CN114891062B - Method for improving functional characteristics of cannabis protein by ultrasonic physical field assisted glycosylation - Google Patents
Method for improving functional characteristics of cannabis protein by ultrasonic physical field assisted glycosylation Download PDFInfo
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Abstract
The invention discloses a method for improving the functional characteristics of cannabis protein by ultrasonic physical field assisted glycosylation, which comprises the steps of firstly extracting cannabis seed protein, adjusting the cannabis seed protein to be neutral, carrying out ultrasonic treatment on the cannabis protein, and controlling the ultrasonic power to be 100-200W/cm 2 And finally, carrying out glycosylation for 3-24 hours at the temperature of 40-90 ℃ for 20-80 minutes to obtain the final product. The emulsifying property of the modified hemp protein isolate prepared by the invention is improved by 356.4 percent, and the surface hydrophobicity is improved by 231.4 percent; the solubility is improved by 47.6%, and the effect is remarkable.
Description
Technical Field
The invention relates to a method for improving the functional characteristics of cannabis protein by ultrasonic physical field assisted glycosylation, and belongs to the technical field of foods.
Background
Hemp is a plant widely planted in China and has important industrial value, and seeds thereof 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 readily eliminated than soy protein. However, in china, cannabis seeds have long been used only for extracting oil, resulting in a large waste of protein. Recently, cannabis seed isolated protein (HPI) has attracted increasing attention as a food ingredient, which has been included in many foods such as dairy products and snack foods. The functional properties of hemp seed proteins are poor compared to soy isolate proteins, which greatly limits their industrial application. Thus, there is a need to improve the undesirable properties of modern technologies and to better utilize such nutritional proteins.
Compared to many other food proteins, cannabis proteins generally have limited functional properties (solubility, emulsifying properties, etc.) due to their compact protein structure. Its use in the food and industry will inevitably be greatly restricted. Therefore, structural modification of the cannabis proteins is necessary to improve their functional properties. Proteins have been subjected to physical, chemical or enzymatic treatments to convert them into stable forms with better functional properties and are better utilized in the food industry. In recent years, many researchers have begun to bind proteins to sugars via maillard reactions. This modification method is reported to be effective in improving the emulsifying property, solubility, antibacterial effect, antioxidant effect and reducing allergy of the protein. In addition, the method utilizes a naturally occurring reaction, and no chemical reagent is used in the method. Thus, 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 in food and product modification. Ultrasonic waves are not only quick, efficient, reliable alternatives to improving food quality, but also have the potential to develop new products with unique functions. At present, the ultrasonic treatment technology is applied to the aspects of auxiliary extraction, physical modification, auxiliary modification and the like. The ultrasonic wave can loosen the structure of protein molecules, increase the solubility of the protein and thus increase the collision chance with sugar molecules, thereby improving the grafting degree of the protein. He Qiushi et al discuss the effect of ultrasonic treatment on the functional properties of red bean proteins, and found that after ultrasonic treatment, the protein solubility is enhanced, the surface hydrophobicity is increased, and the emulsifying property is remarkably improved; martI nez-Velastco, A et al treat fava bean protein with ultrasonic wave, change its structure, improve its emulsifying capacity; li, S et al treat rice protein with ultrasound to increase its solubility.
The hemp proteins mainly consist of edestin (edestin) and albumin (albumin). Edestin accounts for 60-80% of the total protein content, the remainder being albumin. The spherical edestin is positioned in the aleurone particles in a large crystal substructure, and the crystallography technical research finds that the edestin has a structure similar to a glycinin hexamer, and consists of six identical subunits, wherein each subunit is formed by connecting an Acid Subunit (AS) and an alkaline subunit (BS) through a disulfide bond, the compact structure of the edestin leads the functional property of the cannabis protein to be poorer, and the traditional glycosylation process of the cannabis protein to be hindered, so that the particles in the medium generate intense mechanical vibration in the transmission process of ultrasonic waves by utilizing elastic mechanical waves propagated by the ultrasonic waves, and cavitation effect, thermal effect and mechanical effect among the mediums are caused, so that the structure of the cannabis protein is further unfolded, the structure of the protein molecule is loose, the solubility of the protein is increased, the collision opportunity with polysaccharide molecules is increased, and the glycosylation degree of the protein is improved.
