CN108795093A - A kind of preparation of the pitch-dark pigment of marine animal solubility and analysis method - Google Patents
A kind of preparation of the pitch-dark pigment of marine animal solubility and analysis method Download PDFInfo
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- CN108795093A CN108795093A CN201711061461.XA CN201711061461A CN108795093A CN 108795093 A CN108795093 A CN 108795093A CN 201711061461 A CN201711061461 A CN 201711061461A CN 108795093 A CN108795093 A CN 108795093A
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B67/00—Influencing the physical, e.g. the dyeing or printing properties of dyestuffs without chemical reactions, e.g. by treating with solvents grinding or grinding assistants, coating of pigments or dyes; Process features in the making of dyestuff preparations; Dyestuff preparations of a special physical nature, e.g. tablets, films
- C09B67/0096—Purification; Precipitation; Filtration
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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Abstract
The present invention relates to a kind of preparation of the soluble pitch-dark pigment of marine animal and analysis methods, include the following steps：Take Cephalopoda animal prepared Chinese ink, it is centrifuged to obtain the first sediment after prepared Chinese ink is impregnated with isometric purified water, the first sediment is added protein enzyme solution and is hydrolyzed, and solution centrifuges obtain the second sediment after the heating after hydrolysis, second sediment is dried in vacuo, and obtains soluble pitch-dark pigment finished product；Soluble pitch-dark pigment finished product is taken, absorbance is detected after dissolving NaOH solution and heating water bath.Above-mentioned technical proposal extracts the pitch-dark pigment of marine animal using 90 DEG C of orientation heat preservation enzymolysis and high speed centrifugation, can be directed to protein isolate matter and pitch-dark pigment, improves the purity of pitch-dark pigment product；Enzymatic Extraction mild condition is conducive to the stabilization for keeping natural pitch-dark pigment morphosis, and using absorbance as the detection method for judging pitch-dark pigment concentration, accuracy is high, simple and practicable, easy to operate.
Description
Technical field
The present invention relates to the preparation of pitch-dark pigment and analysis method, more particularly to a kind of soluble pitch-dark pigment of marine animal
Preparation and analysis method.
Background technology
Pitch-dark pigment (melanin) word derives from Greece, is proposed first by Swiss chemists Berzelius, is one
Kind large biological molecule polymer not soluble in water or the known native endogenous that can uniquely protect organisms from radiation injury
Biopolymer, it is widely present in animal and plant body.The pitch-dark pigment of marine animal is mainly distributed on Mollusca
(Mollusca) Cephalopoda (Cephalopoda), existing about 650 kinds, as octopus (octopus), cuttlefish are commonly called as inkfish
(cuttlefish), squid is commonly called as squid（Squid）, nautilus (nautilus) etc..From offshore to off-lying sea, surface layer is arrived
4,500 meters or less depths are all distributed.Pitch-dark pigment is in the prepared Chinese ink of the oceans such as inkfish, squid, octopus siphonopods biology
In the generation of contained large biological molecule polymer, claims, and is the known native endogenous that can uniquely protect organisms from radiation injury
Biopolymer.Contain the multiple biological activities ingredient such as polypeptide fragments, acidic polysaccharose, tyrosinase.With remove free radical and
In conjunction with genotoxic potential ion（Such as some transition metal）Characteristic, application potential is huge.It is reported that the big midocean siphonopods in the whole world
Total resources be hundred million tons of 4.2-6.5.
Currently, there are many siphonopods manufacturing and processing enterprises such as squid, octopuses, production generates a large amount of prepared Chinese ink can not be comprehensive
It closes and utilizes, cause largely to waste, it is that siphonopods manufacturing and processing enterprise technology urgently to be resolved hurrily is asked to make full use of siphonopods prepared Chinese ink
Topic.Pitch-dark pigment is while with bioactivity such as enhancing immune function, antitumor, anti-oxidant, anti-aging and antibacterials
There are the shortcomings that not soluble in water, difficulty isolates and purifies, and which greatly limits its applications in fields such as medicine, food.Therefore, it prepares
The pitch-dark pigment of resolvability is a problem instantly urgently to be resolved hurrily.
