CN117044940B - Krill oil haematococcus pluvialis and preparation method thereof - Google Patents
Krill oil haematococcus pluvialis and preparation method thereof Download PDFInfo
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- CN117044940B CN117044940B CN202310868213.5A CN202310868213A CN117044940B CN 117044940 B CN117044940 B CN 117044940B CN 202310868213 A CN202310868213 A CN 202310868213A CN 117044940 B CN117044940 B CN 117044940B
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- 241000168517 Haematococcus lacustris Species 0.000 title claims abstract description 184
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- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- JEBFVOLFMLUKLF-IFPLVEIFSA-N Astaxanthin Natural products CC(=C/C=C/C(=C/C=C/C1=C(C)C(=O)C(O)CC1(C)C)/C)C=CC=C(/C)C=CC=C(/C)C=CC2=C(C)C(=O)C(O)CC2(C)C JEBFVOLFMLUKLF-IFPLVEIFSA-N 0.000 claims abstract description 118
- 239000001168 astaxanthin Substances 0.000 claims abstract description 118
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- JAZBEHYOTPTENJ-JLNKQSITSA-N all-cis-5,8,11,14,17-icosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O JAZBEHYOTPTENJ-JLNKQSITSA-N 0.000 description 4
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- 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
- A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
- A23L17/60—Edible seaweed
-
- 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
- A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
- A23L17/65—Addition of, or treatment with, microorganisms or enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/115—Fatty acids or derivatives thereof; Fats or oils
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
- A23P10/00—Shaping or working of foodstuffs characterised by the products
- A23P10/30—Encapsulation of particles, e.g. foodstuff additives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Marine Sciences & Fisheries (AREA)
- Zoology (AREA)
- Botany (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Microbiology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a krill oil rain red ball algae and a preparation method thereof. The krill oil haematococcus pluvialis consists of mixed nutritional oil and soft capsules wrapping the mixed nutritional oil, wherein the mixed nutritional oil consists of antarctic krill oil and haematococcus pluvialis astaxanthin oil, and the haematococcus pluvialis astaxanthin oil is prepared by extracting haematococcus pluvialis powder with an organic solvent or further combining an enzymolysis method. The rhodococcus pluvialis has the functions of resisting oxidation, resisting free radical, resisting inflammation, etc. It can effectively inhibit active oxygen in vivo, protect cells from oxidative damage, delay aging, and has certain nursing effects on heart disease, diabetes, tumor and other diseases. In addition, can reduce cholesterol in blood, lower cholesterol level in blood, improve blood circulation, and reduce risk of cardiovascular disease.
Description
Technical Field
The invention relates to an edible oil product, in particular to rhodococcus acidilactici for krill oil rain and a preparation method thereof.
Background
The krill oil haematococcus pluvialis is a compound product with high nutritive value and health efficacy, and is mixed nutritive oil mainly composed of antarctic krill oil and haematococcus pluvialis astaxanthin oil, and then the mixed nutritive oil is wrapped and protected by a soft capsule technology so as to be convenient to store and take. Such products are important in the health care market today.
Euphausia superba oil, as the name implies, originates from Euphausia superba. The small shrimps living in extremely cold environments have unique nutritional value and are an important source of omega-3 polyunsaturated fatty acids in phospholipid form, EPA and DHA in phospholipid form. EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) are fatty acids which are essential to the human body but cannot be synthesized by themselves, and have an important role in maintaining heart, brain and retina health. The traditional EPA and DHA supplements are usually mainly fish oil, but the antarctic krill oil can be better digested and absorbed by human bodies due to the characteristic of phospholipid form, so that the health care effect is more remarkable.
Another ingredient that cooperates with antarctic krill oil is haematococcus pluvialis astaxanthin oil. Astaxanthin is a powerful antioxidant which has remarkable effects in maintaining eye health, resisting oxidative stress, delaying aging, and the like. It is distributed in many organisms, however haematococcus pluvialis is considered to be the best source of astaxanthin. On the one hand, the astaxanthin content in haematococcus pluvialis is very high and is as high as 1.5% -3.0%. This is the highest level in all organisms, so haematococcus pluvialis is sometimes considered a "concentrated version" of astaxanthin. The method remarkably improves the efficiency of extracting the astaxanthin from haematococcus pluvialis and is beneficial to the commercial production and application of the astaxanthin. On the other hand, astaxanthin in haematococcus pluvialis has a special molecular structure and is in a levorotatory form. The unique molecular structure makes astaxanthin available in human body cell and has high bioavailability. It can be absorbed and utilized by human body, so that the antioxidant and protecting effects of astaxanthin are more obvious. Astaxanthin of the L-structure is more bioactive than other forms of astaxanthin and is therefore more preferred to be selected when supplementing antioxidants or as a component of health products. Thus, haematococcus pluvialis is an ideal source for extracting astaxanthin, both in terms of content and biological activity. Although the production cost may be higher than other sources, the high bioavailability and the strong antioxidant capacity make astaxanthin extracted from haematococcus pluvialis widely applied to health foods and antioxidant products. This is also why scientists and manufacturers have focused on studying and using Haematococcus pluvialis as a source of astaxanthin.
Therefore, the euphausia superba oil haematococcus pluvialis combines the advantages of euphausia superba oil and haematococcus pluvialis astaxanthin oil, and provides a composite health product with rich nutrition and obvious efficacy. The application of the soft capsule technology ensures that the wrapping protection of the mixed nutritional oil is more thorough, which is beneficial to prolonging the quality guarantee period of the product and preventing the nutritional ingredients from being damaged in the process of storage and taking. In addition, the soft capsule form of the product is easier to swallow and absorb, and is more convenient for various consumers.
