CN105949089B - The method that natural taurine is extracted from mussel - Google Patents
The method that natural taurine is extracted from mussel Download PDFInfo
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- CN105949089B CN105949089B CN201610556023.XA CN201610556023A CN105949089B CN 105949089 B CN105949089 B CN 105949089B CN 201610556023 A CN201610556023 A CN 201610556023A CN 105949089 B CN105949089 B CN 105949089B
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C303/00—Preparation of esters or amides of sulfuric acids; Preparation of sulfonic acids or of their esters, halides, anhydrides or amides
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- C07C303/44—Separation; Purification
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Abstract
The present invention relates to the methods that natural taurine is extracted from mussel, belong to chemical extraction technical field.The method that the present invention extracts natural taurine from mussel comprises the steps of：S1 prepares the clear liquid containing taurine using mussel；S2 is by the clear liquid removing protein；S3 isolates and purifies the product of the step S3.Beneficial effects of the present invention are：Raw material takes the most universal mussel in Dalian Area as raw material, cheap, the problem of belonging to low side marine product and mode is extracted, environmental pollution is not present.And technological operation is simple, yield is big.It is extracted using from containing the highest marine product of amount of taurine, temperature then after the operation of a row, accomplish naturally to extract by organic solvent-free residual.
Description
Technical field
The invention belongs to chemical extraction technical field, especially a kind of method for extracting natural taurine from mussel.
Background technology
Taurine is a kind of beta amino acids of sulfur-bearing, has extensive biological function, mainly there is promotion brain development, is increased
Strong eyesight adjusts nerve conduction, promotes to absorb, digests fat, participates in the metabolism of cholesterol, safeguard cardiovascular and cerebrovascular, endocrine machine
Energy, anti-oxidant equal effect.Thus with memory is improved, enhance eyesight, fat reducing reduces cholesterol, protects cardiovascular and cerebrovascular, resists
Aging, raising immunity etc. all have effects that certain.With the further investigation of the mechanism of action to taurine, application also will more
Come more extensive.A kind of important nutrient of the taurine as humans and animals, extracts from natural products and has attracted much attention always.
Content of taurine is extremely low or also to be found in common food, but marine product contains very high content.China sea
Foreign living resources are abundant, such as rich content in the marine products such as oyster, scallop, octopus, but product itself price is more expensive, such as
Fruit extracts taurine from them, and then cost is higher.This project is investigated by early period, finds mussel, marine rainbow and shrimp head and shrimp
Leftover bits and pieces in equally contain abundant taurine.These products are widely present in Dalian Area, and cheap.From marine product
It is middle to extract the simple for process and pollution-free of taurine, and there is environmentally friendly meaning again with the leftover bits and pieces of aquatic products extraction taurine
Justice, foreground are very good.
Since at the beginning of nineteen fifty, artificial synthesized taurine is started in the world.Currently, the ox sulphur used on domestic and international market
Acid has chemically synthesized taurine and natural taurine, but is wherein mostly chemical synthesis production.Although synthetic method taurine valence
The problems such as lattice are cheap, but that there are material toxicities is big, complex operation, environmental pollution.With living standards of the people raising and
The progress of Green Chemistry, people increasingly advocate using the taurine naturally extracted.Therefore, taurine is extracted from natural products
As market active demand, have great application prospect.
Invention content
It is an object of the invention to overcome the deficiencies in the prior art, by taurine by removing protein, decolourize, isolate and purify
Method is extracted from mussel.
The present invention solves its technical problem and following technical scheme is taken to realize：Natural taurine is extracted from mussel
Method comprises the steps of：S1 prepares the clear liquid containing taurine using mussel；S2 is by the clear liquid removing protein；S3 is by the step
The product of rapid S2 is isolated and purified.
Preferably, the step S1 is comprised the steps of：Mussel cleaning decladding is taken out mussel meat by S1.1；S1.2 will make a gift of
Shellfish meat is put into tissue mashing machine and is homogenized；Mussel meat after homogenate is weighed and uses the water dilution of 3 times of quality by S1.3；S1.4 is adjusted
PH value；S1.5 is put into progress water extraction in constant incubator；Water extraction product is filtered by S1.6, takes filtrate.
