the protein separated from hemp by ultrasonic physical field includes such steps as treating the hemp by ultrasonic wave
(1) Extracting the hemp protein isolate: firstly, the hemp seeds are fully crushed and degreased by 5 times of normal hexane. Dispersing the degreased powder in 5 times volume of ultrapure water, adjusting pH to 10.0 with 1mol/L NaOH, stirring at 25 deg.C for 2h, and centrifuging at 4 deg.C at 10000g for 30min to obtain supernatant. The pH of the supernatant was adjusted to 4.5 with 1mol/L HCl, and then centrifuged at 10000g for 30min at 4 ℃ to collect the precipitate, which was then washed 3 times with water to remove the salts. The resulting precipitate was redispersed in water, adjusted to pH 7.0 with 1mol/L NaOH and then freeze dried.
(2) Ultrasonic treatment of the hemp protein isolate solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred with a magnetic stirrer at 4 ℃ to ensure adequate hydration of the solution. By ultrasonic generationThe processor processes the protein solution. The temperature is controlled at 45 ℃, and the power is set to be 150W/cm 2 . The ultrasound time was set at 40 minutes.
(3) And (3) glycosylation treatment of the hemp protein isolate solution after ultrasonic treatment: adding D-fructose into the ultrasonic-treated hemp protein solution, fully stirring, and performing glycosylation at 70 ℃ for 24 hours. After the glycosylation treatment, the sample was quickly placed in an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and dialyzing and freeze-drying the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain a cannabis sativa protein isolate powder product subjected to ultrasonic-assisted glycosylation.
Comparative example 7
A process for modifying the protein separated from hemp by ultrasonic physical field includes such steps as treating the hemp by ultrasonic wave
(1) Extracting the hemp protein isolate: firstly, the hemp seeds are fully crushed and degreased by 5 times of normal hexane. Dispersing the degreased powder in 5 times of ultrapure water, adjusting pH to 10.0 with 1mol/L NaOH, stirring at 25 deg.C for 2h, and centrifuging at 4 deg.C at 10000g for 30min to obtain supernatant. The pH of the supernatant was adjusted to 4.5 with 1mol/L HCl, and then centrifuged at 10000g for 30min at 4 ℃ to collect the precipitate, which was then washed 3 times with water to remove the salts. The resulting precipitate was redispersed in water, adjusted to pH 7.0 with 1mol/L NaOH and then freeze dried.
(2) Ultrasonic treatment of the hemp protein isolate solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred with a magnetic stirrer at 4 ℃ to ensure adequate hydration of the solution. The protein solution was treated with an ultrasonic generator. The temperature is controlled at 45 ℃, and the power is set to be 150W/cm 2 . The ultrasound time was set at 40 minutes.
(3) And (3) glycosylation treatment of the hemp protein isolate solution after ultrasonic treatment: adding D-fructose into the ultrasonic-treated hemp protein solution, fully stirring, and glycosylating for 12 hours at 50 ℃. After the glycosylation treatment, the sample was quickly placed in an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and dialyzing and freeze-drying the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain a cannabis sativa protein isolate powder product subjected to ultrasonic-assisted glycosylation.
Comparative example 8
A process for modifying the protein separated from hemp by ultrasonic physical field includes such steps as treating the hemp by ultrasonic wave
(1) Extracting the hemp protein isolate: firstly, the hemp seeds are fully crushed and degreased by 5 times of normal hexane. Dispersing the degreased powder in 5 times volume of ultrapure water, adjusting pH to 10.0 with 1mol/L NaOH, stirring at 25 deg.C for 2h, and centrifuging at 4 deg.C at 10000g for 30min to obtain supernatant. The pH of the supernatant was adjusted to 4.5 with 1mol/L HCl, and then centrifuged at 10000g for 30min at 4 deg.C, and the precipitate was collected and washed 3 times with water to remove salts. The resulting precipitate was redispersed in water, adjusted to pH 7.0 with 1mol/L NaOH and then freeze dried.
(2) Ultrasonic treatment of the hemp protein isolate solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred with a magnetic stirrer at 4 ℃ to ensure adequate hydration of the solution. The protein solution was treated with an ultrasonic generator. The temperature is controlled at 45 ℃, and the power is set to be 150W/cm 2 . The ultrasound time was set at 40 minutes.
(3) And (3) glycosylation treatment of the hemp protein isolate solution after ultrasonic treatment: adding D-fructose into the ultrasonic-treated hemp protein solution, fully stirring, and glycosylating for 12 hours at 60 ℃. After the glycosylation treatment, the sample was quickly placed in an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and dialyzing and freeze-drying the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain a cannabis sativa protein isolate powder product subjected to ultrasonic-assisted glycosylation.
Comparative example 9
A process for modifying the protein separated from hemp by ultrasonic physical field includes such steps as treating the hemp by ultrasonic wave
(1) Extracting the hemp protein isolate: firstly, hemp seeds are fully crushed and degreased by 5 times of normal hexane. Dispersing the degreased powder in 5 times volume of ultrapure water, adjusting pH to 10.0 with 1mol/L NaOH, stirring at 25 deg.C for 2h, and centrifuging at 4 deg.C at 10000g for 30min to obtain supernatant. The pH of the supernatant was adjusted to 4.5 with 1mol/L HCl, and then centrifuged at 10000g for 30min at 4 ℃ to collect the precipitate, which was then washed 3 times with water to remove the salts. The resulting precipitate was redispersed in water, adjusted to pH 7.0 with 1mol/L NaOH and then freeze dried.