However, in the prior art, for preparing the krill oil haematococcus pluvialis, especially for extracting astaxanthin oil from the haematococcus pluvialis, the efficiency and the quality are not high, so that the quality of the haematococcus pluvialis caused by the krill oil haematococcus pluvialis is also problematic.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to solve the technical problems that: provides a rhodococcus rhodochrous for krill oil rain.
The invention aims at optimizing the preparation process of the rhodococcus pluvialis astaxanthin oil which is a core raw material of the rhodococcus pluvialis, improving the yield and the quality. Specifically, the preparation process of haematococcus pluvialis astaxanthin oil adopts organic solvent extraction, and the optimized extraction is realized by combining the organic solvent with enzymolysis.
The detailed technical scheme of the invention is as follows:
the krill oil haematococcus pluvialis consists of mixed nutritional oil and soft capsules wrapping the mixed nutritional oil, wherein the mixed nutritional oil consists of antarctic krill oil and haematococcus pluvialis astaxanthin oil;
The haematococcus pluvialis astaxanthin oil is prepared by an extraction method of haematococcus pluvialis powder organic solvent.
Preferably, the mixed nutritional oil consists of euphausia superba oil and haematococcus pluvialis astaxanthin oil according to the mass ratio of (50-90) to (10-50).
Preferably, the mixed nutritional oil consists of euphausia superba oil and haematococcus pluvialis astaxanthin oil according to the mass ratio of (55-85) to (25-45).
Preferably, the mixed nutritional oil consists of euphausia superba oil and haematococcus pluvialis astaxanthin oil according to the mass ratio of (70-80) to (30-40).
Specifically, the invention discloses a method for extracting haematococcus pluvialis astaxanthin oil, which comprises the following steps:
1, pretreatment: grinding Haematococcus pluvialis into powder to obtain Haematococcus pluvialis powder;
2, extracting: adding an organic solvent to extract haematococcus pluvialis algae powder;
3, separating: filtering and separating the organic solvent from haematococcus pluvialis algae powder;
4, distilling.
Preferably, the extraction method of haematococcus pluvialis astaxanthin oil comprises the following steps:
1, pretreatment: grinding Haematococcus pluvialis into powder to obtain Haematococcus pluvialis powder;
2, extracting: adding an organic solvent, stirring at 30-60rpm at 18-25 ℃ for 1-3 hours, and standing at 18-25 ℃ for 2-4 hours, wherein the dosage of the organic solvent is 3-5 times of the mass of haematococcus pluvialis powder;
3, separating: filtering and separating the organic solvent from haematococcus pluvialis algae powder;
4, distilling: and (5) distilling under reduced pressure to obtain haematococcus pluvialis astaxanthin oil.
Preferably, the haematococcus pluvialis astaxanthin oil is prepared by combining organic solvent extraction and enzymolysis of haematococcus pluvialis powder.
Further preferably, the extraction method of haematococcus pluvialis astaxanthin oil comprises the following steps:
1, pretreatment: grinding Haematococcus pluvialis into powder to obtain Haematococcus pluvialis powder;
2, enzymolysis: adding lipase into haematococcus pluvialis algae powder for enzymolysis;
3, extracting: adding an organic solvent to extract haematococcus pluvialis algae powder;
4, separating: filtering and separating the organic solvent from haematococcus pluvialis algae powder;
And 5, distilling.
Most preferably, the extraction method of haematococcus pluvialis astaxanthin oil comprises the following steps:
1, pretreatment: grinding Haematococcus pluvialis into powder to obtain Haematococcus pluvialis powder;
2, enzymolysis: adding 1-2 times of water and candida antarctica lipase B into haematococcus pluvialis powder, keeping the temperature at 50-60 ℃ and stirring at the speed of 30-60rpm for 4-6 hours to obtain a mixture, wherein the dosage of the candida antarctica lipase B is 5000-20000 units per gram of haematococcus pluvialis powder;
3, secondary enzymolysis and emulsification: adding cholesterol esterase and emulsifier lecithin into the mixture, and continuously stirring at 30-40 ℃ at a speed of 30-60rpm for 1-3 hours, wherein the cholesterol esterase is 5000-20000 units per gram of haematococcus pluvialis powder, and the emulsifier is 0.5-2% of the haematococcus pluvialis powder;
4, solvent extraction: then adding organic solvent ethanol, keeping at 18-25 ℃, stirring at 30-60rpm for 1-3 hours, and then standing for 2-4 hours, wherein the dosage of the organic solvent ethanol is 3-5 times of the quality of haematococcus pluvialis algae powder;
5, separating: filtering and separating the mixed system in the step 4, and performing rotary evaporation to obtain an oily ethanol solution;
6, reduced pressure distillation: and (3) distilling the oily ethanol solution under reduced pressure to obtain haematococcus pluvialis astaxanthin oil.
The invention also discloses haematococcus pluvialis astaxanthin oil which is prepared by adopting the method.
The antarctic krill oil is prepared by a conventional method in the field. Obviously, the optimization of the preparation method of the antarctic krill oil also greatly improves the quality of the product, and the inventor can conduct discussion, experiments and analysis in the subsequent research and development. The present invention may be accomplished by selecting antarctic krill oil from applicant company.
The invention also provides a preparation method of the krill oil rain haematococcus, which comprises the following steps:
uniformly mixing antarctic krill oil and haematococcus pluvialis astaxanthin oil to obtain mixed nutrition oil; the mixed nutritional oil is pressed into soft capsules.