Preferably, the step S2 is comprised the steps of：S2.1 removes acidic protein；S2.2 removes basic protein.
Preferably, the pH of the step S1 products is adjusted to 3-4 except acidic protein by the step S2.1 using hydrochloric acid, then
It is centrifuged 20 minutes at 4500r/min, and collects supernatant.
Preferably, the pH of the product of the step S2.1 is adjusted to using sodium hydroxide except basic protein by the step S2.2
9-10 is centrifuged 20 minutes at 4500r/min, collects supernatant.
Preferably, the step S3 the specific steps are：S3.1 adjusts pH to the 4-5 of the step S2 products；S3.2 will
The product of the step S3.1 injects strong acidic ion resin exchange column, and is eluted using distilled water；S3.3, which is collected, to be inhaled
The part efflux of luminosity peak-peak 0.55 or so.
Preferably, there is the step of decolourizing to the step S2 products between the step S2 and the step S3.
Preferably, the decolorization process includes specifically：Activated carbon is added into the product of the step S2 to decolourize.
Preferably, 2~3g activated carbons are added into the product of step S2 described in every 100mL.
The advantages and positive effects of the present invention are：The problem of advantages of nontoxic raw materials benefit obtains, and there is no environmental pollutions.And technique
Easy to operate, yield is big.It is extracted using from containing the highest marine product of amount of taurine, takes the most common mussel in Dalian Area and make
For raw material.Mild mode is extracted, and then after the operation of a row, organic solvent-free residual accomplishes naturally to extract.
Description of the drawings
Fig. 1 is the canonical plotting of taurine；
Fig. 2 is that pH extracts influence curve figure to taurine；
Fig. 3 is that enzymolysis time extracts influence curve figure to taurine；
Fig. 4 is that hydrolysis temperature extracts influence curve figure to taurine；
Fig. 5 is each factorial effect curve graph；
Fig. 6 is taurine standard items infrared spectrogram；
Fig. 7 is the infrared spectrogram of experiment extraction taurine；
Fig. 8 is taurine standard items hydrogen nuclear magnetic resonance spectrogram；
Fig. 9 is taurine sample product hydrogen nuclear magnetic resonance spectrogram.
Specific implementation mode
Below in conjunction with the accompanying drawings, by specific embodiment, the invention will be further described.Following embodiment is descriptive
, it is not restrictive, protection scope of the present invention cannot be limited with this.Experimental method described in embodiment is such as without special theory
It is bright, it is conventional method；Unless otherwise specified, the reagent and equipment, commercially obtain.
Experiment reagent
Laboratory apparatus
The determination of standard curve regression equation
10mmol/L taurine titer 0mL, 1mL, 2mL, 4mL, 6mL, 8mL, 10mL are taken respectively, are settled to 10mL, respectively
One milliliter is taken, 1ml color developing agents, 8ml 1mol/L sodium acetates is added, OD values are measured at 400nm, obtains standard curve (as schemed
1)。
Color developing agent is settled to 25ml with deionized water by 10ml 1mol/L sodium acetates, 0.4ml acetylacetone,2,4-pentanediones, 1ml formaldehyde and matches
System need to face with now matching.The reason of color reaction behind, is, in the solution containing sodium acetate, taurine and acetylacetone,2,4-pentanedione and first
Aldehyde can generate 1,4 dihydropyridine of N- substituent group 2,6- dimethyl 3,5- diacetyls under the high temperature conditions.This complex energy
Apparent yellow is enough showed, and content of taurine is higher, then color is more apparent, a length of 390~400nm of maximum absorption wave.
Taurine colour developing principle used in the present invention is：In the presence of sodium acetate, taurine and acetylacetone,2,4-pentanedione and formaldehyde
Heated reaction generates 1,4 dihydropyridine complex of N- substituent group 2,6- dimethyl 3,5- diacetyls.The complex displaing yellow,
Its absorbance is directly proportional in a certain range to content of taurine, a length of 390~400nm of maximum absorption wave.
