CN109734822A - A kind of comprehensive method for extracting abalone internal organs bioactive substance - Google Patents
A kind of comprehensive method for extracting abalone internal organs bioactive substance Download PDFInfo
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Abstract
The invention discloses a kind of comprehensive methods for extracting abalone internal organs bioactive substance; including water-bath extraction, primary centrifugation, the processing of pulse alternating electric field, secondary centrifuging, alcohol precipitation, distillation, membrane filtration, isolated polypeptide, concentration, crystallization, four kinds of target products of crude protein, polysaccharide, polypeptide, taurine of feed are obtained in entire extraction process.It is the integrated extraction technique for extracting target that the present invention provides a kind of by raw material, collection various bioactivators of abalone internal organs waste, the rational exploitation and utilization bioactive ingredients of abalone internal organs, and content of beary metal is low in the polysaccharide product obtained, extraction efficiency is high, input cost is low, it is effective way that is most scientific, being most reasonably reality potential productivity in current industry by transformation of scientific findings, there is very high economic benefit and social value.
Description
Technical field
The present invention relates to marine product waste utilization studying technological domains, and in particular to a kind of comprehensive extraction abalone internal organs biology
The method of active material.
Background technique
Abalone fine and tender taste, the title delicious, full of nutrition for being endowed " hat of seafood delights ", the edible and processing of abalone mainly with
Based on abdominal foot.And account for about the abalone internal organs of abalone weight 20% or so, it is not yet received makes full use of in process, usually make
Waste abandons, while giving the affected pollution of environment.In fact, nutriment rich in and special ocean in abalone internal organs
Bioactive ingredients, including taurine, fish oil, phosphatide, polysaccharide, polypeptide, amino acid, enzyme etc., Efficient Development are dirty using abalone
Device resource significance is great.
Currently, abalone internal organs active material scientific research has obtained good progress.Researcher utilizes modern study
Technology has obtained a variety of abalone internal organs active constituents using different research methods, such as: polysaccharide, fish oil, phosphatide, taurine,
Polypeptide, enzyme etc..More comprehensive research, knot have been done to each activity substance content, composition, bioactive functions on this basis
Fruit shows that abalone internal organs active material has important physiological activity.But up to the present, these researchs also rest on experiment
In the stage, the problem of being primarily present the following aspects of tracing it to its cause: 1, extraction process products therefrom is more single, is mainly manifested in
A set of extraction process can only extract polysaccharide, fish oil, phosphatide, taurine, polypeptide, one or two kinds of in enzyme, extract
The economic benefit that substance generates is very little；2, effective component extraction rate is lower, while can generate a large amount of secondary emission pollution；
3, complex process, production equipment cost input are big, low in economic efficiency.It is effective that the above problem seriously constrains extraction abalone internal organs
The industrialized production of ingredient.In addition, research shows that in abalone internal organ containing very high heavy metal, especially cadmium content it is especially high,
The content of beary metal that finished product how is reduced when extracting polysaccharide is greatly to challenge, such as patent ZL201610187090 .9 is utilized
The heavy metal of the method abalone internal organs Polysaccharide removing of resin adsorption, although removal effect is good, process efficiency is low, and operation is multiple
It is miscellaneous, environment easy to pollute；It is removed in ZL201110123164.X using high-pressure pulse electric processing abalone polysaccharide combination electroosmose process
Heavy metal, but there are the danger of certain high voltage, and wherein electric field strength is up to 12 ~ 35kv/cm, and equipment manufacturing costs are excessively high, and
It is electrodialytic process low efficiency, at high cost, it is difficult into industrial practical application.
Therefore, it is highly desirable to research and develop a kind of novel technology for extracting for integrating various bioactivators in industry, close
The bioactive ingredients of reason development and utilization abalone internal organs, are reality potential productivity by transformation of scientific findings, are gone out for social creativity higher
Economic value and social value.
Summary of the invention
The object of the present invention is to provide a kind of comprehensive methods for extracting abalone internal organs bioactive substance, to solve existing mention
Taking technique extract content of beary metal in abalone internal organs bioactive substance there are extract component single, polysaccharide is high, extraction efficiency is low,
The low problem of high production cost, productivity effect.