Obviously, the krill oil haematococcus pluvialis is a soft capsule, is an edible oil product, and contains the mixed nutritional oil consisting of antarctic krill oil and haematococcus pluvialis astaxanthin oil.
The rhodococcus pluvialis has the functions of resisting oxidation, resisting free radical, resisting inflammation, etc. It can effectively inhibit active oxygen in vivo, protect cells from oxidative damage, delay aging, and has certain nursing effects on heart disease, diabetes, tumor and other diseases. In addition, the rhodococcus rhodochrous astaxanthin produced by the euphausia superba oil can reduce cholesterol in blood, lower the cholesterol level in blood, improve blood circulation and reduce the risk of cardiovascular diseases. It also can inhibit the growth of cancer cells, prevent cancer, improve liver function, enhance immunity, and enhance disease resistance.
Detailed Description
The extraction method of haematococcus pluvialis astaxanthin oil comprises the following steps:
1, pretreatment: grinding Haematococcus pluvialis into powder to obtain Haematococcus pluvialis powder; grinding Haematococcus pluvialis into powder can increase the specific surface area of the material, so as to improve the efficiency of the subsequent extraction step. Grinding Haematococcus pluvialis into powder can break the cell wall of the algae, so that enzymes and solvents can be more easily contacted with astaxanthin in cells, and the extraction efficiency is also improved.
2, Enzymolysis: adding 1-2 times of water and candida antarctica lipase B into haematococcus pluvialis powder, keeping the temperature at 50-60 ℃ and stirring at the speed of 30-60rpm for 4-6 hours to obtain a mixture, wherein the dosage of the candida antarctica lipase B is 5000-20000 units per gram of haematococcus pluvialis powder; in this process, the primary function of the enzymatic hydrolysis step is to disrupt the structure of the algae cells in order to release astaxanthin from the interior of the cells. In particular, in the microalgae species having a strong pressure-resistant ability such as haematococcus pluvialis, the cell wall is hard and is not easily broken, and therefore, the cell wall needs to be broken by the action of enzymes, and astaxanthin in the cells is released.
3, Secondary enzymolysis and emulsification: adding cholesterol esterase and emulsifier lecithin into the mixture, and continuously stirring at 30-40 ℃ at a speed of 30-60rpm for 1-3 hours, wherein the cholesterol esterase is 5000-20000 units per gram of haematococcus pluvialis powder, and the emulsifier is 0.5-2% of the haematococcus pluvialis powder; in the extraction process of astaxanthin, the step of secondary enzymolysis and emulsification plays a key role. This step occurs after the first enzymatic hydrolysis and has the main purpose of continuing to break down and disrupt the structure of the algae cells, thereby releasing more astaxanthin. Cholesterol esterase is used in the secondary enzymatic hydrolysis process, which further breaks down the remaining lipids and destroys the structure of the algae cells. The goal of this approach is to maximize astaxanthin release, thereby making it easier to extract from the algal cells. In addition, the addition of the emulsifier lecithin plays a very important role. The emulsifier can enhance the compatibility of the fat-soluble substance (e.g., astaxanthin) with the aqueous phase, so that the dispersibility of astaxanthin in the aqueous environment is enhanced. This allows more efficient extraction of astaxanthin from the algal cells, as it reduces the interfacial tension between water and astaxanthin, so that astaxanthin can be released from the cells and into the aqueous phase more easily.
4, Solvent extraction: then adding organic solvent ethanol, keeping at 18-25 ℃, stirring at 30-60rpm for 1-3 hours, and then standing for 2-4 hours, wherein the dosage of the organic solvent ethanol is 3-5 times of the quality of haematococcus pluvialis algae powder; in extracting the bioactive substance haematococcus pluvialis astaxanthin oil, solvent extraction generally involves dissolving the target compound haematococcus pluvialis astaxanthin oil from a solid matrix, and then separating the solvent and solute by filtration or centrifugation or the like, ethanol has good solubility and relatively high safety, and its volatility also makes it relatively easy to operate in a subsequent solvent recovery step.
5, Separating: filtering and separating the mixed system in the step 4, and performing rotary evaporation to obtain an oily ethanol solution; the step is that the ethanol solution containing haematococcus pluvialis astaxanthin oil is separated from haematococcus pluvialis powder, and the solvent is removed by rotary evaporation to obtain the more concentrated haematococcus pluvialis astaxanthin oil ethanol solution.
6, Reduced pressure distillation: and (3) distilling the oily ethanol solution under reduced pressure to obtain haematococcus pluvialis astaxanthin oil.
A preparation method of rhodococcus pluvialis for krill oil rain comprises the following steps:
1.1, adding antarctic krill oil and haematococcus pluvialis astaxanthin oil with proper parameters into a mixing tank in advance according to requirements;
1.2, starting a mixer, mixing two kinds of oil for 1-24 hours, sampling and detecting at different time intervals, and uniformly mixing after the detection result is stable to obtain mixed nutrient oil;
1.3 evenly mixing gelatin, glycerol and water according to the mass ratio of 1 (0.2-0.6) (0.6-1), heating to 60-90 ℃ and keeping for 1-3 hours;
1.4 after no gelatin particles are sensed, vacuumizing is started, the negative pressure value is 0.04-0.06Mpa, and the time is 0.5-2 hours;
1.5 pressing the mixed nutritional oil into the soft capsules by using soft capsule production equipment;
shaping at 20-40deg.C and humidity of 15-40% for 0.5-2 hr;
1.7, drying at 20-40 ℃ and humidity of 15-40% for 2-36 hours;
1.8, and controlling the weight of the soft capsule to be 0.2-0.3 g and the weight of the mixed oil to be 0.4-0.6 g in each capsule.