Embodiment 1
Step 1：The preparation of the clear liquid containing taurine
Mussel meat is crushed, pure water is added, stirs 10min, agitator speed 30r/min, by the mussel meat by homogenate
With adjusting pH value after the water dilution of equivalent volumes to neutrality.
Step 2：Ultrasonication
It preferably incorporates in homogenate for convenience of taurine, according to the difference of experimental group, is distinguished using sonicator
The broken of 5min, 10min, 15min, 20min, 25min is carried out, homogenate is filtered using water cycle suction filtration machine, takes filter
Liquid.
Step 3：Enzyme process slightly carries taurine
The homogenate 30ml being crushed is taken, 0.5g neutrality papains are added thereto, pH is adjusted after mixing to specified
Value is put into the pot of assigned temperature constant temperature waters, stands 20min, centre agitation is several times.The homogenate that extraction is completed, is put into 100
Degree Celsius water-bath in heat 15min, so that papain is lost activity under high temperature action.
During experiment of single factor, three variables are taken, are removed the ultrasonic disruption time mentioned above, also the temperature of enzyme extraction
Degree and pH, each variable respectively set five levels.Through inquiry, test used papain is 6~7 using pH, is used
Temperature is 55~65 DEG C, I expands its pH range to 5.5~7.5, and carries out experiment of single factor with every difference 0.5 for a shelves；
Its temperature range is expanded to 45~65 DEG C, and with every 5 DEG C for a shelves.
In orthogonal experiment process, according to bell curve, four conditions are selected from five condition settings, carry out three factors
Four horizontal orthogonal experiments.
Step 4：Removing protein
The clam meat homogenate (extracting solution) after enzyme deactivation is taken, 2~3 times are filtered repeatedly extremely using recirculated water Vacuum filtration device
Extracting solution is in the clear solution whitened slightly.
Extracting solution pH to 4~5 is adjusted with 5%HCl (mass ratio, below-mentioned all percentages are mass ratio),
Room temperature, 4500r/min centrifuge 15min.Stay supernatant.
The supernatant obtained in previous step with a concentration of 5%NaOH adjust pH to 9~10, and continue under normal temperature condition,
15min is centrifuged with the speed of 4500r/min.Stay supernatant.
Step 5：Decoloration
Suitable activated carbon is added into the supernatant of collection to decolourize, preferred embodiment is that 2~3g is added per 100mL,
Filtered pH value of solution is adjusted to 4.5 with 5%HCl, for use.
Step 6：It isolates and purifies
Draw 8.0mL collect solution inject 7cm × 60cm strong acidic ion resin exchange columns, and using distilled water into
Row elution, elution speed collect the part efflux of absorbance peak-peak or so in 2mL/min.
Step 7：The measurement of extracted amount
The extracted amount of taurine is measured using formaldehyde-acetylacetone method.1mL effluxes or its dilution are taken, 1mol/L is added
Sodium acetate solution 8mL is added 1mL formaldehyde-acetylacetone,2,4-pentanedione as color developing agent, prepares 10mL solution, 15 are kept the temperature in 100 DEG C of water-baths
Minute, it is cooled to room temperature, absorbance is measured at 400nm wavelength, by taurine standard solution absorption curve (such as Fig. 1 institutes
Show), determine the extracted amount of taurine.
Step 8：Detection crystallization
Component containing taurine is detected using ultraviolet spectrophotometry, puts it in 4 DEG C of refrigerators and places 2h, obtain ox
Sulfonic acid crude crystalline.Crude product is redissolved in water, after filtering, adds absolute ethyl alcohol, 4 DEG C of recrystallizations are positioned over, by solid-liquid
It is detached, is so crystallized, then recrystallize repeatedly, you can obtain pure taurine crystal.