Invention content
For this reason, it may be necessary to provide a kind of preparation method of the pitch-dark pigment of solubility of high-purity.To achieve the above object, it invents
People provides a kind of preparation method of the soluble pitch-dark pigment of marine animal, the described method comprises the following steps：
S1：Cephalopoda animal abdomen is splitted, ink sac is taken out, cuts off cyst membrane, prepared Chinese ink is squeezed out, by the isometric purified water of prepared Chinese ink
Impregnate 12 hours or more, obtain the first mixed solution;
S2：First mixed solution is centrifuged, centrifugal speed 12000r/min, centrifugation time is 10 minutes, is abandoned after centrifugation
Except supernatant, the first sediment is obtained；
S3：First sediment addition protein enzyme solution is hydrolyzed, at 4 ~ 8 hours, hydrolysis temperature controlled for hydrolysis time control
At 40 ~ 60 DEG C, the second mixed solution is obtained；
S4：Second mixed solution is heated 10 minutes in 90 DEG C of water-bath, obtains third mixed solution；
S5：Third mixed solution is centrifuged, centrifugal speed 12000r/min, centrifugation time is 10 minutes, is abandoned after centrifugation
Except supernatant, the second sediment is obtained；
S6：Second sediment is dried in vacuo, drying temperature is controlled at 65 ~ 75 DEG C, obtains soluble pitch-dark pigment finished product；
S7：Soluble pitch-dark pigment finished product 2mg is taken, 2% NaOH solution 25ml is added, in 65 ~ 75 DEG C of heating water baths 10 minutes,
Obtain the 4th mixed solution；
S8：4th mixed solution is subjected to absorbance detection, the concentration of the soluble pitch-dark pigment of detection.
Further, in the S1 steps, the Cephalopoda animal is squid, octopus or cuttlefish.
Further, in the S1 steps, the temperature of the purified water is controlled at 0 ~ 4 DEG C.
Further, in the S3 steps, the protein enzyme solution is alkaline protease solution, and the proteinase activity is
4200U/g, hydrolysis time are 6 hours, and hydrolysis temperature is 50 DEG C.
Further, in the S3 steps, the weight ratio of the first sediment and protein enzyme solution is 1：50.
Further, in the S3 steps, use 0.1mol/L HCl or NaOH solution control hydrolytic process pH value for
8.0。
Further, in the S6 steps, the second drying precipitate is carried out using vacuum belt type drying equipment.
Further, the Detection wavelength of the S8 steps is 206nm.
The pitch-dark pigment of solubility prepared by the present invention, in the application of drug, health food, nutraceutical and cosmetic field.
It is different from the prior art, above-mentioned technical proposal is using 90 DEG C of heating 10min orientation heat preservation enzymolysis and high speed centrifugation
（12000r/min, 10min）The pitch-dark pigment of marine animal is extracted, protein isolate matter and pitch-dark pigment are can be directed to, improves ink black
The purity of plain product；Enzymatic Extraction mild condition is conducive to the stabilization for keeping natural pitch-dark pigment morphosis, can also reduce biography
The consumption of system extraction method chemical reagent；Shorten the pitch-dark pigment enzymatic isolation method preparation time of ocean cephalopod, improves pitch-dark pigment
Purity, the vacuum belt type drying equipment of use is dried at 65 ~ 75 DEG C, keeps pitch-dark depigmenting activity and stability, improves
Dry yield.Finally use absorbance as the detection method for judging pitch-dark pigment concentration, accuracy is high, simple and practicable, is easy
Operation.
Specific implementation mode
For the technology contents of technical solution, construction feature, the objects and the effects are described in detail, below in conjunction with specific reality
Example is applied to be explained in detail.