Candida antarctica Lipase B supplied by Shanghai Kangdi Biotechnology Co., ltd. Chiralzyme IM-100 Candida antarctica Lipase B.
The cholesterol esterase is 10KU cholesterol esterase provided by Synbiotics biosciences, inc.
Lecithin, which is provided by Hebei Pengyu biotechnology Co., ltd, is selected and has a content of 99%.
Example 1
The extraction method of haematococcus pluvialis astaxanthin oil comprises the following steps:
1, pretreatment: grinding Haematococcus pluvialis to obtain Haematococcus pluvialis powder;
2, enzymolysis: adding 1.5 times of water and candida antarctica lipase B into haematococcus pluvialis powder, keeping the temperature at 55 ℃ and stirring at 50rpm for 5 hours to obtain a mixture, wherein the dosage of the candida antarctica lipase B is 10000 units per gram of haematococcus pluvialis powder;
3, secondary enzymolysis and emulsification: adding candida antarctica lipase B and emulsifier lecithin into the mixture, keeping the temperature at 35 ℃ and stirring the mixture at a speed of 50rpm for 2 hours, wherein the dosage of the candida antarctica lipase B is 10000 units per gram of haematococcus pluvialis powder, and the dosage of the emulsifier is 1% of the mass of the haematococcus pluvialis powder;
4, solvent extraction: then adding organic solvent ethanol, keeping at 20 ℃ and stirring at 50rpm for 2 hours, and then standing for 3 hours, wherein the dosage of the organic solvent ethanol is 4 times of the mass of haematococcus pluvialis algae powder;
5, separating: filtering and separating the mixed system in the step 4 with a pore diameter of 0.2mm, and performing rotary evaporation (heating temperature is 50 ℃, and rotary evaporation speed is set at 150 rpm) to obtain an oily ethanol solution;
6, reduced pressure distillation: the oily ethanol solution was distilled under reduced pressure, the distillation temperature was set at 75℃and the distillation pressure was set at 30mmHg, to obtain Haematococcus pluvialis astaxanthin oil.
Example 2
The extraction method of haematococcus pluvialis astaxanthin oil comprises the following steps:
1, pretreatment: grinding Haematococcus pluvialis to obtain Haematococcus pluvialis powder;
2, enzymolysis: adding 1.5 times of water and cholesterol esterase into haematococcus pluvialis powder, keeping the temperature at 55 ℃ and stirring at 50rpm for 5 hours to obtain a mixture, wherein the cholesterol esterase is used in an amount of 10000 units per gram of haematococcus pluvialis powder;
3, secondary enzymolysis and emulsification: adding cholesterol esterase and emulsifier lecithin into the mixture, keeping the temperature at 35 ℃ and stirring the mixture at a speed of 50rpm for 2 hours, wherein the cholesterol esterase is 10000 units per gram of haematococcus pluvialis powder, and the emulsifier is 1% of the haematococcus pluvialis powder in mass;
4, solvent extraction: then adding organic solvent ethanol, keeping at 20 ℃ and stirring at 50rpm for 2 hours, and then standing for 3 hours, wherein the dosage of the organic solvent ethanol is 4 times of the mass of haematococcus pluvialis algae powder;
5, separating: filtering and separating the mixed system in the step 4 with a pore diameter of 0.2mm, and performing rotary evaporation (heating temperature is 50 ℃, and rotary evaporation speed is set at 150 rpm) to obtain an oily ethanol solution;
6, reduced pressure distillation: the oily ethanol solution was distilled under reduced pressure, the distillation temperature was set at 75℃and the distillation pressure was set at 30mmHg, to obtain Haematococcus pluvialis astaxanthin oil.
Example 3
The extraction method of haematococcus pluvialis astaxanthin oil comprises the following steps:
1, pretreatment: grinding Haematococcus pluvialis to obtain Haematococcus pluvialis powder;
2, enzymolysis: adding 1.5 times of water and candida antarctica lipase B into haematococcus pluvialis powder, keeping the temperature at 55 ℃ and stirring at 50rpm for 5 hours to obtain a mixture, wherein the dosage of the candida antarctica lipase B is 10000 units per gram of haematococcus pluvialis powder;
3, secondary enzymolysis and emulsification: adding cholesterol esterase and emulsifier lecithin into the mixture, keeping the temperature at 35 ℃ and stirring the mixture at a speed of 50rpm for 2 hours, wherein the cholesterol esterase is 10000 units per gram of haematococcus pluvialis powder, and the emulsifier is 1% of the haematococcus pluvialis powder in mass;
4, solvent extraction: then adding organic solvent ethanol, keeping at 20 ℃ and stirring at 50rpm for 2 hours, and then standing for 3 hours, wherein the dosage of the organic solvent ethanol is 4 times of the mass of haematococcus pluvialis algae powder;
5, separating: filtering and separating the mixed system in the step 4 with a pore diameter of 0.2mm, and performing rotary evaporation (heating temperature is 50 ℃, and rotary evaporation speed is set at 150 rpm) to obtain an oily ethanol solution;
6, reduced pressure distillation: the oily ethanol solution was distilled under reduced pressure, the distillation temperature was set at 75℃and the distillation pressure was set at 30mmHg, to obtain Haematococcus pluvialis astaxanthin oil.
