CN113367227A - Method for simultaneously extracting nannochloropsis oculata protein and dietary fiber by using ultrasonic wave assistance - Google Patents
Method for simultaneously extracting nannochloropsis oculata protein and dietary fiber by using ultrasonic wave assistance Download PDFInfo
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- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 4
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/009—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from unicellular algae
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/015—Inorganic compounds
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/06—Enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
- A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
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- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
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- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
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- A23L5/30—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation
- A23L5/32—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation using phonon wave energy, e.g. sound or ultrasonic waves
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Abstract
The invention discloses a method for simultaneously extracting nannochloropsis oculata protein and dietary fiber by utilizing ultrasonic assistance, and relates to the technical field of extraction of protein and dietary fiber. Comprises the steps of degreasing, wall breaking, double-enzyme enzymolysis, enzyme deactivation, solid-liquid separation, protein extraction, soluble dietary fiber separation, heavy metal removal and the like; the method has the advantages of simple operation, high extraction rate of protein and dietary fiber, and low heavy metal content, and three products of nannochloropsis oculata protein, nannochloropsis oculata soluble dietary fiber and nannochloropsis oculata insoluble dietary fiber can be simultaneously obtained in one technological process.
Description
Technical Field
The invention relates to the technical field of extraction of protein and dietary fiber, in particular to a method for simultaneously extracting nannochloropsis oculata protein and dietary fiber by utilizing ultrasonic assistance.
Background
Nannochloropsis sp is a unicellular marine algae, and has high ecological and economic values.
The nannochloropsis oculata is rich in DHA, EPA, beta-carotene, etc., and has effects of promoting infantile brain and vision development, reducing blood lipid, resisting cancer, resisting inflammation, and delaying aging. The nannochloropsis has wide application prospect as a sustainable renewable energy source. At present, nannochloropsis is mainly used for producing biodiesel, and a large amount of algae residues which account for 50-70% of raw materials can be produced after oil extraction of microalgae. These algal residues also contain a large amount of dietary fiber and protein, which account for 20% and 50% of algal residues, respectively. However, the nannochloropsis cell has thick wall and compact wall, and the combination between the dietary fiber and the protein is tight, so that the nannochloropsis dietary fiber and the protein have low extraction rate and low purity. In the past, the algae residues are not fully utilized, so that great resource waste and environmental pollution are caused. On the other hand, due to the water body pollution, the characteristics of marine organisms and the like, the content of heavy metals in the marine organisms and derivatives exceeds the standard inevitably, and potential safety hazards are brought to the application of marine products.
Dietary fiber mainly exists in grains, fruits, vegetables and algae, has special physiological functions, plays an important role in daily life of people, and is known as 'the seventh major nutrient'. A large number of studies show that the dietary fiber can reduce the postprandial blood sugar level and has good effect on controlling the blood sugar of the diabetic; can also adsorb cholesterol, cholate, nitrite, etc., and promote discharge of cholesterol and harmful substances. In addition, the dietary fiber has good water absorption and expansibility, can generate satiety, has a certain effect on controlling diet of obesity patients, and also has the effects of improving intestinal flora, regulating intestinal functions and preventing intestinal diseases.
The essential amino acid content of the nannochloropsis oculata protein accounts for 40 percent of the total amino acids, the EAAI accounts for about 94 percent, and the nannochloropsis oculata protein is a high-quality protein and has good application prospect in the aspects of food, medicine and the like.
Most of the current methods remove protein as an impurity when extracting dietary fiber, and remove dietary fiber as an impurity when extracting protein. For example, chinese patent CN201811172799.7 reports a preparation method and application of phycocyanin from spirulina, and chinese patent CN111988997A reports a method for obtaining protein or protein-rich extract from algae, extract and application thereof, all of which are single component extraction. Chinese invention patent CN109593128B discloses a method for industrially co-producing phycocyanin, spirulina polysaccharide and protein feed by using fresh spirulina, compared with the method, the method does not consider the problem that heavy metal in the product exceeds the standard, the adsorption characteristic of the spirulina makes the spirulina easily adsorb the heavy metal in the growth environment, and if the spirulina is not removed in the processing, the edible safety of the final product is affected.