Embodiment 1:The selection of protease
Select five kinds of pepsin, papain, neutral proteinase, trypsase, alkali protease protease, hydrolysising condition
In the mass ratio of the first sediment and protein enzyme solution be 1:50, protein enzyme solution activity is 4200 U/g, hydrolysis
Time is 6 hours, under the conditions of each protease is most suitable for temperature and pH, is carried out respectively to ocean cephalopod prepared Chinese ink
Heat preservation enzymolysis, with the light absorption value of product melanin（260nm）For index, the albumen best to prepared Chinese ink hydrolysis effect is therefrom selected
Enzyme.According to Beer law, light absorption value is directly proportional to the concentration of extinction material.Melanin concentration is high, and corresponding light absorption value is also big.
Therefore, this research is using absorbance as the relative indicatrix for weighing melanin content height.The pitch-dark pigment of marine animal is in ultraviolet light
In region (190~400 nm) and visible light region, respectively there is an obvious absorption peaks (206 and 557 nm), wherein
There is larger absorption at about 206 nm.Excellent determining be used as using 206 nm is selected to measure wavelength.
Concrete outcome see the table below
From result, we can significantly find out, the light absorption value in 206nm of basic protein is 0.3696, is higher than other eggs
White enzyme illustrates that alkali protease is best to the hydrolysis effect of pitch-dark pigment.If other conditions are the same, alkali protease
The concentration highest of pitch-dark pigment in protease preparations.Therefore we select alkali protease as the degrading enzyme of pitch-dark pigment.
Embodiment 2：Optimization of process conditions：
Experiment of single factor is carried out to 4 temperature of enzymolysis, pH, enzyme dosage and time factors, to determine correlative factor and each factor
OK range.On the basis of extracting single factor experiment, using orthogonal experiment Optimizing Technical.With hydrolysis temperature, pH
As investigation object, each factor selects 3 levels, and arranges to test, Optimizing Technical for value, enzyme activity amount, hydrolysis time.
Temperature is 50 DEG C, enzyme dosage is 4200 U/g, enzymolysis time 6 hours under the conditions of, select different pH values
6.8, it 7.4,8.0,8.6,9.2 is tested, inquires into influences of the pH to enzymolysis process.When the pH of enzyme solutions is 6.8
Between~8.0, the light absorption value of reaction product when pH reaches 8.0, reaches a maximum, then there are one slowly being promoted
Present apparent downward trend.
In conclusion we may safely draw the conclusion：When using alkali protease, and 50 DEG C of hydrolysis temperature, pitch-dark pigment
It is 1 with alkaline protease solution mass ratio:50, PH values are 8.0, enzyme activity 4200U/g, are that marine animal solubility is pitch-dark
Optimised process prepared by pigment.
Embodiment 3：Process conditions are verified：
The optimal processing parameter that orthogonal experiment obtains carries out melanin work obtained under confirmatory experiment, with not enzyme equal conditions
Control, as a result compared with not enzyme water extraction, the melanin light absorption value of the Enzymatic Extraction through orthogonal experiment optimization is significantly higher,
Absorbance is promoted to 0.6265 by 0.3841.
Embodiment 4：It is prepared by the soluble pitch-dark pigment finished product of marine animal
Squid abdomen is splitted, ink sac is taken out, cuts off cyst membrane, squeezes out prepared Chinese ink.Above-mentioned prepared Chinese ink is collected, by the 0 of prepared Chinese ink same volume ~
4 DEG C of purified waters are impregnated 12 hours;Obtain the first mixed solution；First mixed solution is centrifuged, centrifugal speed 12000r/
Min, centrifugation time are 10 minutes, and reject supernatant after centrifugation obtains the pitch-dark pigment of insolubility；Insoluble pitch-dark pigment is added
The proteinase activity for entering 50 times of quality is that 4200U/g alkaline protease solutions are hydrolyzed, and hydrolysis time is 6 hours, hydrolyzed
Solution ph is 8.0 with NaOH and the HCl control of 0.1mol/L in journey, and 50 DEG C of hydrolysis temperature obtains the second mixed solution；By
Two mixed solutions heat 10 minutes in 90 DEG C of water-bath, obtain third mixed solution；Third mixed solution is centrifuged,
Centrifugal speed is 12000r/min, and centrifugation time is 10 minutes, and reject supernatant after centrifugation obtains the second sediment；By second
Sediment is dried in vacuo using vacuum belt type drying equipment, and drying temperature is controlled at 70 DEG C, obtains soluble pitch-dark pigment
Finished product.