Example 4
The extraction method of haematococcus pluvialis astaxanthin oil comprises the following steps:
1, pretreatment: grinding Haematococcus pluvialis to obtain Haematococcus pluvialis powder;
2, enzymolysis: adding 1.5 times of water and candida antarctica lipase B into haematococcus pluvialis powder, keeping the temperature at 55 ℃ and stirring at 50rpm for 5 hours to obtain a mixture, wherein the dosage of the candida antarctica lipase B is 10000 units per gram of haematococcus pluvialis powder;
3, secondary enzymolysis: adding cholesterol esterase into the mixture, keeping the temperature at 35 ℃ and stirring the mixture at a speed of 50rpm for 2 hours, wherein the cholesterol esterase is used in an amount of 10000 units per gram of haematococcus pluvialis powder;
4, solvent extraction: then adding organic solvent ethanol, keeping at 20 ℃ and stirring at 50rpm for 2 hours, and then standing for 3 hours, wherein the dosage of the organic solvent ethanol is 4 times of the mass of haematococcus pluvialis algae powder;
5, separating: filtering and separating the mixed system in the step 4 with a pore diameter of 0.2mm, and performing rotary evaporation (heating temperature is 50 ℃, and rotary evaporation speed is set at 150 rpm) to obtain an oily ethanol solution;
6, reduced pressure distillation: the oily ethanol solution was distilled under reduced pressure, the distillation temperature was set at 75℃and the distillation pressure was set at 30mmHg, to obtain Haematococcus pluvialis astaxanthin oil.
Example 5
The extraction method of haematococcus pluvialis astaxanthin oil comprises the following steps:
1, pretreatment: grinding Haematococcus pluvialis to obtain Haematococcus pluvialis powder;
2, enzymolysis: adding 1.5 times of water and candida antarctica lipase B into haematococcus pluvialis powder, keeping the temperature at 55 ℃ and stirring at 50rpm for 5 hours, wherein the dosage of the candida antarctica lipase B is 10000 units per gram of haematococcus pluvialis powder;
3, solvent extraction: then adding organic solvent ethanol, keeping at 20 ℃ and stirring at 50rpm for 2 hours, and then standing for 3 hours, wherein the dosage of the organic solvent ethanol is 4 times of the mass of haematococcus pluvialis algae powder;
4, separating: filtering and separating the mixed system in the step 3 with a pore diameter of 0.2mm, and performing rotary evaporation (heating temperature is 50 ℃, and rotary evaporation speed is set at 150 rpm) to obtain an oily ethanol solution;
5, reduced pressure distillation: the oily ethanol solution was distilled under reduced pressure, the distillation temperature was set at 75℃and the distillation pressure was set at 30mmHg, to obtain Haematococcus pluvialis astaxanthin oil.
Example 6
The extraction method of haematococcus pluvialis astaxanthin oil comprises the following steps:
1, pretreatment: grinding Haematococcus pluvialis to obtain Haematococcus pluvialis powder;
2, solvent extraction: adding organic solvent ethanol, keeping at 20 ℃ and stirring at 50rpm for 2 hours, and then standing for 3 hours, wherein the dosage of the organic solvent ethanol is 4 times of the mass of haematococcus pluvialis algae powder;
3, separating: filtering and separating the mixed system in the step 2 with a pore diameter of 0.2mm, and performing rotary evaporation (heating temperature is 50 ℃, and rotary evaporation speed is set at 150 rpm) to obtain an oily ethanol solution;
4, reduced pressure distillation: the oily ethanol solution was distilled under reduced pressure, the distillation temperature was set at 75℃and the distillation pressure was set at 30mmHg, to obtain Haematococcus pluvialis astaxanthin oil.
Example 7
The extraction method of haematococcus pluvialis astaxanthin oil comprises the following steps:
1, pretreatment: grinding Haematococcus pluvialis to obtain Haematococcus pluvialis powder;
2, enzymolysis: adding 1.5 times of water and candida antarctica lipase B into haematococcus pluvialis powder, keeping the temperature at 35 ℃ and stirring at 50rpm for 5 hours to obtain a mixture, wherein the dosage of the candida antarctica lipase B is 10000 units per gram of haematococcus pluvialis powder;
3, secondary enzymolysis and emulsification: adding cholesterol esterase and emulsifier lecithin into the mixture, keeping the temperature at 35 ℃ and stirring the mixture at a speed of 50rpm for 2 hours, wherein the cholesterol esterase is 10000 units per gram of haematococcus pluvialis powder, and the emulsifier is 1% of the haematococcus pluvialis powder in mass;
4, solvent extraction: then adding organic solvent ethanol, keeping at 20 ℃ and stirring at 50rpm for 2 hours, and then standing for 3 hours, wherein the dosage of the organic solvent ethanol is 4 times of the mass of haematococcus pluvialis algae powder;
5, separating: filtering and separating the mixed system in the step 4 with a pore diameter of 0.2mm, and performing rotary evaporation (heating temperature is 50 ℃, and rotary evaporation speed is set at 150 rpm) to obtain an oily ethanol solution;
6, reduced pressure distillation: the oily ethanol solution was distilled under reduced pressure, the distillation temperature was set at 75℃and the distillation pressure was set at 30mmHg, to obtain Haematococcus pluvialis astaxanthin oil.