The purpose of the present invention is what is be achieved through the following technical solutions: a kind of comprehensive extraction abalone internal organs bioactive substance
Method, comprising the following steps:
(a) water-bath extracts: fresh abalone internal organs being mixed by weight 1:0.2 ~ 0.5 with water, 60 ~ 75 DEG C is heated to and keeps 30
~ 50min obtains abalone internal organs water-bath leaching liquor；
(b) primary centrifugation: the abalone internal organs water-bath leaching liquor is centrifuged by horizontal spiral centrifuge, separation obtains crude protein
Sediment and separating liquid A；
(c) pulse alternating electric field is handled: the separating liquid A being handled by pulse alternating electric field, obtains flocculent liquid；Institute
The process for stating the processing of pulse alternating electric field is continous way, and processing voltage is 220 ~ 380V, process chamber internal electric field field strength for 50 ~
80V/cm；Handle 2 ~ 4s of time；
(d) secondary centrifuging: by flocculent liquid obtained by step (c) by disk centrifugal separator or tube centrifuge carry out from
The heart obtains crude protein sediment and supernatant B；
(e) alcohol precipitation: with ethyl alcohol being that 1:2.5 ~ 3.3 mix by the obtained supernatant B of step (d) by volume, and sedimentation centrifugation obtains
Polysaccharide precipitation object and supernatant C；
(f) distill: the supernatant C distillation removal ethyl alcohol that step (e) is obtained obtains liquid D；
(g) membrane filtration: step (f) is obtained into liquid D through nanofiltration UF membrane, obtains trapped fluid E and permeate F；
(h) isolated polypeptide: with ethyl alcohol being that 1:4 ~ 5.5 mix by trapped fluid E that step (g) obtains, centrifugation obtains by volume
Clear liquid G and sediment abalone polypeptide；
(i) the obtained permeate F of step (g) is concentrated, and crystallization obtains taurine crystal.
The revolving speed that horizontal spiral centrifuge is centrifuged in the step (b) is 3000 ~ 4000 r/min, and treatment process is continuous
Formula, 1500 ~ 2000kg/h for the treatment of capacity.
Step (c) the pulse alternating electric field processing refers to that the alternating current of sine wave is turned off by the moment of solid-state relay
With so that continuous alternating current is become pulse current, duty ratio is controlled by PLC, and leaching liquor is continued through by pumping
Pulse alternating electric field obtains flocculent liquid under the action of electric field.
The revolving speed that disk centrifugal separator is centrifuged in the step (d) is 10000 ~ 14000r/min, and treatment process is continous way,
Treating capacity is 1000 ~ 1500kg/h；The revolving speed of tube centrifuge centrifugation is 12000 ~ 15000r/min, and treatment process is continuous
Formula, treating capacity are 400 ~ 1000kg/h.
The revolving speed of tube centrifuge centrifugation is 4000 ~ 5000r/min in the step (e) and step (h), and treatment process is
Continous way, treating capacity are 400 ~ 1000kg/h.
The molecular cut off of step (f) the nanofiltration UF membrane is 250-1000, operating pressure 0.3-0.8Mpa.
The present invention provides a kind of comprehensive methods for extracting abalone internal organs bioactive substance of simple possible, make full use of
Abalone internal organs resource passes through abalone internal organs water-bath extraction, primary centrifugation, the processing of alternating impulse electric field, secondary centrifuging, alcohol precipitation, steaming
Evaporate, film worry, separate, crystallizing and etc., obtain abalone internal organs polysaccharide, four kinds of polypeptide, taurine and feed crude protein Objective extractions
Object.The present invention turns waste into wealth abalone internal organs resource, has accomplished zero-emission, solves existing extraction process and designs unreasonable, product
Problem single, extraction cost is high, extraction efficiency is low.Compared with prior art, the present invention its Heterosis exists:
(1) comprehensive extraction efficiency is high, and products therefrom is more；
(2) content of beary metal is low in the polysaccharide extracted；
(3) extract obtained purity is high；
(4) all physical methods of extraction process, safe operation process are easy to operate；
(5) internal organs resource is turned waste into wealth, increases added value by no pollution to the environment；
(6) equipment input cost needed for technique is low, is easy to realize industrial production.
It is the integration for extracting target that the present invention, which is realized by raw material, collection various bioactivators of abalone internal organs waste,
Extraction process, the rational exploitation and utilization bioactive ingredients of abalone internal organs, and the polysaccharide product content of beary metal obtained is low, mentions
Take that high-efficient, input cost is low, be in current industry it is most scientific, by transformation of scientific findings be reasonably most actual productivity
Approach can create huge economic value and social benefit.
Detailed description of the invention
Fig. 1 is 1 extract abalone polysaccharide component A infared spectrum of embodiment.
Fig. 2 is 1 extract abalone polysaccharide component B infared spectrum of embodiment.
Fig. 3 is 1 extract abalone polysaccharide component C infared spectrum of embodiment.
Fig. 4 is 1 extract abalone proteins SDS-PAGE band of embodiment.
Fig. 5 is 1 extract taurine infrared spectrogram of embodiment.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method unless otherwise specified, can be from unless otherwise specified, experimental facilities used in embodiment, test material, reagent etc.
Commercial sources obtain.