The soluble pitch-dark pigment finished product interpretation of result of 5 marine animal of embodiment
Soluble pitch-dark pigment finished product 2mg is taken, 2% NaOH solution 25ml is added, in 70 DEG C of heating water baths after ten minutes by solution
Absorbance detection is carried out, detection solution and then obtains the concentration of pitch-dark pigment in the finished product in the light absorption value of 260nm.
According to Beer law, light absorption value is directly proportional to the concentration of the amount of extinction material.Melanin concentration is high, corresponding to inhale
Light value is also big.Therefore, this research is using absorbance as the relative indicatrix for weighing melanin content height.The pitch-dark pigment of marine animal
In UV light region (190~400 nm) and visible light region, respectively there is an obvious absorption peaks (206 and 557 nm),
Wherein there is larger absorption at 206 nm.Excellent determining be used as using 206 nm is selected to measure wavelength.
6 bioactivity research of embodiment
Soluble pitch-dark pigment finished product 2mg is taken, 2% NaOH solution 25ml is added, mixed solution is crossed to the ultrafiltration membrane of different pore size
It is detached；Detect the accounting of the various pitch-dark pigments of different molecular weight in pitch-dark pigment solution；To pitch-dark pigment solution and separation
The pitch-dark pigment of the different molecular weight gone out carries out bioactivity research, so that it is in drug, health food, nutraceutical and makeup
The application in product field.
It should be noted that herein, relational terms such as first and second and the like be used merely to an entity or
Person operates to be distinguished with another entity or operation, is appointed without necessarily requiring or implying existing between these entities or operation
What this actual relationship or sequence.Moreover, the terms "include", "comprise" or its any other variant are intended to non-row
His property includes, so that process, method, article or terminal device including a series of elements are not only wanted including those
Element, but also include other elements that are not explicitly listed, or further include for this process, method, article or terminal
The intrinsic element of equipment.In the absence of more restrictions, being wanted by what sentence " including ... " or " including ... " limited
Element, it is not excluded that there is also other elements in process, method, article or the terminal device including the element.In addition,
Herein, " being more than ", " being less than ", " being more than " etc. are interpreted as not including this number；" more than ", " following ", " within " etc. be interpreted as
Including this number.
Although the various embodiments described above are described, once a person skilled in the art knows basic wounds
The property made concept, then additional changes and modifications can be made to these embodiments, so example the above is only the implementation of the present invention,
It is not intended to limit the scope of patent protection of the present invention, it is every to utilize equivalent structure or equivalent process made by description of the invention
Transformation, be applied directly or indirectly in other relevant technical fields, similarly be included in the present invention scope of patent protection it
It is interior.