Disclosure of Invention
Technical problems:
the invention provides a method for improving the functional property of cannabis protein aiming at the problem of poor functional property of the cannabis protein.
The technical scheme is as follows:
the invention relates to a method for modifying hemp protein isolate, which is realized by the following technical proposal, and comprises the following steps of
(1) Protein extraction: extracting cannabis sativa seed protein, adjusting to neutrality, and freeze drying;
(2) And (3) ultrasonic treatment: dissolving the cannabis protein prepared in the step 1) in phosphate buffer solution, performing ultrasonic treatment at the temperature of 35-65 ℃ and the ultrasonic power of 100-200W/cm 2 Ultrasonic time is 20-80 minutes;
(3) Glycosylation treatment: adding D-fructose into the Cannabin protein solution after ultrasonic treatment, fully stirring, carrying out glycosylation at 40-90 ℃ for 3-24 hours, and rapidly placing a sample into an ice-water mixture to terminate the reaction after glycosylation;
(4) And (3) freeze drying: dialyzing and freeze-drying the product obtained in the step 3) to obtain a final product.
One specific embodiment of the invention is:
(1) Extraction of cannabis isolated protein: hemp seeds were first fully crushed and defatted with 5-fold n-hexane. The defatted powder was dispersed in 5 volumes of ultrapure water, the pH was adjusted to 10.0 with 1mol/L NaOH, stirred at 25℃for 2 hours at room temperature, and centrifuged at 10000g for 30 minutes at 4℃to obtain a 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 washed with water 3 times to remove the salt. The resulting precipitate was redispersed in water, its pH was adjusted back to 7.0 with 1mol/L NaOH, and then freeze-dried. The protein content of the dry powder was 82.25% (N.times.6.25) as determined by Kjeldahl method.
(2) Sonication of the cannabis isolated protein solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred at 4 ℃ with a magnetic stirrer to ensure adequate hydration of the solution. The protein solution was treated with an ultrasonic generator. The temperature is controlled at 35-65 ℃ and the power is set at 100-200W/cm 2 . The ultrasonic time is set to 20-80 minutes.
(3) Glycosylation of cannabis isolated protein solution after sonication: adding D-fructose into the Cannabin protein solution after ultrasonic treatment, stirring thoroughly, and glycosylating at 40-90deg.C for 3-12 hr. After glycosylation, the sample was quickly placed into an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and (3) performing dialysis and freeze drying on the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain the cannabis sativa isolated protein powder product obtained through ultrasonic-assisted glycosylation.
In one embodiment of the present invention, the ultrasonic treatment process in step 2) is as follows: the temperature is 35-65 ℃ and the ultrasonic power is 100-200W/cm 2 The ultrasonic time is 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 Ultrasonic time was 40min.
In one embodiment of the present invention, the step 3) glycosylation treatment process is: glycosylation is carried out for 3-24 hours at 50-80 ℃.
Preferably, the glycosylation treatment process of 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 emulsifying activity of the cannabis isolated protein powder product of the ultrasound-assisted glycosylation treatment in step (4) is increased by 356.4%.
In the present invention, preferably, the surface hydrophobicity of the cannabis isolated protein powder product subjected to the ultrasonic-assisted glycosylation treatment in the step (4) is improved by 231.4%.
In the present invention, preferably, the solubility of the cannabis isolated protein powder product of the ultrasound-assisted glycosylation treatment of step (4) is increased by 47.6%.
It is another object of the present invention to provide a modified cannabis protein prepared by the above method.