Embodiment 1
(1) water-bath of abalone internal organs extracts: 500kg fresh abalone internal organs are mixed with 100kg water (by solid and liquid weight ratio 1:0.2)
75 DEG C of holding 30min are heated to, abalone internal organs water-bath leaching liquor is obtained；
(2) horizontal screw centrifugal: abalone internal organs water-bath leaching liquor obtained by step (1) is separated by horizontal spiral centrifuge, if
Setting horizontal spiral centrifuge revolving speed is 4000r/min, and continuous sample introduction is handled, and treating capacity 2000kg/h, initial gross separation obtains insoluble
Property sediment and separating liquid A；The main component for being detected the infusible precipitate is crude protein；
(3) pulse alternating electric field is handled: separating liquid A obtained by step (2) being continued through the processing of pulse alternating electric field, handles voltage
For 380V, time 2s is handled, process chamber electric field intensity inside high 80V/cm obtains flocculent liquid；
(4) disk centrifugal separator is centrifugated: flocculent liquid being centrifuged by disk centrifugal separator, disk centrifugal separator revolving speed is
10000r/min, continuous sample introduction processing, treating capacity 1000kg/h obtain insoluble precipitate and supernatant B；It is insoluble to detect this
Property precipitating main component be crude protein, by gained insoluble precipitate in gained insoluble precipitate crude protein and step (2)
Merge, through drying, be pulverized into powder, is i.e. acquisition crude protein powder；
(5) alcohol precipitation: 1:2.5 is mixed the supernatant B that step (4) is obtained by volume with ethyl alcohol, is filled immediately with tube centrifuge
Nitrogen centrifugation, centrifugal rotational speed 5000r/min, continuous sample introduction, treating capacity 800kg/h obtain sediment and supernatant C；Through examining
Surveying the sediment is mainly abalone polysaccharide, and infrared detection map is shown in Fig. 1, Fig. 2 and Fig. 3；
(6) distill: the supernatant C distillation removal ethyl alcohol that step (5) is obtained obtains liquid D；Its process conditions distilled: true
Reciprocal of duty cycle -0.08Mpa, 70 DEG C of heating temperature；
(7) membrane filtration: step (6) is obtained into liquid D through nanofiltration UF membrane, nanofiltration retaining molecular weight is 800, and operating pressure is
0.5Mpa obtains trapped fluid E and permeate F；
(8) isolated polypeptide: the trapped fluid E and ethyl alcohol that step (7) is obtained, 1:4 is mixed by volume, and passes through tube centrifuge
Nitrogen charging centrifugation, centrifugal rotational speed 4000r/min, continuous sample introduction, treating capacity 500kg/h obtain supernatant G and sediment, should
Sediment is detected as abalone polypeptide, detects amino acid composition in the polypeptide and is shown in Table 2；
(9) the obtained permeate F of step (7) is concentrated, crystallization sets vacuum degree -0.08Mpa, 62 DEG C of evaporating temperature, dense
It is reduced to 20% then gradually to cool down, taurine crystal is constantly precipitated, being detected the crystal is taurine crystal, and testing result is shown in figure
5。
Embodiment 2
(1) water-bath of abalone internal organs extracts: 500kg fresh abalone internal organs are mixed with 250kg water (by solid and liquid weight ratio 1:0.5)
60 DEG C of holding 50min are heated to, abalone internal organs water-bath leaching liquor is obtained；
(2) horizontal screw centrifugal: abalone internal organs water-bath leaching liquor obtained by step (1) is separated by horizontal spiral centrifuge, if
Setting horizontal spiral centrifuge revolving speed is 4000r/min, and continuous sample introduction is handled, and treating capacity 1500kg/h, initial gross separation obtains insoluble
The crude protein and separating liquid A of property；
(3) pulse alternating electric field is handled: separating liquid A obtained by step (2) being continued through the processing of pulse alternating electric field, handles voltage
For 220V, time 4s is handled, process chamber electric field intensity inside high 50V/cm obtains flocculent liquid；
(4) disk centrifugal separator is centrifugated: flocculent liquid being centrifuged by disk centrifugal separator, disk centrifugal separator revolving speed is
14000r/min, continuous sample introduction processing, treating capacity 1500kg/h obtain insoluble precipitate crude protein and supernatant B；By institute
It obtains insoluble precipitate crude protein to merge with gained insoluble precipitate in step (2), through drying, is pulverized into powder, that is, obtains
Obtain crude protein powder；
(5) alcohol precipitation: 1:3.3 is mixed the supernatant B that step (4) is obtained by volume with ethyl alcohol, carries out tube centrifuge immediately
Nitrogen charging centrifugation, revolving speed 4000r/min, continuous sample introduction, treating capacity 800kg/h obtain sediment and supernatant C；The precipitating
Object is abalone polysaccharide；
(6) distill: the supernatant C distillation removal ethyl alcohol that step (5) is obtained obtains liquid D；Its distillation technique is the same as embodiment 1；
(7) membrane filtration: step (6) is obtained into liquid D through nanofiltration UF membrane, nanofiltration retaining molecular weight is 250, and operating pressure is
0.3Mpa obtains trapped fluid E and permeate F；
(8) isolated polypeptide: the trapped fluid E and ethyl alcohol that step (7) is obtained, 1:5.5 is mixed by volume, is filled with tube centrifuge
Nitrogen centrifugation, revolving speed 4500r/min, continuous sample introduction, treating capacity 1000kg/h obtain supernatant G and sediment, this is precipitated as Bao
Fish polypeptide；
(9) the obtained permeate F of step (7) is concentrated, crystallization, with embodiment 1, obtaining crystal is for concentration and crystallization processes
Taurine crystal.