Example 8
The extraction method of haematococcus pluvialis astaxanthin oil comprises the following steps:
1, pretreatment: grinding Haematococcus pluvialis to obtain Haematococcus pluvialis powder;
2, enzymolysis: adding 1.5 times of water and candida antarctica lipase B into haematococcus pluvialis powder, keeping the temperature at 55 ℃ and stirring at 50rpm for 5 hours to obtain a mixture, wherein the dosage of the candida antarctica lipase B is 10000 units per gram of haematococcus pluvialis powder;
3, secondary enzymolysis and emulsification: adding cholesterol esterase and emulsifier lecithin into the mixture, keeping the temperature at 55 ℃ and stirring the mixture at a speed of 50rpm for 2 hours, wherein the cholesterol esterase is 10000 units per gram of haematococcus pluvialis powder, and the emulsifier is 1% of the haematococcus pluvialis powder in mass;
4, solvent extraction: then adding organic solvent ethanol, keeping at 20 ℃ and stirring at 50rpm for 2 hours, and then standing for 3 hours, wherein the dosage of the organic solvent ethanol is 4 times of the mass of haematococcus pluvialis algae powder;
5, separating: filtering and separating the mixed system in the step 4 with a pore diameter of 0.2mm, and performing rotary evaporation (heating temperature is 50 ℃, and rotary evaporation speed is set at 150 rpm) to obtain an oily ethanol solution;
6, reduced pressure distillation: the oily ethanol solution was distilled under reduced pressure, the distillation temperature was set at 75℃and the distillation pressure was set at 30mmHg, to obtain Haematococcus pluvialis astaxanthin oil.
Example 9
The extraction method of haematococcus pluvialis astaxanthin oil comprises the following steps:
1, pretreatment: grinding Haematococcus pluvialis to obtain Haematococcus pluvialis powder;
2, enzymolysis: adding 1.5 times of water and candida antarctica lipase B into haematococcus pluvialis powder, keeping the temperature at 35 ℃ and stirring at 50rpm for 5 hours to obtain a mixture, wherein the dosage of the candida antarctica lipase B is 10000 units per gram of haematococcus pluvialis powder;
3, secondary enzymolysis and emulsification: adding cholesterol esterase and emulsifier lecithin into the mixture, keeping the temperature at 55 ℃ and stirring the mixture at a speed of 50rpm for 2 hours, wherein the cholesterol esterase is 10000 units per gram of haematococcus pluvialis powder, and the emulsifier is 1% of the haematococcus pluvialis powder in mass;
4, solvent extraction: then adding organic solvent ethanol, keeping at 20 ℃ and stirring at 50rpm for 2 hours, and then standing for 3 hours, wherein the dosage of the organic solvent ethanol is 4 times of the mass of haematococcus pluvialis algae powder;
5, separating: filtering and separating the mixed system in the step 4 with a pore diameter of 0.2mm, and performing rotary evaporation (heating temperature is 50 ℃, and rotary evaporation speed is set at 150 rpm) to obtain an oily ethanol solution;
6, reduced pressure distillation: the oily ethanol solution was distilled under reduced pressure, the distillation temperature was set at 75℃and the distillation pressure was set at 30mmHg, to obtain Haematococcus pluvialis astaxanthin oil.
Comparing example 3 with examples 1-2, example 3 was formulated with candida antarctica lipase B and cholesterol esterase, example 1 was single candida antarctica lipase B, example 2 was single cholesterol esterase, and the astaxanthin extraction rate of example 3 was significantly higher than that of examples 1 and 2, and a synergistic effect was seen.
Candida antarctica lipase B and cholesterol esterase are two lipases with different catalytic activities. The invention is used for destroying the cell wall of haematococcus pluvialis and releasing astaxanthin. Candida antarctica lipase B is a lipase mainly used for hydrolyzing fatty acid ester bonds, thereby destroying the structure of fatty acids. Cholesterol esterase has more specific activity and can specifically hydrolyze cholesterol ester. The two enzymes form a synergistic effect when hydrolyzing cholesterol esters and other lipid esters, so that the enzymolysis efficiency is greatly improved. First, candida antarctica lipase B may first disrupt the lipid structure of the cell wall, so that cholesterol esterase can more easily contact and hydrolyze cholesterol esters. Second, cholesterol esterases may also help to some extent disrupt the structure of the cell wall, so that candida antarctica lipase B can more effectively hydrolyze the lipids of the cell wall. This synergistic effect resulted in significantly higher astaxanthin extraction in example 3 than in examples 1 and 2, which also demonstrates that the use of candida antarctica lipase B in combination with cholesterol esterase is more effective in extracting astaxanthin.
Comparative example 3 and example 4:
In both examples, the enzymatic hydrolysis step is performed on Haematococcus pluvialis using Candida antarctica Lipase B and cholesterol esterase. However, a major difference between example 3 and example 4 is that example 3 adds the emulsifier lecithin during the secondary enzymolysis and emulsification steps. The use of emulsifiers is generally done to increase the stability of the solution, reduce the polymerization of the oil droplets, increase the surface area of the oil droplets for more enzyme to contact and thus increase the efficiency of the reaction. The emulsifier lecithin can reduce the surface tension between the water and the oil, so that the water phase and the oil phase can be better mixed. During this process, the emulsifier lecithin can help form small oil droplets that disperse in the aqueous phase forming an emulsion. In this case the surface area of the oil droplets is greatly increased, which means that more enzyme can contact the oil droplets, resulting in a greatly improved efficiency of the enzymatic reaction. In addition, the emulsifier lecithin also stabilizes the oil droplets, preventing them from polymerizing together. This is because the emulsifier lecithin has both hydrophilic and hydrophobic portions, the hydrophilic portion extending toward the aqueous phase and the hydrophobic portion extending toward the oil phase, forming a protective film, making the oil droplets less likely to polymerize. In example 4, since no emulsifier was used, although the enzymatic hydrolysis process could destroy the algae cell wall and release astaxanthin, the surface area of the oil droplets was reduced due to the polymerization of the oil droplets, resulting in less enzyme being able to contact the oil droplets, thereby reducing the efficiency of the enzymatic hydrolysis reaction. Meanwhile, the polymerization of oil drops can also influence the mixing degree of the oil drops and the water phase, and the efficiency of enzymolysis reaction is further reduced. Thus, it can be seen that the addition of the emulsifier lecithin significantly improved the extraction efficiency of astaxanthin in example 3. This also demonstrates the importance of the emulsification process in enzymatic reactions, especially in extraction processes involving hydrophobic substances such as astaxanthin.