Claims (9)
1. preparation and the analysis method of a kind of soluble pitch-dark pigment of marine animal, which is characterized in that the method includes following
Step：
S1：Cephalopoda animal abdomen is splitted, ink sac is taken out, cuts off cyst membrane, prepared Chinese ink is squeezed out, by the isometric purified water of prepared Chinese ink
Impregnate 12 hours or more, obtain the first mixed solution;
S2：First mixed solution is centrifuged, centrifugal speed 12000r/min, centrifugation time is 10 minutes, is abandoned after centrifugation
Except supernatant, the first sediment is obtained；
S3：First sediment addition protein enzyme solution is hydrolyzed, at 4 ~ 8 hours, hydrolysis temperature controlled for hydrolysis time control
At 40 ~ 60 DEG C, the second mixed solution is obtained；
S4：Second mixed solution is heated 10 minutes in 90 DEG C of water-bath, obtains third mixed solution；
S5：Third mixed solution is centrifuged, centrifugal speed 12000r/min, centrifugation time is 10 minutes, is abandoned after centrifugation
Except supernatant, the second sediment is obtained；
S6：Second sediment is dried in vacuo, drying temperature is controlled at 65 ~ 75 DEG C, obtains soluble pitch-dark pigment finished product；
S7：Soluble pitch-dark pigment finished product 2mg is taken, 2% NaOH solution 25ml is added, in 65 ~ 75 DEG C of heating water baths 10 minutes,
Obtain the 4th mixed solution；
S8：4th mixed solution is subjected to absorbance detection, the concentration of the soluble pitch-dark pigment of detection.
2. preparation according to claim 1 and analysis method, which is characterized in that in the S1 steps, the Cephalopoda is dynamic
Object is squid, octopus or cuttlefish.
3. preparation according to claim 1 and analysis method, which is characterized in that in the S1 steps, the purified water
Temperature is controlled at 0 ~ 4 DEG C.
4. preparation according to claim 1 and analysis method, which is characterized in that in the S3 steps, the protease is molten
Liquid is alkaline protease solution, and proteinase activity 4200U/g, hydrolysis time is 6 hours, and hydrolysis temperature is 50 DEG C.
5. preparation according to claim 1 and analysis method, which is characterized in that in the S3 steps, the first sediment and
The weight ratio of protein enzyme solution is 1：50.
6. preparation according to claim 1 and analysis method, which is characterized in that in the S3 steps, using 0.1mol/L
HCl or NaOH solution control hydrolytic process pH value be 8.0.
7. preparation according to claim 1 and analysis method, which is characterized in that in the S6 steps, using vacuum belt
Drying equipment carries out the second drying precipitate.
8. preparation described in claim 1 and analysis method, which is characterized in that the Detection wavelength of the S8 steps is 206nm.
9. the pitch-dark pigment of solubility described in claim 1, in answering for drug, health food, nutraceutical and cosmetic field
With.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114433243A (en) * | 2022-01-31 | 2022-05-06 | 广东工业大学 | Multi-enzyme active metal-based cuttlefish juice melanin composite material and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100999616A (en) * | 2006-12-22 | 2007-07-18 | 中国海洋大学 | Preparation process of squid ink black pigment and application thereof |
| CN104544321A (en) * | 2013-10-21 | 2015-04-29 | 大连市沙河口区中小微企业服务中心 | Soluble squid inkiness pigment |
| CN105111781A (en) * | 2015-09-25 | 2015-12-02 | 谢友亮 | Industrial production method of cephalopod melanin |
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2017
- 2017-11-02 CN CN201711061461.XA patent/CN108795093A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100999616A (en) * | 2006-12-22 | 2007-07-18 | 中国海洋大学 | Preparation process of squid ink black pigment and application thereof |
| CN104544321A (en) * | 2013-10-21 | 2015-04-29 | 大连市沙河口区中小微企业服务中心 | Soluble squid inkiness pigment |
| CN105111781A (en) * | 2015-09-25 | 2015-12-02 | 谢友亮 | Industrial production method of cephalopod melanin |
Non-Patent Citations (1)
| Title |
|---|
| 李晓 等: ""酶法提取金乌贼墨汁中黑色素的工艺条件研究"", 《天然产物研究与开发》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114433243A (en) * | 2022-01-31 | 2022-05-06 | 广东工业大学 | Multi-enzyme active metal-based cuttlefish juice melanin composite material and preparation method thereof |
| CN114433243B (en) * | 2022-01-31 | 2023-11-24 | 广东工业大学 | Multi-enzyme-activity metal-based black carp juice melanin composite material and preparation method thereof |
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Application publication date: 20181113 |