Experiment of single factor
50 grams of mussel meats are taken, 150 grams of deionized waters are added, tissue smashing machine is put into and homogenate is made；Homogenate is divided into
Impartial 5 parts, and homogenate pH value is tuned into 5,5.5,6,6.5,7 successively, temperature is controlled at 55 DEG C, is added a concentration of 0.6%
Papain, ultrasonic cell disruption instrument effect under carry out clasmatosis extract 15min.Show that pH extracts taurine
Influence the results are shown in Figure 2.
Taurine extracts yield highest when according to fig. 2 and data analysis obtains pH=5.5, after pH5.5, taurine
Rate is declining successively.Therefore determine that Optimal pH condition is 5.5.
50 grams of mussel meats are taken, 150 grams of deionized waters are added, tissue smashing machine is put into and homogenate is made；Homogenate is divided into
Impartial 5 parts, and set gradually ultrasonic wave _ enzymolysis it is broken when enzymolysis time be 5min, 10min, 15min, 20min, 25min,
5.5, hydrolysis temperature is 55 DEG C for pH controls.A concentration of 0.6% papain is added, is acted in ultrasonic cell disruption instrument
Lower progress clasmatosis extraction.
For enzymolysis time in 20min, the extraction rate reached of taurine digests ox sulphur after 20min to peak value as can be seen from FIG. 3
Sour yield is on a declining curve, therefore determines that the peak enzymolysis-ability time is 20min.
50 grams of mussel meats are taken, 150 grams of deionized waters are added, tissue smashing machine is put into and homogenate is made；Homogenate is divided into
Impartial 5 parts, and set gradually hydrolysis temperature when ultrasonic wave enzymolysis is crushed and be followed successively by 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, pH
A concentration of 0.6% papain is added 5.5 in control, and carrying out clasmatosis under ultrasonic cell disruption instrument effect carries
Take 15min.Hydrolysis temperature to influence that taurine extracts, the results are shown in Figure 4.
As can be seen from FIG. 4, initial extraction time gradually continues, and the extraction yield of taurine is extremely aobvious also with the continuity of time
Write improve, and before 60 DEG C taurine extraction yield increase trend it is fairly obvious, upon extracting between at 60 DEG C, the extraction of taurine
Yield is maximum, hereafter the time continue again, the extraction yield of taurine is below yield at 60 DEG C, therefore selects 60 DEG C most preferably to carry
Take the time.
Orthogonal experiment
1 empirical factor table of table
It is operated according to the orthogonal design table of design, obtains orthogonal result table 2.
The intuitive analytical table of 2 Orthogonal experiment results of table
The extracted amount that taurine is measured according to spectrophotometry determines that calibration curve equation is Y=2.2257x-
0.01857, R2=0.99118, it may be determined that the single factor test condition selected in material clam meets linear rule, can be used as orthogonal
The level conditions of experiment determine that information is as shown in Figure 5.
According to data in intuitive analytical table it is found that hydrolysis temperature, pH, broken the very poor of time are sequentially reduced, illustrate to digest
Temperature, pH, broken time are followed successively by hydrolysis temperature to the influence sequence of taurine extraction experiment>pH>The broken time.It is true according to table
It is 5.5 that determine optimum extraction condition, which be pH, and it is 20min that ultrasonic wave-enzymolysis, which is crushed the time, and breaking temperature is 60 DEG C.
Infrared detection
In the infrared detection experiment of taurine, pellet technique is used, the implementation of specific experiment is with reference to country
Standard GB/T 6040-2002《Infrared spectrum analysis general rule》It carries out.Such as Fig. 6,7, sample and taurine standard items are compared
Infrared absorption peak picture, would know that through absolute ethyl alcohol handle be precipitated white needle-like crystals be exactly high-purity natural taurine.
Nuclear-magnetism detects
It is shown such as Fig. 8,9, occur three signals in nmr spectrum, illustrates that there are three types of different types of H, non-is not δ
4.646, for δ 3.296 and δ 3.132 [27] through comparing, these data are similar with standard items, illustrate to obtain from mussel homogenate
White needle-like crystals be high-purity natural taurine crystal.