Has the beneficial effects of
(1) The emulsifying property of the hemp separation protein subjected to ultrasonic assisted glycosylation is improved by 356.4 percent;
(2) The surface hydrophobicity of the cannabis sativa isolated protein subjected to ultrasonic-assisted glycosylation is improved by 231.4%;
(3) The solubility of the cannabis isolated protein of the ultrasonic assisted glycosylation treatment is improved by 47.6 percent.
Drawings
Fig. 1: SDS-PAGE of ultrasound-assisted glycosylated cannabis isolated proteins
Note that: 1: a marker;2, separating protein from hemp; 3: carrying out ultrasonic treatment for 20min;4: performing ultrasonic treatment for 40min;5: carrying out ultrasonic treatment for 60min;6: and (5) carrying out ultrasonic treatment for 80min.
Fig. 2: emulsifying analysis of cannabis isolated proteins with ultrasound-assisted glycosylation
Fig. 3: analysis of protein solubility of cannabis isolates with ultrasound-assisted glycosylation
Fig. 4: ultrasonic assisted glycosylation treated cannabis isolate protein surface hydrophobicity analysis
Detailed Description
The following description of the preferred embodiments of the present invention is provided for better illustration of the invention, and should not be construed as limiting the invention.
Hemp seeds: purchased from guangxi Bama county.
Functional characteristic analysis of hemp separation protein 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 voltages of the concentrated gel and the separation gel were 80V and 120V, respectively. 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. Determination of emulsifying Activity
The sample was dissolved in phosphate buffer (10 mM, pH 7.0) to obtain a final protein content of 2 mg/ml. For emulsion formation, soybean oil and protein solution (1:3, v/v) were homogenized at 24000rpm for 1min. Immediately after homogenization for 0min and 10min, 50ll of the emulsion was removed from the bottom of the beaker and diluted 1:100 with 0.1% SDS solution. The absorbance of the diluted emulsion was recorded at 500 nm. The equation for EAI and ESI is as follows:
wherein EAI is the emulsion area per gram of protein (m 2 ESI is the emulsion stability index, DF is the dilution factor (100), C is the protein concentration (g/ml), u is the optical path (1 cm), h is the oil volume fraction (0.25), and A0 and a10 are the absorbance of the emulsion at 0 and 10 minutes, respectively.
3. Determination of solubility
Protein solubility was determined by dispersing the sample 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 model centrifuge. The soluble protein content was determined by Lowry before and after centrifugation.
4. Measurement of surface hydrophobicity
The samples were dispersed in phosphate buffer (50 mM, pH 7.4) at a final concentration of 0.1-0.5mg/mL. 20 μLANS (8.0 mM) was added to 4mL of the protein buffer, and fluorescence intensity was measured at 390nm (excitation) and 470nm (emission) using a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, japan), to calculate surface hydrophobicity.
Example 1
A method for modifying the protein isolate of hemp after ultrasonic physical field treatment comprises the following steps
(1) Extraction of cannabis isolated protein: hemp seeds were first fully crushed and defatted with 5-fold n-hexane. The defatted powder was dispersed in 5 volumes of ultrapure water, the pH was adjusted to 10.0 with 1mol/L NaOH, stirred at 25℃for 2 hours at room temperature, and centrifuged at 10000g for 30 minutes at 4℃to obtain a 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 washed with water 3 times to remove the salt. The resulting precipitate was redispersed in water, its pH was adjusted back to 7.0 with 1mol/L NaOH, and then freeze-dried.
(2) Sonication of the cannabis isolated protein solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred at 4 ℃ with a magnetic stirrer 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 at 150W/cm 2 . The ultrasonic time was set to 20 minutes.
(3) Glycosylation of cannabis isolated protein solution after sonication: the ultrasound treated cannabis protein solution was added with D-fructose and glycosylated at 70 ℃ for 12 hours after sufficient agitation. After glycosylation, the sample was quickly placed into an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and (3) performing dialysis and freeze drying on the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain the cannabis sativa isolated protein powder product obtained through ultrasonic-assisted glycosylation.