Embodiment 3
(1) water-bath of abalone internal organs extracts: 500kg fresh abalone internal organs are mixed with 250kg water (by solid and liquid weight ratio 1:0.5)
60 DEG C of holding 50min are heated to, abalone internal organs water-bath leaching liquor is obtained；
(2) horizontal screw centrifugal: abalone internal organs water-bath leaching liquor obtained by step (1) is separated by horizontal spiral centrifuge, if
Setting horizontal spiral centrifuge revolving speed is 3500r/min, and continuous sample introduction is handled, and treating capacity 1800kg/h, initial gross separation obtains insoluble
The crude protein and separating liquid A of property；
(3) pulse alternating electric field is handled: separating liquid A obtained by step (2) being continued through the processing of alternating impulse electric field, handles voltage
For 300V, time 3s, process chamber electric field intensity inside high 70V/cm are handled, obtain flocculent liquid；
(4) disk centrifugal separator is centrifugated: flocculent liquid being centrifuged by disk centrifugal separator, disk centrifugal separator revolving speed is
12000r/min, continuous sample introduction processing, treating capacity 1250kg/h obtain insoluble precipitate crude protein and supernatant B；By institute
It obtains insoluble precipitate crude protein to merge with gained insoluble precipitate in step (2), through drying, is pulverized into powder, that is, obtains
Obtain crude protein powder；
(5) alcohol precipitation: 1:3 is mixed the supernatant B that step (4) is obtained by volume with ethyl alcohol, is carried out tube centrifuge immediately and is filled
Nitrogen centrifugation, centrifugal rotational speed 5000r/min, continuous sample introduction, treating capacity 1000kg/h obtain sediment and supernatant C；This is heavy
Starch is abalone polysaccharide；
(6) distill: the supernatant C distillation removal ethyl alcohol that step (5) is obtained obtains liquid D；Its distillation technique is the same as embodiment 1；
(7) membrane filtration: step (6) is obtained into liquid D through nanofiltration UF membrane, nanofiltration retaining molecular weight is 750, and operating pressure is
0.6Mpa obtains trapped fluid E and permeate F；
(8) isolated polypeptide: the trapped fluid E and ethyl alcohol that step (7) is obtained, 1:5 is mixed by volume, is filled by tube centrifuge
Nitrogen centrifugation, revolving speed 5000r/min, continuous sample introduction, treating capacity 1000kg/h obtain supernatant G and sediment, which is
Abalone polypeptide；
(9) the obtained permeate F of step (7) is concentrated, crystallization, concentration and crystallization processes obtain crystal with embodiment 1,
It is detected as taurine crystal.
Embodiment 4
(1) water-bath of abalone internal organs extracts: 500kg fresh abalone internal organs are mixed with 200kg water (by solid and liquid weight ratio 1:0.4)
60 DEG C of holding 35min are heated to, abalone internal organs water-bath leaching liquor is obtained；
(2) horizontal screw centrifugal: abalone internal organs water-bath leaching liquor obtained by step (1) is separated by horizontal spiral centrifuge, if
Setting horizontal spiral centrifuge revolving speed is 3800r/min, and continuous sample introduction is handled, and treating capacity 1650kg/h, initial gross separation obtains insoluble
The crude protein and separating liquid A of property；
(3) pulse alternating electric field is handled: separating liquid A obtained by step (2) being continued through the processing of alternating impulse electric field, handles voltage
For 350V, time 4s is handled, process chamber electric field intensity inside high 75V/cm obtains flocculent liquid；
(4) tube centrifuge is centrifugated: flocculent liquid being centrifuged by tube centrifuge, tube centrifuge turns
Speed is 12000r/min, and continuous sample introduction is handled, and treating capacity 400kg/h obtains insoluble precipitate crude protein and supernatant B；
Gained insoluble precipitate crude protein is merged with gained insoluble precipitate in step (2), through drying, is pulverized into powder,
Obtain crude protein powder；
(5) alcohol precipitation: 1:3 is mixed the supernatant B that step (4) is obtained by volume with ethyl alcohol, is carried out tube centrifuge immediately and is filled
Nitrogen centrifugation, centrifugal rotational speed 4000r/min, continuous sample introduction, treating capacity 400kg/h obtain sediment and supernatant C；This is heavy
Starch is abalone polysaccharide；
(6) distill: the supernatant C distillation removal ethyl alcohol that step (5) is obtained obtains liquid D；Distillation technique is the same as embodiment 1；
(7) membrane filtration: step (6) is obtained into liquid D through nanofiltration UF membrane, nanofiltration retaining molecular weight is 500, and operating pressure is
0.5Mpa obtains trapped fluid E and permeate F；
(8) isolated polypeptide: the trapped fluid E and ethyl alcohol that step (7) is obtained, 1:5 is mixed by volume, and carries out tube centrifuge
Nitrogen charging centrifugation, revolving speed 4000r/min, continuous sample introduction, treating capacity 600kg/h obtain supernatant G and sediment, the sediment
For abalone polypeptide；
(9) the obtained permeate F of step (7) is concentrated, crystallization, concentration and crystallization processes obtain crystal with embodiment 1,
It is detected as taurine crystal.