(2) Ultrasonic treatment of the hemp protein isolate solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred with a magnetic stirrer at 4 ℃ to ensure adequate hydration of the solution. The protein solution was treated with an ultrasonic generator. The temperature is controlled at 45 ℃, and the power is set to be 150W/cm 2 . The ultrasound time was set at 40 minutes.
(3) And (3) glycosylation treatment of the hemp protein isolate solution after ultrasonic treatment: adding D-fructose into the ultrasonic-treated hemp protein solution, fully stirring, and glycosylating for 12 hours at 80 ℃. After the glycosylation treatment, the sample was quickly placed in an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and dialyzing and freeze-drying the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain a cannabis sativa protein isolate powder product subjected to ultrasonic-assisted glycosylation.
(4) And (3) freeze drying: and (3) dialyzing and freeze-drying the HPI sample subjected to ultrasonic-assisted glycosylation to obtain a cannabis sativa protein isolate powder product subjected to ultrasonic-assisted glycosylation.
The protein powders and the hemp proteins prepared in examples 1 to 3 and comparative examples 1 to 9 were analyzed for emulsifiability, emulsion stability, surface hydrophobicity and solubility, which are shown in Table 1.
The results of the above specific embodiments and experimental examples are combined to show that ultrasonic-assisted glycosylation treatment of the hemp protein isolate is an effective way for modifying the functional properties of the hemp protein isolate, wherein the donor D-fructose is more critical. By controlling the ultrasonic process, the temperature is 45 ℃, the ultrasonic time is 40min, and the ultrasonic intensity is 150W/cm 2 Then separating the protein from the hempThe glycosylation reaction is carried out at 70 ℃ for 12h to obtain the most novel hemp protein.
TABLE 1
Claims (10)
1. A method for improving the functional characteristics of hemp protein by ultrasonic physical field assisted glycosylation comprises the following steps
(1) Protein extraction: extracting fructus Cannabis protein, adjusting to neutrality, and freeze drying;
(2) ultrasonic treatment: dissolving the hemp protein prepared in the step 1) in a phosphate buffer solution, and carrying out ultrasonic treatment at the temperature of 35-65 ℃ and the ultrasonic power of 100-200W/cm 2 Ultrasonic treatment is carried out for 20-80 minutes;
(3) and (3) glycosylation treatment: adding D-fructose into the ultrasonic-treated hemp protein solution, fully stirring, glycosylating for 3-24 hours at 40-90 ℃, and quickly placing the sample into an ice-water mixture to stop the reaction after the reaction is finished;
(4) and (3) freeze drying: dialyzing and freeze-drying the product obtained in the step 3) to obtain the final product.
2. The method of claim 1, wherein the protein extraction step comprises: firstly, fully crushing the hemp seeds, degreasing the crushed hemp seeds by using 5 times of normal hexane, dispersing the degreased powder in 5 times of volume of ultrapure water, adjusting the pH to 10.0 by using 1mol/L NaOH, stirring the mixture at the room temperature of 25 ℃ for 2 hours, and centrifuging the mixture at the rotation speed of 10000g at the temperature of 4 ℃ for 30 minutes to obtain a supernatant; adjusting the pH of the supernatant to 4.5 with 1mol/L HCl, centrifuging at 4 deg.C at 10000g for 30min, collecting precipitate, and washing the precipitate with water for 3 times to remove salt; the precipitate obtained finally was redispersed in water, the pH adjusted back to 7.0 with 1mol/L NaOH and then freeze-dried.
3. The method of claim 1 or 2, wherein the step 2) sonication process is:the temperature is 35-65 ℃, and the ultrasonic power is 100-200W/cm 2 And carrying out ultrasonic treatment for 40-60 min.
4. The method according to claim 3, wherein the step (2) ultrasonic treatment process is: the temperature is 45 ℃, and the ultrasonic power is 150W/cm 2 And the ultrasonic treatment time is 40 min.
5. The method according to claim 1 or 2, wherein the glycosylation process of step 3) is: glycosylation is carried out for 3-24 hours at 50-80 ℃.
6. The method according to claim 4, wherein the glycosylation process of the step 3) is: glycosylation is carried out for 6-12 hours at 60-70 ℃.
7. The method of claim 1, wherein in step (3) glycosylation is selected at 70 ℃ for 12 hours.
8. The modified hemp protein produced by the process of claim 1, which is produced by the following steps:
(1) protein extraction: extracting fructus Cannabis protein, adjusting to neutrality, and freeze drying;
(2) ultrasonic treatment: dissolving the hemp protein obtained in the step 1) in a phosphate buffer solution, and performing ultrasonic treatment at the temperature of 35-65 ℃ and the ultrasonic power of 100-200W/cm 2 Ultrasonic treatment is carried out for 20-80 minutes;
(3) and (3) glycosylation treatment: adding D-fructose into the ultrasonic-treated hemp protein solution, fully stirring, glycosylating for 3-24 hours at 40-90 ℃, and quickly putting the sample into an ice-water mixture to stop the reaction after glycosylation;
(4) and (3) freeze drying: dialyzing and freeze-drying the product obtained in the step 3) to obtain the final product.
9. The modified hemp protein of claim 6, wherein the ultrasonic treatment process of step 2) is as follows: the temperature is 35-65 DEG CUltrasonic power of 100-200W/cm 2 And carrying out ultrasonic treatment for 40-60 min.
10. The modified kenaf protein of claim 7 or 8, wherein the glycosylation process of step 3) is: glycosylation is carried out for 3-24 hours at 50-80 ℃.
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