Claims (1)
1. the method for extracting natural taurine from mussel, which is characterized in that comprise the steps of：
S1 prepares the clear liquid containing taurine using mussel；
Mussel cleaning decladding is taken out mussel meat by S1.1；
Mussel meat is put into tissue mashing machine and is homogenized by S1.2；
Mussel meat after homogenate is weighed and uses the water dilution of 3 times of quality by S1.3；
S1.4 adjusts pH value to neutrality；
S1.5 is put into progress water extraction in constant incubator；
Water extraction product is filtered by S1.6, takes filtrate；
Before by the clear liquid removing protein, it is crushed using sonicator, homogenate is filtered, take filtrate；It takes
The homogenate 30ml being crushed is added 0.5g neutrality papains, adjusts pH=5.5 after mixing, be put into 60 DEG C of perseverance thereto
In warm water bath, 20min is stood, several times, the homogenate that extraction is completed is put into 100 degrees Celsius of water-bath for centre agitation
15min is heated, papain is made to lose activity under high temperature action；
S2 is by the clear liquid removing protein；
S2.1 removes acidic protein；
The pH of the step S1 products is adjusted to 3-4 by the step S2.1 except acidic protein using hydrochloric acid, then in 4500r/min
Lower centrifugation 20 minutes, and collect supernatant；
S2.2 removes basic protein；
The pH of the product of the step S2.1 is adjusted to 9-10 by the step S2.2 except basic protein using sodium hydroxide,
It is centrifuged 20 minutes under 4500r/min, collects supernatant；
The activated carbon that 2~3g is added in the supernatant collected per 100mL decolourizes, by 5% HCl tune of filtered pH value of solution
It saves to 4.5；
S3 isolates and purifies the product of the step S2；
It draws the solution collected and injects strong acidic ion resin exchange column, draw the solution that 8.0mL is collected and inject 7cm × 60cm
Strong acidic ion resin exchange column, and eluted using distilled water, elution speed is collected in 2mL/min and inhales absorbance most
The part efflux of peak value 0.55 or so；
The measurement of step S4 extracted amounts；
The extracted amount that taurine is measured using formaldehyde-acetylacetone method, is taken 1mL effluxes or its dilution, adds 1mol/L acetic acid
Sodium solution 8mL is added 1mL formaldehyde-acetylacetone,2,4-pentanedione as color developing agent, prepares 10mL solution, 15 points are kept the temperature in 100 DEG C of water-baths
Clock is cooled to room temperature, and absorbance is measured at 400 nm wavelength, taurine is determined by taurine standard solution absorption curve
Extracted amount；
Step 8：Detection crystallization；
Component containing taurine is detected using ultraviolet spectrophotometry, puts it in 4 DEG C of refrigerators and places 2h, obtain taurine
Crude product is redissolved in water, after filtering, adds absolute ethyl alcohol by crude crystalline, is positioned over 4 DEG C of recrystallizations, solid-liquid is carried out
Separation is so crystallized, then is recrystallized repeatedly, you can obtains pure taurine crystal.
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| CN111153835B (en) * | 2020-01-17 | 2021-03-30 | 吉林大学 | Method for preparing taurine through ultrahigh pressure assisted enzymolysis of freshwater mussel meat |
| CN113200889A (en) * | 2020-12-18 | 2021-08-03 | 浙江经贸职业技术学院 | Method for preparing taurine from mussels |
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| CN102643368B (en) * | 2012-05-07 | 2014-04-09 | 连云港职业技术学院 | Method for synchronously extracting taurine, chitin and polypeptide from shrimp heads and shrimp leftovers |
| CN102807511B (en) * | 2012-06-18 | 2013-12-25 | 浙江省海洋开发研究院 | Method for extracting taurine from mussel |
| CN103408473B (en) * | 2013-07-22 | 2016-01-06 | 中国科学院海洋研究所 | A kind of method extracting natural taurine from scallop splanchna |
| CN104725284B (en) * | 2015-03-06 | 2016-06-29 | 集美大学 | A kind of natural taurine preparation method |
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