Embodiment 5
(1) water-bath of abalone internal organs extracts: 500kg fresh abalone internal organs are mixed with 250kg water (by solid and liquid weight ratio 1:0.5)
60 DEG C of holding 50min are heated to, abalone internal organs water-bath leaching liquor is obtained；
(2) horizontal screw centrifugal: abalone internal organs water-bath leaching liquor obtained by step (1) is separated by horizontal spiral centrifuge, if
Setting horizontal spiral centrifuge revolving speed is 3500r/min, and continuous sample introduction is handled, and treating capacity 1800kg/h, initial gross separation obtains insoluble
The crude protein and separating liquid A of property；
(3) pulse alternating electric field is handled: separating liquid A obtained by step (2) being continued through the processing of pulse alternating electric field, handles voltage
For 300V, time 3s is handled, process chamber electric field intensity inside high 70V/cm obtains flocculent liquid；
(4) tube centrifuge is centrifugated: flocculent liquid being centrifuged by tube centrifuge, tube centrifuge turns
Speed is 15000r/min, and continuous sample introduction is handled, and treating capacity 1000kg/h obtains insoluble precipitate crude protein and supernatant B；
Gained insoluble precipitate crude protein is merged with the insoluble crude protein of gained in step (2), through drying, is pulverized into powder,
Obtain crude protein powder；
(5) alcohol precipitation: 1:2.8 is mixed the supernatant B that step (4) is obtained by volume with ethyl alcohol, carries out tube centrifuge immediately
Nitrogen charging centrifugation, centrifugal rotational speed 5000r/min, continuous sample introduction, treating capacity 1000kg/h obtain sediment and supernatant C；It should
Sediment is abalone polysaccharide；
(6) distill: the supernatant C distillation removal ethyl alcohol that step (5) is obtained obtains liquid D；Distillation technique is the same as embodiment 1；
(7) membrane filtration: step (6) is obtained into liquid D through nanofiltration UF membrane, nanofiltration retaining molecular weight is 300, and operating pressure is
0.5Mpa obtains trapped fluid E and permeate F；
(8) isolated polypeptide: the trapped fluid E and ethyl alcohol that step (7) is obtained, 1:4.5 is mixed by volume, and carries out tubular type centrifugation
The centrifugation of machine nitrogen charging, revolving speed 5000r/min, continuous sample introduction, treating capacity 1000kg/h, obtains supernatant G and sediment abalone is more
Peptide；
(9) the obtained permeate F of step (7) is concentrated, crystallization, concentration and crystallization processes obtain crystal with embodiment 1,
It is detected as taurine crystal.
Comparative example 1
Fresh abalone internal organs → freeze-drying → pulverize and sieve → and abalone visceral meal → addition pH buffer → addition is a certain amount of
Enzyme stirs evenly → digests certain time → 90 DEG C enzyme deactivation 10min → cooling, adjust neutrality → centrifugation (4000r/min) 10min → on
Clear liquid adds 3 times of volumes, 95% second ethyl alcohol → alcohol precipitation 12h → centrifugation → acquisition polysaccharide, and polysaccharide yield is 6.5%(yield %=extract
Dry weight/raw material dry weight * 100%).Bibliography: Cheng Tingting, Li Dongmei, Li Tao, Zhu Beiwei abalone internal organs Polyose extraction item
Research [J] the food industry science and technology of part, 2008, (6): 208-210,213.
Comparative example 2
The abalone internal organ of freezing are thawed, are cleaned, vacuum freeze drying, pulverizer crushes, and crosses 30 meshes, (instant with solid-liquid ratio
The ratio of matter quality (g) and solvent volume (mL)) 1: 20 abalone internal organ dry powder is dissolved in distilled water, 60 DEG C of reaction 1h,
Be centrifuged 10 min, discard precipitating, supernatant add the 95%(volume point of 3 times of volumes similarly hereinafter) 4 DEG C of alcohol precipitations of ethyl alcohol stay overnight, centrifugation 10
Min discards supernatant liquid, precipitates vacuum freeze drying, obtains abalone polysaccharide, and polysaccharide yield is dry for 6.66%(yield %=extract
Weight/raw material dry weight * 100%).Bibliography: the extraction of Zhang Ruijuan, Ke Lina, Zheng Jing, Shi Yan, Wang Qin abalone internal organ polysaccharide, pure
Change and its anti-oxidant and bacteriostatic activity [J] Xiamen University's journal (natural science edition), 2018,57 (01): 58-64.