Disclosure of Invention
In order to solve the problems of low utilization rate of nannochloropsis oculata residue, high production cost of nannochloropsis oculata dietary fiber and protein, long extraction time, high loss rate, excessive heavy metal content of the obtained dietary fiber and the like at present, the invention provides a method for simultaneously extracting nannochloropsis oculata protein and dietary fiber by utilizing ultrasonic wave assistance, and the purposes of improving the extraction rate of the dietary fiber and protein and shortening the extraction time are achieved by ultrasonic-assisted alkaline extraction; organic acid is adopted to remove heavy metals in the soluble dietary fiber and the insoluble dietary fiber, and the removal effect is good. The obtained dietary fiber and protein have high purity and high safety, thereby improving the additional value of nannochloropsis oculata, prolonging the production chain of nannochloropsis oculata and reducing the production cost.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for simultaneously extracting nannochloropsis oculata protein and dietary fiber by using ultrasonic wave assistance comprises the following steps:
s1, degreasing: removing residual fat from nannochloropsis oculata powder with supercritical CO2 at extraction pressure of 20MPa, extraction temperature of 37.5 deg.C, separation pressure of 4MPa, separation temperature of 40 deg.C, and extraction time of 2 hr to obtain defatted nannochloropsis oculata powder;
s2, wall breaking: adding degreased nannochloropsis oculata powder into water, wherein the mass ratio of the degreased nannochloropsis oculata powder to the water is 1:10, and performing ultrasonic wall breaking for 20 minutes; the energy-gathering ultrasonic wave for wall breaking has frequency of 20kHz, power of 1000W, and wall breaking time of 30 min;
s3, double-enzyme enzymolysis: heating the degreased nannochloropsis oculata powder suspension subjected to wall breaking to 50 ℃, preserving heat, adding neutral protease according to 3% of the mass ratio of an enzyme substrate, adding cellulase according to 3% of the mass ratio of the enzyme substrate without controlling pH, and carrying out ultrasonic-assisted enzymolysis reaction for 15min, wherein the frequency of the used divergent ultrasonic is 40kHz, and the power is 300W;
s4 enzyme deactivation: ultrasonic enzyme deactivation is carried out for 10min, and energy-gathered ultrasonic frequency for enzyme deactivation is 20kHz and power is 2000W;
s5, carrying out solid-liquid separation, and carrying out 3000rmp centrifugal separation;
s6 protein extraction: s5, adding ammonia water into the obtained supernatant to adjust the pH value to 11, carrying out ultrasonic extraction for 30min, carrying out centrifugation for 20min at the frequency of 20kHz of the used dispersed ultrasonic and the power of 1200W to obtain the supernatant, adding 1M organic acid mixture to adjust the pH value to 4.3, carrying out centrifugation at 3000rmp to obtain a precipitate, washing and drying to obtain nannochloropsis oculata protein;
s7 isolating soluble dietary fiber: adding 95% ethanol of one tenth volume into the supernatant obtained in S6, centrifuging to obtain precipitate, washing and drying to obtain soluble dietary fiber;
s8 heavy metal removal: and (S5) mixing the obtained precipitate according to the mass ratio of 1: 3 adding water for dispersion, adding 1M organic acid mixture to adjust the pH to 6, 100KHz, and carrying out 300W ultrasonic treatment for 10 min; adding 1M desalting agent according to the volume ratio of 10%, 100KHz, and performing ultrasonic treatment at 300W for 10 min; centrifuging, and precipitating to obtain insoluble dietary fiber;
further, in step S6, the organic acid is one or more of citric acid, acetic acid, lactic acid, and the like.
Further, in step S8, the organic acid is one or more of citric acid, acetic acid, lactic acid, and the like.
Further, the salt removing agent in step S8 is CaO and NaHCO3And FeS and the like.