Example 2
A method for modifying the protein isolate of hemp after ultrasonic physical field treatment comprises the following steps
(1) Extraction of cannabis isolated protein: hemp seeds were first fully crushed and defatted with 5-fold n-hexane. The defatted powder was dispersed in 5 volumes of ultrapure water, the pH was adjusted to 10.0 with 1mol/L NaOH, stirred at 25℃for 2 hours at room temperature, and centrifuged at 10000g for 30 minutes at 4℃to obtain a 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 washed with water 3 times to remove the salt. The resulting precipitate was redispersed in water, its pH was adjusted back to 7.0 with 1mol/L NaOH, and then freeze-dried.
(2) Sonication of the cannabis isolated protein solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred at 4 ℃ with a magnetic stirrer 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 at 150W/cm 2 . The ultrasonic time was set at 40 minutes.
(3) Glycosylation of cannabis isolated protein solution after sonication: the ultrasound treated cannabis protein solution was added with D-fructose and glycosylated at 70 ℃ for 12 hours after sufficient agitation. After glycosylation, the sample was quickly placed into an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and (3) performing dialysis and freeze drying on the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain the cannabis sativa isolated protein powder product obtained through ultrasonic-assisted glycosylation.
Example 3
A method for modifying the protein isolate of hemp after ultrasonic physical field treatment comprises the following steps
(1) Extraction of cannabis isolated protein: hemp seeds were first fully crushed and defatted with 5-fold n-hexane. The defatted powder was dispersed in 5 volumes of ultrapure water, the pH was adjusted to 10.0 with 1mol/L NaOH, stirred at 25℃for 2 hours at room temperature, and centrifuged at 10000g for 30 minutes at 4℃to obtain a 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 washed with water 3 times to remove the salt. The resulting precipitate was redispersed in water, its pH was adjusted back to 7.0 with 1mol/L NaOH, and then freeze-dried.
(2) Sonication of the cannabis isolated protein solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred at 4 ℃ with a magnetic stirrer 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/cm2. The ultrasonic time was set at 60 minutes.
(3) Glycosylation of cannabis isolated protein solution after sonication: the ultrasound treated cannabis protein solution was added with D-fructose and glycosylated at 70 ℃ for 12 hours after sufficient agitation. After glycosylation, the sample was quickly placed into an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and (3) performing dialysis and freeze drying on the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain the cannabis sativa isolated protein powder product obtained through ultrasonic-assisted glycosylation.
Example 4
A method for modifying the protein isolate of hemp after ultrasonic physical field treatment comprises the following steps
(1) Extraction of cannabis isolated protein: hemp seeds were first fully crushed and defatted with 5-fold n-hexane. The defatted powder was dispersed in 5 volumes of ultrapure water, the pH was adjusted to 10.0 with 1mol/L NaOH, stirred at 25℃for 2 hours at room temperature, and centrifuged at 10000g for 30 minutes at 4℃to obtain a 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 washed with water 3 times to remove the salt. The resulting precipitate was redispersed in water, its pH was adjusted back to 7.0 with 1mol/L NaOH, and then freeze-dried.
(2) Sonication of the cannabis isolated protein solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred at 4 ℃ with a magnetic stirrer 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/cm2. The ultrasonic time was set at 80 minutes.
(3) Glycosylation of cannabis isolated protein solution after sonication: the ultrasound treated cannabis protein solution was added with D-fructose and glycosylated at 70 ℃ for 12 hours after sufficient agitation. After glycosylation, the sample was quickly placed into an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and (3) performing dialysis and freeze drying on the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain the cannabis sativa isolated protein powder product obtained through ultrasonic-assisted glycosylation.
SDS-PAGE analysis of cannabis isolated proteins with ultrasound-assisted glycosylation
SDS-PAGE analysis of the cannabis isolated protein samples prepared in examples 1-4 was performed as shown in FIG. 1: the results show that ultrasound helps to accelerate the glycosylation of the cannabis proteins, thereby increasing the molecular weight of the proteins and gradually lightening the bands.