Comparative example 3
By the taurine crystal of solid content high-purity obtained by drying after suction filtration.1L water is added into 1kg abalone internal organ, in 100 DEG C
Under boil 60min, collect soup.95% ethyl alcohol 2L is added into soup, is being placed at room temperature for 4 days, is sunk after 2000g centrifugation 20min
Starch and supernatant.It is 49% that supernatant, which is concentrated into soluble solid content through Rotary Evaporators,.It is added into concentrate
250mL dehydrated alcohol is placed at 4 DEG C and stands 12h, discards supernatant and takes precipitating.100mL80 DEG C of hot water, dissolution are added into precipitating
80 DEG C of stirring 30min of 5g active carbon are added afterwards, filters and removes active carbon, obtains filtrate, 3 times of volumes is added in Xiang Shangshu filtrate
Removal of impurities is filtered after dehydrated alcohol immediately, filtrate, which is placed at 4 DEG C, to be stood still for crystals more than for 24 hours, as a result: being extracted in 1kg abalone internal organ
To natural taurine 3.07g, the content of taurine 3.07 ‰ of acquisition is extracted in every kilogram of abalone internal organ.From document: Zhang Qian,
Zheng Fulai, Weng Ling wait extraction and detection [J] food safety quality testing journal of natural taurine in abalone internal organ,
2014, 5(1):70-76。
Embodiment 6 detects the ingredient and content for the extract that above-described embodiment obtains
Detection method: each extract is detected by taking embodiment 1 as an example
(1) crude protein detects: collecting 1 step of embodiment (4) crude protein powder obtained, weighs, using (GB5009.5-2016
First method) method it is detected, using calculation formula: the content of protein is calculated as follows in crude protein powder:
In formula: X --- the content of protein in sample, unit are gram every hectogram (g/100g)；
V1--- test solution consumes the volume of sulfuric acid or normal hydrochloric acid titrating solution, and unit is milliliter (mL)；
V2--- reagent blank consumes the volume of sulfuric acid or normal hydrochloric acid titrating solution, and unit is milliliter (mL)；
C --- sulfuric acid or Hydrochloric Standard Titration concentration, unit are mole every liter (mol/L)；
0.0140 --- 1.0mL sulfuric acid [c (1/2H2SO4)=1.000mol/L] or hydrochloric acid [c (HCl)=1.000mol/L] standard
The quality of the comparable nitrogen of titration solution, unit are gram (g)；
The quality of m --- sample, unit are gram (g)；
V3--- the volume of digestive juice is drawn, unit is milliliter (mL)；
F --- nitrogen is scaled the coefficient of protein, and 6.25；
100 --- conversion coefficient.
The crude protein powder can be used as feed use.
(2) polysaccharide detects: the polysaccharide precipitation object obtained to step (5) weighs, and contained polysaccharide uses in sediment
Phend-sulphuric acid is detected.Phend-sulphuric acid is a kind of most common method in polysaccharide determination.It is more under concentrated sulfuric acid effect
Sugar is hydrolyzed into monosaccharide, and the furfural that rapid dehydration generates can play chromogenic reaction with phenol, be condensed into a kind of orange red compound,
There is maximum absorption band at 490nm wavelength.Testing result is shown in Table 1.
Gained polysaccharide is purified to obtain three components, respectively component A, component B, component through DEAE-52 fibre column chromatography
C.Map as shown in Figs. 1-3 is respectively obtained through infra-red sepectrometry detection；
Fig. 1 is abalone polysaccharide component A infared spectrum, show in figure respectively 3436.53,2933.48,1650.40,1417.48,
1376.62、1237.68、1147.79、1078.32、1033.37、874.01、812.71、763.68cm-1There is apparent polysaccharide at place
Key band.1237.68 cm-1Nearby it is S=O stretching vibration, shows component A containing SO4 2-Group.763.68cm-1It inhales vicinity
Receiving peak is α-D- xylopyranose characteristic absorption peak, 812.71 cm-1Locate the characteristic absorption peak of faint absorption peak mannopyranose；
874.01 cm-1Place is the characteristic absorption peak of mannopyranose, gala pyranose；1078.32 and 1033.37 cm-1The suction at place
Receiving peak is generated by pyranoid ring ehter bond C-O-C stretching vibration and the vibration of O-H angle；1417.48 and 1376.62 cm-1It is polysaccharide
Middle ﹦ CH2Deformation absorption peak；1650.40cm-1Place is the absorption peak that C=O asymmetric stretching vibration of-CHO generates；
2933.48cm-1Neighbouring acromion is C-H stretching vibration absworption peak；3436.53cm-1The absorption peak at place is that the O-H on polysaccharide stretches
Caused by contracting vibration；It is confirmed that containing mannose, galactolipin, xylose in component A.