Comparative example 3 and example 5:
There are two main differences in the procedure of example 3 and example 5: enzymolysis step and emulsification step. Example 3 was run twice, first with candida antarctica lipase B and second with cholesterol esterase. In example 5, only one enzymatic hydrolysis was performed, and only candida antarctica lipase B was used. The combined use of two enzymes (as shown in example 3) allows specific enzymolysis of different lipids, resulting in more efficient release of astaxanthin from haematococcus pluvialis algae powder. In this case, candida antarctica lipase B is mainly used to degrade fatty acid ester bonds, thereby releasing fatty acids, while cholesterol esterase is mainly used to degrade cholesterol esters, thereby releasing cholesterol and fatty acids. The combined action of the two enzymes can improve enzymolysis efficiency, thereby increasing the extraction amount of astaxanthin. Example 3 was emulsified after enzymatic hydrolysis, whereas example 5 was absent. The emulsification treatment can reduce the surface tension of the oil-water interface, so that the oil phase (containing astaxanthin) can be dispersed in the water phase in the form of tiny oil drops, thereby increasing the surface area of the oil drops and providing more action sites for enzymes. This is important for further improving the enzymolysis efficiency and the extraction rate of astaxanthin. Meanwhile, the hydrophobic layer formed by the emulsifier lecithin in the water phase can prevent the polymerization of oil drops, and keep the stability of an emulsifying system, which is also beneficial to the enzymolysis process. Thus, example 3 employed finer processing steps than example 5, including secondary enzymolysis and emulsification, which all helped to increase the astaxanthin extraction efficiency. This is the main reason that example 3 is significantly superior to example 5 in terms of astaxanthin extraction yield.
Comparative example 3 and example 6:
there are two main differences in the procedure of example 3 and example 6: enzymolysis step and emulsification step.
First, example 3 was run twice, first with candida antarctica lipase B and second with cholesterol esterase. Whereas example 6 does not have an enzymatic hydrolysis step at all. Next, example 3 was subjected to emulsification treatment after enzymolysis, whereas example 6 was not. The procedure of example 3, including secondary enzymolysis and emulsification, all helped to increase the astaxanthin extraction efficiency. This is the main reason that example 3 is significantly superior to example 6 in terms of astaxanthin extraction yield. Example 6 due to the lack of enzymatic hydrolysis and emulsification steps, astaxanthin may be more difficult to release from haematococcus pluvialis algae powder, thereby affecting the extraction efficiency.
Comparative example 3 and examples 7-9:
The first enzymatic hydrolysis of example 3 was carried out at 55℃and the second enzymatic hydrolysis and emulsification were carried out at 35 ℃. The temperature setting of this two-step enzymatic hydrolysis process is to maximize the use of the activity of two different enzymes. In the invention, the optimal activity temperature of candida antarctica lipase B is 55 ℃, and the optimal activity temperature of cholesterol esterase is 35 ℃. The first enzymolysis process mainly depends on candida antarctica lipase B, and the temperature is set to 55 ℃ at the moment, so that the activity of the lipase is the highest, and the hydrolysis reaction of the lipid is catalyzed better. In the second enzymolysis and emulsification process, the cholesterol esterase and the emulsifier lecithin have more prominent effects, and the temperature is adjusted to 35 ℃ at the moment so as to enable the cholesterol esterase to exert the maximum activity. By the arrangement, the catalytic effect of the two enzymes can be fully exerted, the enzymolysis efficiency is enhanced, and finally the extraction rate of astaxanthin is improved. In examples 7 to 9, however, the astaxanthin extraction efficiency was low because the setting of the enzymatic hydrolysis temperature may not be fully adapted to the optimal activity temperatures of the two enzymes.
Examples 10 to 18
A preparation method of rhodococcus pluvialis for krill oil rain comprises the following steps:
1.1 adding antarctic krill oil and haematococcus pluvialis astaxanthin oil corresponding to examples 1-9 into a mixing tank in advance according to requirements; the mass ratio of the antarctic krill oil to the haematococcus pluvialis astaxanthin oil is 75:35;
wherein example 10 corresponds to example 1; example 11 corresponds to example 2; ....until example 18 corresponds to example 9;
1.2, starting a mixer, mixing two kinds of oil for 3 hours, sampling and detecting at different time intervals, and uniformly mixing after the detection result is stable to obtain mixed nutrient oil;
1.3 evenly mixing gelatin, glycerol and water according to the mass ratio of 1:0.4:0.8, heating to 80 ℃, and keeping for 2 hours;
1.4 starting vacuumizing after no gelatin particles exist in the sense, wherein the negative pressure value is 0.05Mpa, and the time is 1 hour;
1.5 pressing the mixed nutritional oil into the soft capsules by using soft capsule production equipment;
Shaping at 30 deg.c and 30% humidity for 1 hr;
1.7, drying at 30 ℃ and 30% humidity for 10 hours;
1.8 is filled, and each capsule is controlled to be 0.25 g, and the mass of the mixed oil is controlled to be 0.50 g.