Examples 1-4 were analyzed as follows:
emulsifying analysis of cannabis isolated proteins with ultrasound-assisted glycosylation
The results of the analysis of the emulsifiability of the hemp separation protein samples at 150W/cm2 and 45 ℃ under different ultrasonic treatment conditions (0, 20, 40, 60 and 80 min) are shown in the figure 2, wherein the emulsifiability is improved and then reduced along with the extension of the ultrasonic time, and the emulsifiability is strongest at the ultrasonic time of 40min.
Analysis of protein solubility of cannabis isolates with ultrasound-assisted glycosylation
The hemp isolate protein samples prepared in examples 1-4 were analyzed for solubility as shown in fig. 3: the results show that the solubility increases and then decreases with increasing ultrasound time, with the highest solubility at 40min ultrasound time.
Ultrasonic assisted glycosylation treated cannabis isolate protein surface hydrophobicity analysis
The surface hydrophobicity analysis of the cannabis isolated protein samples prepared in examples 1-4 was performed as shown in figure 4: the results show that ultrasonic treatment of the cannabis sativa isolated protein at different times can increase the surface hydrophobicity of the cannabis sativa isolated protein, the hydrophobicity of the cannabis sativa isolated protein gradually increases along with the increase of blood over time, and the surface hydrophobicity is strongest at 40min of ultrasonic time.
Comparative example 1
A method for modifying the protein isolate of hemp after ultrasonic physical field treatment comprises the following steps
(1) Extraction of cannabis isolated protein: hemp seeds were first fully crushed and defatted with 5-fold n-hexane. The defatted powder was dispersed in 5 volumes of ultrapure water, the pH was adjusted to 10.0 with 1mol/L NaOH, stirred at 25℃for 2 hours at room temperature, and centrifuged at 10000g for 30 minutes at 4℃to obtain a 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 washed with water 3 times to remove the salt. The resulting precipitate was redispersed in water, its pH was adjusted back to 7.0 with 1mol/L NaOH, and then freeze-dried.
(2) Sonication of the cannabis isolated protein solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred at 4 ℃ with a magnetic stirrer 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 at 150W/cm 2 . The ultrasonic time was set at 40 minutes.
(3) Glycosylation of cannabis isolated protein solution after sonication: the ultrasonic treated cannabis protein solution was added with fructooligosaccharides and glycosylated at 70 ℃ for 12 hours after sufficient agitation. After glycosylation, the sample was quickly placed into an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and (3) performing dialysis and freeze drying on the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain the cannabis sativa isolated protein powder product obtained through ultrasonic-assisted glycosylation.
Comparative example 2
A method for modifying the protein isolate of hemp after ultrasonic physical field treatment comprises the following steps
(1) Extraction of cannabis isolated protein: hemp seeds were first fully crushed and defatted with 5-fold n-hexane. The defatted powder was dispersed in 5 volumes of ultrapure water, the pH was adjusted to 10.0 with 1mol/L NaOH, stirred at 25℃for 2 hours at room temperature, and centrifuged at 10000g for 30 minutes at 4℃to obtain a 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 washed with water 3 times to remove the salt. The resulting precipitate was redispersed in water, its pH was adjusted back to 7.0 with 1mol/L NaOH, and then freeze-dried.
(2) Sonication of the cannabis isolated protein solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred at 4 ℃ with a magnetic stirrer 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 at 150W/cm 2 . The ultrasonic time was set at 40 minutes.
(3) Glycosylation of cannabis isolated protein solution after sonication: d sucrose is added into the hemp protein solution after ultrasonic treatment, and glycosylation is carried out for 12 hours at 70 ℃ after full stirring. After glycosylation, the sample was quickly placed into an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and (3) performing dialysis and freeze drying on the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain the cannabis sativa isolated protein powder product obtained through ultrasonic-assisted glycosylation.