Fig. 2 is extract abalone polysaccharide component B infared spectrum, in figure component B respectively 3448.35,2917.14,
1642.22, there is apparent polysaccharide key band at 1421.57,1249.94,1151.87,1078.32,1029.28,775.94.
1078.32 and 1029.28 cm-1The absorption peak at place is generated by pyranoid ring ehter bond C-O-C stretching vibration and the vibration of O-H angle
's；1151.87 cm-1The absorption peak at place is the characteristic absorption peak of C-O on ring；3448.35cm-1The absorption peak at place is on polysaccharide
Caused by O-H stretching vibration；1642.22cm-1Place is the absorption peak that C=O asymmetric stretching vibration of-CHO generates；1421.57 being
﹦ CH in polysaccharide2Deformation absorption peak；2917.14cm-1It is nearby the absorption peak generated by C-H stretching vibration；1249.94 cm-1
Nearby it is S=O stretching vibration, shows component B containing SO4 2-Group.
Fig. 3 is extract abalone polysaccharide component C infared spectrum, in figure component C respectively 3468.79,2933.48,
1646.31、1421.57、1360.27、1258.12、1147.79、1086.49、1029.28、849.49、788.20cm-1Place has
Apparent polysaccharide key band.1086.49 and 1029.28 cm-1The absorption peak at place is by pyranoid ring ehter bond C-O-C stretching vibration
It is generated with the vibration of O-H angle；1147.79 cm-1The absorption peak at place is the characteristic absorption peak of C-O on ring；1360.27 cm-1Place
Absorption peak is that C-H bending vibration generates；1421.57 cm-1It is ﹦ CH in polysaccharide2Deformation absorption peak；1646.31cm-1Place is-
The absorption peak that C=O asymmetric stretching vibration of CHO generates；2933.48cm-1Neighbouring acromion is C-H stretching vibration absworption peak；
3468.79cm-1Caused by the absorption peak at place is the O-H stretching vibration on polysaccharide；1258.12cm-1It is nearby S=O stretching vibration,
Show component C containing SO4 2-Group；849.49 cm-1Place is the characteristic absorption peak of α-type glycosidic bond, shows that component C is α-type polysaccharide.
(3) polypeptide detects: carrying out weighing detection to the sediment that step (8) obtain, Fig. 4 is 1 extract abalone of embodiment
Protein SDS-PAGE strips S DS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), is had found by protein gel,
Various concentration: 5mg/ml, 10mg/ml, 15mg/ml, 20mg/ml, 25 mg/ml, 30 mg/ml, 35 mg/ml, 40 mg/ml,
45 mg/ml, 50 mg/ml sample show deep mixed band；Have near 18kDa and 70 kDa, 80kDa obvious more
Peptide band.
It is tested and analyzed using amino acid detector, composition of amino acid is shown in Table 2 in polypeptide.
Composition of the table 2 through amino-acid analyzer measurement polypeptide
(4) taurine detects: the liquid obtained to step (10) weighs, and is detected using the second method of GB5009.169-2016
And FTIR spectrum method identifies taurine, testing result is shown in Fig. 5.Shown in Fig. 5, preparation gained abalone taurine and standard ox
Sulfonic acid infrared spectrogram, by FTIR spectrum method, scanning obtains the map hair of taurine recrystallised sample and standard items
Both existing absorption peak is consistent, confirms that the crystal is taurine crystal.
(5) heavy metal analysis in polysaccharide: to step (5) obtain sediment polysaccharide in heavy metal cadmium and lead examine
It surveys, using the content of national standard GB the first method of 5009.12-2017 detection lead；Cadmium is detected using the first method of GB 5009.15-2014
Content.Testing result is shown in Table 1.
Other embodiments detection method is same as above, contained content of beary metal inspection in the weight and content and polysaccharide of each extract
The results are shown in Table 1 for survey.
Contained content of beary metal testing result in the weight of each extract and content and polysaccharide in 1 embodiment of table
Through detecting, raw material abalone internal organ water content of the present invention is 80%, the i.e. dry matter containing 100kg in 500kg abalone internal organ.