By the technical scheme, the extraction method provided by the invention can respectively obtain the dietary fiber and the protein without heavy metals, and the dietary fiber and the protein are respectively or cooperatively applied according to different functional characteristics of the dietary fiber and the protein, so that the aim of reasonably utilizing resources is fulfilled.
Compared with the prior art, the invention has the beneficial effects that:
1. the wall breaking by ultrasonic waves and the enzymolysis of the double enzymes are beneficial to the destruction of the inherent association relationship between the microalgae dietary fibers and the protein, the separation of the microalgae dietary fibers and the protein is facilitated, and the extraction yield and the purity of the protein and the dietary fibers are improved;
2. the acid used in the enzymolysis process is organic acid, ammonia water is used as alkali, sodium ions are not introduced, and the desalting burden in the subsequent purification process is reduced;
3. organic acid ultrasonic wave assists heavy metal removal, improves product safety. The unique adsorption performance of the microalgae enables the microalgae to naturally enrich heavy metals in the growth process, and the heavy metals are removed by ultrasonic assistance, so that the content of the heavy metals in the final product is reduced;
4. ultrasonic enzyme deactivation is adopted, so that damage of heating to product activity is reduced;
5. the method can simultaneously obtain three products of nannochloropsis oculata protein, soluble dietary fiber and insoluble dietary fiber in one technological process, and has the advantages of simple technological operation and high production efficiency.
Detailed Description
The technical solution of the present invention will be clearly and completely described in the following embodiments of the present invention, but the application is not limited to the embodiments.
Examples 1 to 6
S1, degreasing: removing residual fat from nannochloropsis oculata powder with supercritical CO2 at extraction pressure of 20MPa, extraction temperature of 37.5 deg.C, separation pressure of 4MPa, separation temperature of 40 deg.C, and extraction time of 2 hr to obtain defatted nannochloropsis oculata powder; s2, wall breaking: adding degreased nannochloropsis oculata powder into water, wherein the mass ratio of the degreased nannochloropsis oculata powder to the water is 1:10, and performing ultrasonic wall breaking for 20 minutes; the energy-gathering ultrasonic frequency for breaking cell wall is 20KHz, power is 1000W, and cell wall breaking time is 30 min; s3, double-enzyme enzymolysis: heating the degreased nannochloropsis oculata powder suspension after wall breaking to 50 ℃, preserving heat, adding neutral protease according to 3% of the mass ratio of an enzyme substrate, adding cellulase according to 3% of the mass ratio of the enzyme substrate without controlling pH, and carrying out ultrasonic-assisted enzymolysis reaction for 15min, wherein the frequency of the used divergent ultrasonic is 40kHz, and the power is 300W; s4 enzyme deactivation: ultrasonic enzyme deactivation is carried out for 10min, and energy-gathered ultrasonic frequency for enzyme deactivation is 20kHz and power is 2000W; s5, carrying out solid-liquid separation, and carrying out 3000rmp centrifugal separation; s6 protein extraction: s5 adding ammonia water into the obtained supernatant to adjust the pH value to 11, carrying out ultrasonic extraction for 30min, using a divergent ultrasonic frequency of 20kHz and a power of 1200W, centrifuging for 20min to obtain a supernatant, adding a 1M organic acid mixture to adjust the pH value to 4.3, centrifuging at 3000rmp to obtain a precipitate, washing and drying to obtain nannochloropsis oculata protein; s7 isolating soluble dietary fiber: adding 95% ethanol of one tenth volume into the supernatant obtained in S6, centrifuging to obtain precipitate, washing and drying to obtain soluble dietary fiber; s8 heavy metal removal: and (S5) mixing the obtained precipitate according to the mass ratio of 1: 3 adding water for dispersion, adding 1M organic acid mixture to adjust the pH to 6, 100KHz, and carrying out 300W ultrasonic treatment for 10 min; adding 1M desalting agent according to the volume ratio of 10%, and performing ultrasonic treatment at 100KHz under 300W for 10 min; centrifuging, and precipitating to obtain insoluble dietary fiber;
other parameters of examples 1 to 6 are shown in Table 1.