Comparative example 3
A method for modifying the protein isolate of hemp after ultrasonic physical field treatment comprises the following steps
(1) Extraction of cannabis isolated protein: hemp seeds were first fully crushed and defatted with 5-fold n-hexane. The defatted powder was dispersed in 5 volumes of ultrapure water, the pH was adjusted to 10.0 with 1mol/L NaOH, stirred at 25℃for 2 hours at room temperature, and centrifuged at 10000g for 30 minutes at 4℃to obtain a 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 washed with water 3 times to remove the salt. The resulting precipitate was redispersed in water, its pH was adjusted back to 7.0 with 1mol/L NaOH, and then freeze-dried.
(2) Sonication of the cannabis isolated protein solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred at 4 ℃ with a magnetic stirrer 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 at 150W/cm 2 . The ultrasonic time was set at 40 minutes.
(3) Glycosylation of cannabis isolated protein solution after sonication: adding D stachyose into the solution of Cannabin protein after ultrasonic treatment, stirring thoroughly, and glycosylation at 70deg.C for 12 hr. After glycosylation, the sample was quickly placed into an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and (3) performing dialysis and freeze drying on the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain the cannabis sativa isolated protein powder product obtained through ultrasonic-assisted glycosylation.
Comparative example 4
A method for modifying the protein isolate of hemp after ultrasonic physical field treatment comprises the following steps
(1) Extraction of cannabis isolated protein: hemp seeds were first fully crushed and defatted with 5-fold n-hexane. The defatted powder was dispersed in 5 volumes of ultrapure water, the pH was adjusted to 10.0 with 1mol/L NaOH, stirred at 25℃for 2 hours at room temperature, and centrifuged at 10000g for 30 minutes at 4℃to obtain a 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 washed with water 3 times to remove the salt. The resulting precipitate was redispersed in water, its pH was adjusted back to 7.0 with 1mol/L NaOH, and then freeze-dried.
(2) Sonication of the cannabis isolated protein solution: HPI was dissolved in 50mM phosphate buffer pH 7.4 and then stirred at 4℃with a magnetic stirrer to ensure dissolutionThe liquid was fully hydrated. The protein solution was treated with an ultrasonic generator. The temperature is controlled at 45 ℃ and the power is set at 150W/cm 2 . The ultrasonic time was set at 40 minutes.
(3) Glycosylation of cannabis isolated protein solution after sonication: adding D-fructose into the Cannabin protein solution after ultrasonic treatment, stirring thoroughly, and glycosylation at 70deg.C for 3 hr. After glycosylation, the sample was quickly placed into an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and (3) performing dialysis and freeze drying on the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain the cannabis sativa isolated protein powder product obtained through ultrasonic-assisted glycosylation.
Comparative example 5
A method for modifying the protein isolate of hemp after ultrasonic physical field treatment comprises the following steps
(1) Extraction of cannabis isolated protein: hemp seeds were first fully crushed and defatted with 5-fold n-hexane. The defatted powder was dispersed in 5 volumes of ultrapure water, the pH was adjusted to 10.0 with 1mol/L NaOH, stirred at 25℃for 2 hours at room temperature, and centrifuged at 10000g for 30 minutes at 4℃to obtain a 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 washed with water 3 times to remove the salt. The resulting precipitate was redispersed in water, its pH was adjusted back to 7.0 with 1mol/L NaOH, and then freeze-dried.
(2) Sonication of the cannabis isolated protein solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred at 4 ℃ with a magnetic stirrer 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 at 150W/cm 2 . The ultrasonic time was set at 40 minutes.
(3) Glycosylation of cannabis isolated protein solution after sonication: adding D-fructose into the Cannabin protein solution after ultrasonic treatment, stirring thoroughly, and glycosylation at 70deg.C for 6 hr. After glycosylation, the sample was quickly placed into an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and (3) performing dialysis and freeze drying on the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain the cannabis sativa isolated protein powder product obtained through ultrasonic-assisted glycosylation.