Test example 1:
the test was carried out with reference to the method provided in patent CN103232375A, in particular as follows:
Determination of the astaxanthin content of Haematococcus pluvialis powder:
About 25mg of pretreated haematococcus pluvialis powder is weighed, placed into a 10ml centrifuge tube, 3g of quartz sand is added, 5ml of DMSO is added into the centrifuge tube, and the mixture is placed in a water bath at 50 ℃ for 15min, and is oscillated for 3 times. Centrifuging at 4000rpm for 3min to precipitate insoluble substances, transferring supernatant into 25ml volumetric flask, adding 5ml n-hexane into centrifuge tube, shaking by vortex for 30s, centrifuging to separate precipitate, transferring supernatant into 25m1 volumetric flask, and repeatedly extracting n-hexane to precipitate until supernatant is colorless (absorbance less than 0.05). N-hexane was used for constant volume, diluted appropriately, and light absorption (between 0.25 and 0.75) was measured at 474nm with n-hexane as a blank. Calculated as follows:
algae powder astaxanthin content (%) =a×25×dilution ≡2100×algae powder mass (g) ×80%
In the formula: 2100 is the absorbance at 474nm of a standard solution of astaxanthin in n-hexane of 1% (m/v).
Determination of astaxanthin content in haematococcus pluvialis astaxanthin oil:
About 25mg of haematococcus pluvialis astaxanthin oil is weighed into a 100ml volumetric flask, dissolved in normal hexane to a constant volume, diluted appropriately, and the absorbance value (between 0.25 and 0.75) is measured at 474nm with normal hexane as a blank. Calculated as follows:
Astaxanthin content (%) =a×25×dilution ≡ ≡2100×haematococcus pluvialis astaxanthin oil mass (g) ×80% in haematococcus pluvialis astaxanthin oil
In the formula: 2100 is the absorbance at 474nm of a standard solution of astaxanthin in n-hexane of 1% (m/v).
The extraction rate refers to the extraction rate of astaxanthin in haematococcus pluvialis, and is calculated by the following formula:
astaxanthin extraction yield (%) =mass of haematococcus pluvialis astaxanthin oil×mass of astaxanthin in haematococcus pluvialis astaxanthin oil ≡astaxanthin powder ≡astaxanthin content×100%
Table 1: astaxanthin extraction rate test table for haematococcus pluvialis astaxanthin oil
Test example 2:
the performance test of the krill oil haematococcus pluvialis capsule prepared in example 10 is specifically as follows: a step of
| Sequence number | Detecting items | Unit (B) | Detection result | Detection method |
| 1 | Tissue morphology | / | Meets the requirements | Q/NJWK0004S-2022 |
| 2 | Color of | / | Meets the requirements | Q/NTWK0004S-2022 |
| 3 | Smell and taste | / | Meets the requirements | Q/NJWK0004S-2022 |
| 4 | Impurity(s) | / | Meets the requirements | Q/NJWK0004S-2022 |
| 5 | Moisture and volatiles | g/100g | 5.44 | GB 5009.3-2016 second method |
| 6 | Astaxanthin content | mg/kg | 12089 | GB/T 31520-2015 |
| 7 | Total phospholipid | g/100g | 22.4 | GB/T 5537-2008 |
| 8 | Colony count | CFU/g | 10，10，20，20，30 | GB4789.2-2016 |
| 9 | Coliform group bacteria | CFU/g | <10，<10，<10，<10，<10 | GB 4789.3-2016 second method |
| 10 | Net content of | g | 0.50 | JJF 1070-2005 |
Claims (1)
1. The preparation method of the rhodococcus pluvialis for krill oil rain is characterized by comprising the following steps of:
uniformly mixing antarctic krill oil and haematococcus pluvialis astaxanthin oil to obtain mixed nutrition oil; pressing the mixed nutritional oil into soft capsules; the haematococcus pluvialis astaxanthin oil is prepared by combining organic solvent extraction of haematococcus pluvialis powder with an enzymolysis method;
The haematococcus pluvialis astaxanthin oil is prepared by the following method:
Pretreatment: grinding Haematococcus pluvialis to obtain Haematococcus pluvialis powder;
Enzymolysis: adding 1.5 times of water and candida antarctica lipase B into haematococcus pluvialis powder, keeping the temperature at 55 ℃ and stirring at 50rpm for 5 hours to obtain a mixture, wherein the dosage of the candida antarctica lipase B is 10000 units per gram of haematococcus pluvialis powder;
Secondary enzymolysis and emulsification: adding cholesterol esterase and emulsifier lecithin into the mixture, keeping the temperature at 35 ℃ and stirring the mixture at a speed of 50rpm for 2 hours, wherein the cholesterol esterase is 10000 units per gram of haematococcus pluvialis powder, and the emulsifier is 1% of the haematococcus pluvialis powder in mass;
Extracting a solvent: then adding ethanol, keeping at 20 ℃ and stirring at 50rpm for 2 hours, and then standing for 3 hours, wherein the dosage of the ethanol is 4 times of the mass of haematococcus pluvialis powder;
separating: filtering and separating the mixed system, and performing rotary evaporation to obtain an oily ethanol solution;
And (3) reduced pressure distillation: and (3) distilling the oily ethanol solution under reduced pressure to obtain haematococcus pluvialis astaxanthin oil.
Priority Applications (1)
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