Therefore, in above embodiments, abalone polysaccharide yield %=abalone polysaccharide purity %* polysaccharide precipitation object dry weight/raw material is dry
Weight * 100%)；Wherein abalone polysaccharide purity %=measure polysaccharide weight/polysaccharide precipitation object dry weight * 100%.With comparative example and existing skill
Art is compared, we can be found that extracting method provided by the present application can from a abalone internal organ and meanwhile extract 4 kinds of objects, and
Extraction process in the prior art all only solely extracts polysaccharide or a kind of object of taurine；And under equal conditions,
It is higher compared with the yield of the prior art that the application extracts target product；In addition, the extraction process of the application is easier, the polysaccharide of extraction
Middle cadmium, lead content meet national food safety standard pollutants in food limitation GB2762-2017(aquatic livestock and its system
Product Mesogastropoda Cadmium Pollutants On The Chinese≤2mg/kg, land pollutant≤1.0mg/kg), more existing common Polyose extraction technology obtains more
Content of beary metal significantly reduces in sugar.
The above is only several specific embodiments of the invention.Present invention is not limited to the above embodiments, can also there are many
Deformation.All deformations that those skilled in the art directly can export or associate from present disclosure, should all
It is considered protection scope of the present invention.
Claims (6)
1. a kind of comprehensive method for extracting abalone internal organs bioactive substance, which comprises the following steps:
(a) water-bath extracts: fresh abalone internal organs being mixed by weight 1:0.2 ~ 0.5 with water, 60 ~ 75 DEG C is heated to and keeps 30
~ 50min obtains abalone internal organs water-bath leaching liquor；
(b) primary centrifugation: the abalone internal organs water-bath leaching liquor is centrifuged by horizontal spiral centrifuge, separation obtains crude protein
Sediment and separating liquid A；
(c) pulse alternating electric field is handled: the separating liquid A being handled by pulse alternating electric field, obtains flocculent liquid；Institute
The process for stating the processing of pulse alternating electric field is continous way, and processing voltage is 220 ~ 380V, process chamber internal electric field field strength for 50 ~
80V/cm；Handle 2 ~ 4s of time；
(d) secondary centrifuging: by flocculent liquid obtained by step (c) by disk centrifugal separator or tube centrifuge carry out from
The heart obtains crude protein sediment and supernatant B；
(e) alcohol precipitation: with ethyl alcohol by volume it is that 1:2.5 ~ 3.3 mix by the obtained supernatant B of step (d), is centrifuged with tubular type
The centrifugation of machine nitrogen charging, obtains polysaccharide sediment and supernatant C；
(f) distill: the supernatant C distillation removal ethyl alcohol that step (e) is obtained obtains liquid D；
(g) membrane filtration: step (f) is obtained into liquid D through nanofiltration UF membrane, obtains trapped fluid E and permeate F；
(h) isolated polypeptide: with ethyl alcohol by volume it is that 1:4 ~ 5.5 mix by trapped fluid E that step (g) obtains, is centrifuged with tubular type
The centrifugation of machine nitrogen charging, obtains supernatant G and sediment abalone polypeptide；
(i), by the obtained permeate F concentration of step (g), crystallization, taurine crystal is obtained.
2. the comprehensive method for extracting abalone internal organs bioactive substance according to claim 1, which is characterized in that the step
Suddenly the revolving speed that horizontal spiral centrifuge is centrifuged in (b) is 3000 ~ 4000 r/min, and treatment process is continous way, and treating capacity is
1500~2000kg/h。
3. the comprehensive method for extracting abalone internal organs bioactive substance according to claim 1, which is characterized in that the step
Suddenly the processing of (c) pulse alternating electric field refers to that the alternating current of sine wave is turned off by the moment of solid-state relay and makes continuously to exchange
Electric current becomes pulse current, and duty ratio is controlled by PLC, and leaching liquor continues through pulse alternating electric field by pumping,
Flocculent liquid is obtained under the action of electric field.
4. the comprehensive method for extracting abalone internal organs bioactive substance according to claim 1, which is characterized in that the step
Suddenly the revolving speed that disk centrifugal separator is centrifuged in (d) is 10000 ~ 14000r/min, and treatment process is continous way, treating capacity is 1000 ~
1500kg/h；The revolving speed of tube centrifuge centrifugation is 12000 ~ 15000r/min, and treatment process is continous way, treating capacity is 400 ~
1000kg/h。
5. the comprehensive method for extracting abalone internal organs bioactive substance according to claim 1, which is characterized in that the step
Suddenly the revolving speed of the tube centrifuge centrifugation in (e) and step (h) is 4000 ~ 5000r/min, and treatment process is continous way, processing
Amount is 400 ~ 1000kg/h.
6. according to claim 1, the comprehensive method for extracting abalone internal organs bioactive substance, feature described in 2,3,4 or 5 exist
In the molecular cut off of step (f) the nanofiltration UF membrane is 250-1000, operating pressure 0.3-0.8Mpa.
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Denomination of invention: A method for comprehensively extracting biologically active substances from abalone organs Effective date of registration: 20220916 Granted publication date: 20210629 Pledgee: Zhao'an County Sub-branch of Postal Savings Bank of China Co.,Ltd. Pledgor: ZHAO'AN HAILIAN FOOD Co.,Ltd. Registration number: Y2022350000120 |