Comparative example 6
A method for modifying the protein isolate of hemp after ultrasonic physical field treatment comprises the following steps
(1) Extraction of cannabis isolated protein: hemp seeds were first fully crushed and defatted with 5-fold n-hexane. The defatted powder was dispersed in 5 volumes of ultrapure water, the pH was adjusted to 10.0 with 1mol/L NaOH, stirred at 25℃for 2 hours at room temperature, and centrifuged at 10000g for 30 minutes at 4℃to obtain a 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 washed with water 3 times to remove the salt. The resulting precipitate was redispersed in water, its pH was adjusted back to 7.0 with 1mol/L NaOH, and then freeze-dried.
(2) Sonication of the cannabis isolated protein solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred at 4 ℃ with a magnetic stirrer 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 at 150W/cm 2 . The ultrasonic time was set at 40 minutes.
(3) Glycosylation of cannabis isolated protein solution after sonication: the ultrasonic treated cannabis protein solution was added with D-fructose and glycosylated at 70℃for 24 hours after sufficient agitation. After glycosylation, the sample was quickly placed into an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and (3) performing dialysis and freeze drying on the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain the cannabis sativa isolated protein powder product obtained through ultrasonic-assisted glycosylation.
Comparative example 7
A method for modifying the protein isolate of hemp after ultrasonic physical field treatment comprises the following steps
(1) Extraction of cannabis isolated protein: hemp seeds were first fully crushed and defatted with 5-fold n-hexane. The defatted powder was dispersed in 5 volumes of ultrapure water, the pH was adjusted to 10.0 with 1mol/L NaOH, stirred at 25℃for 2 hours at room temperature, and centrifuged at 10000g for 30 minutes at 4℃to obtain a 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 washed with water 3 times to remove the salt. The resulting precipitate was redispersed in water, its pH was adjusted back to 7.0 with 1mol/L NaOH, and then freeze-dried.
(2) Sonication of the cannabis isolated protein solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred at 4 ℃ with a magnetic stirrer 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 at 150W/cm 2 . The ultrasonic time was set at 40 minutes.
(3) Glycosylation of cannabis isolated protein solution after sonication: adding D-fructose into the Cannabin protein solution after ultrasonic treatment, stirring thoroughly, and glycosylation at 50deg.C for 12 hr. After glycosylation, the sample was quickly placed into an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and (3) performing dialysis and freeze drying on the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain the cannabis sativa isolated protein powder product obtained through ultrasonic-assisted glycosylation.
Comparative example 8
A method for modifying the protein isolate of hemp after ultrasonic physical field treatment comprises the following steps
(1) Extraction of cannabis isolated protein: hemp seeds were first fully crushed and defatted with 5-fold n-hexane. The defatted powder was dispersed in 5 volumes of ultrapure water, the pH was adjusted to 10.0 with 1mol/L NaOH, stirred at 25℃for 2 hours at room temperature, and centrifuged at 10000g for 30 minutes at 4℃to obtain a 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 washed with water 3 times to remove the salt. The resulting precipitate was redispersed in water, its pH was adjusted back to 7.0 with 1mol/L NaOH, and then freeze-dried.
(2) Sonication of the cannabis isolated protein solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred at 4 ℃ with a magnetic stirrer 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 at 150W/cm 2 . The ultrasonic time was set at 40 minutes.
(3) Glycosylation of cannabis isolated protein solution after sonication: adding D-fructose into the Cannabin protein solution after ultrasonic treatment, stirring thoroughly, and glycosylation at 60deg.C for 12 hr. After glycosylation, the sample was quickly placed into an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and (3) performing dialysis and freeze drying on the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain the cannabis sativa isolated protein powder product obtained through ultrasonic-assisted glycosylation.