TABLE 1 table of parameters of examples 1 to 6
Comparative example
The production process is the same as the embodiment, and the difference is that the extraction process is not assisted by ultrasound, and the heavy metal removal step is not carried out.
The yield, purity and heavy metal content of the protein and the dietary fiber prepared by the examples and the comparative examples are shown in Table 2.
TABLE 2 results of protein and dietary fiber yield, purity and heavy metal content determination in examples
Note: in the table SDF stands for soluble dietary fiber and IDF for insoluble dietary fiber.
Claims (4)
1. A method for simultaneously extracting nannochloropsis oculata protein and dietary fiber by using ultrasonic wave assistance is characterized by comprising the following steps:
s1 degreasing: removing residual fat from nannochloropsis oculata powder with supercritical CO2 at extraction pressure of 20MPa, extraction temperature of 37.5 deg.C, separation pressure of 4MPa, separation temperature of 40 deg.C, and extraction time of 2 hr to obtain defatted nannochloropsis oculata powder;
s2 wall breaking: adding degreased nannochloropsis oculata powder into water, wherein the mass ratio of the degreased nannochloropsis oculata powder to the water is 1:10, and performing ultrasonic wall breaking for 20 minutes; the energy-gathering ultrasonic wave for wall breaking has frequency of 20kHz, power of 1000W, and wall breaking time of 30 min;
s3 double enzyme enzymolysis: heating the degreased nannochloropsis oculata powder suspension subjected to wall breaking to 50 ℃, preserving heat, adding neutral protease according to 3% of the mass ratio of enzyme to substrate, adding cellulase according to 3% of the mass ratio of enzyme to substrate without controlling pH, and carrying out ultrasonic-assisted enzymolysis reaction for 15min, wherein the frequency of the used divergent ultrasonic wave is 40kHz, and the power is 300W;
s4 enzyme deactivation: ultrasonic enzyme deactivation is carried out for 10min, and energy-gathered ultrasonic frequency for enzyme deactivation is 20kHz and power is 2000W;
s5, carrying out solid-liquid separation, and carrying out 3000rmp centrifugal separation;
s6 protein extraction: s5 adding ammonia water into the obtained supernatant to adjust the pH value to 11, carrying out ultrasonic extraction for 30min, using a divergent ultrasonic frequency of 20kHz and a power of 1200W, centrifuging for 20min to obtain a supernatant, adding a 1M organic acid mixture to adjust the pH value to 4.3, centrifuging at 3000rmp to obtain a precipitate, washing and drying to obtain nannochloropsis oculata protein;
s7 isolating soluble dietary fiber: adding 95% ethanol of one tenth volume into the supernatant obtained in S6, centrifuging to obtain precipitate, washing and drying to obtain soluble dietary fiber;
s8 heavy metal removal: and (S5) mixing the obtained precipitate according to the mass ratio of 1: 3 adding water for dispersion, adding 1M organic acid mixture to adjust the pH to 6, 100KHz, and carrying out 300W ultrasonic treatment for 10 min; adding 1M desalting agent according to the volume ratio of 10%, 100KHz, and performing ultrasonic treatment at 300W for 10 min; centrifuging, and precipitating to obtain insoluble dietary fiber.
2. The method for simultaneously extracting nannochloropsis oculata protein and dietary fiber with the aid of ultrasonic waves as claimed in claim 1, wherein in said step S6, the organic acid is one or more of citric acid, acetic acid, lactic acid, etc.
3. The method for simultaneously extracting nannochloropsis oculata protein and dietary fiber with the aid of ultrasonic waves as claimed in claim 1, wherein in said step S8, the organic acid is one or more of citric acid, acetic acid, lactic acid, etc.
4. The method for simultaneously extracting nannochloropsis oculata protein and dietary fiber with ultrasonic wave assistance as claimed in claim 1, wherein the desalting agent in step S8 is CaO, NaHCO3And FeS and the like.
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