Comparative example 9
A method for modifying the protein isolate of hemp after ultrasonic physical field treatment comprises the following steps
(1) Extraction of cannabis isolated protein: hemp seeds were first fully crushed and defatted with 5-fold n-hexane. The defatted powder was dispersed in 5 volumes of ultrapure water, the pH was adjusted to 10.0 with 1mol/L NaOH, stirred at 25℃for 2 hours at room temperature, and centrifuged at 10000g for 30 minutes at 4℃to obtain a 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 washed with water 3 times to remove the salt. The resulting precipitate was redispersed in water, its pH was adjusted back to 7.0 with 1mol/L NaOH, and then freeze-dried.
(2) Sonication of the cannabis isolated protein solution: HPI was dissolved in 50mM phosphate buffer at pH 7.4 and then stirred at 4 ℃ with a magnetic stirrer 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 at 150W/cm 2 . The ultrasonic time was set at 40 minutes.
(3) Glycosylation of cannabis isolated protein solution after sonication: adding D-fructose into the Cannabin protein solution after ultrasonic treatment, stirring thoroughly, and glycosylation at 80deg.C for 12 hr. After glycosylation, the sample was quickly placed into an ice-water mixture to terminate the reaction.
(4) And (3) freeze drying: and (3) performing dialysis and freeze drying on the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain the cannabis sativa isolated protein powder product obtained through ultrasonic-assisted glycosylation.
(4) And (3) freeze drying: and (3) performing dialysis and freeze drying on the HPI sample subjected to ultrasonic-assisted glycosylation treatment to obtain the cannabis sativa isolated protein powder product obtained through ultrasonic-assisted glycosylation.
The protein powders and the cannabis proteins prepared in examples 1 to 3 and comparative examples 1 to 9 were subjected to analysis of emulsifying property, emulsion stability, surface hydrophobicity and solubility, and are specifically shown in Table 1.
From the above detailed description and the analysis results of the experimental examples, it can be seen that the ultrasonic-assisted glycosylation treatment of the cannabis sativa isolated protein is an effective way to modify the functional properties of the cannabis sativa isolated protein, wherein the donor D-fructose is critical. By controlling the ultrasonic process, the temperature is 45 ℃, the ultrasonic time is 40min, and the ultrasonic intensity is 150W/cm 2 Then, the hemp protein isolate is subjected to glycosylation reaction at 70 ℃ for 12 hours to obtain the hemp protein with the best special new quality.
Table 1: emulsifying property, surface hydrophobicity and solubility of cannabis isolated protein treated by ultrasonic assisted glycosylation
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Claims (3)
1. A method for improving functional characteristics of cannabis protein by ultrasonic physical field assisted glycosylation comprises the following steps
(1) Protein extraction: extracting cannabis sativa seed protein, adjusting to neutrality, and freeze drying;
(2) And (3) ultrasonic treatment: dissolving the cannabis protein prepared in the step 1) in phosphate buffer solution, performing ultrasonic treatment at 45 ℃ and ultrasonic power of 150W/cm 2 Ultrasonic time is 40min;
(3) Glycosylation treatment: adding D-fructose into the Cannabin protein solution after ultrasonic treatment, fully stirring, carrying out glycosylation at 70 ℃ for 12 hours, and rapidly placing a sample into an ice-water mixture to terminate 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 a final product.
2. The method of claim 1, wherein the protein extraction step is as follows: firstly, fully crushing hemp seeds, degreasing with 5 times of n-hexane, dispersing the degreased powder into 5 times of volume of ultrapure water, adjusting the pH to 10.0 with 1mol/L NaOH, stirring for 2 hours at room temperature at 25 ℃, and centrifuging for 30 minutes at 10000g at 4 ℃ to obtain supernatant; adjusting pH of the supernatant to 4.5 with 1mol/L HCl, centrifuging at 4deg.C at 10000g for 30min, collecting precipitate, and washing the precipitate with water for 3 times to remove salt; the resulting precipitate was redispersed in water, its pH was adjusted back to 7.0 with 1mol/L NaOH, and then freeze-dried.
3. A modified cannabis protein prepared by the method of claim 1.
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