CN104531642A - Method for extracting and separating bromelain from pineapple peels with reverse micelle having Gemini surfactant - Google Patents

Method for extracting and separating bromelain from pineapple peels with reverse micelle having Gemini surfactant Download PDF

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CN104531642A
CN104531642A CN201510028564.0A CN201510028564A CN104531642A CN 104531642 A CN104531642 A CN 104531642A CN 201510028564 A CN201510028564 A CN 201510028564A CN 104531642 A CN104531642 A CN 104531642A
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reverse micelle
extraction
damping fluid
bromelain
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郭霞
万静
缪枝彤
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Yangzhou University
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/63—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/22004—Bromelain (3.4.22.4)

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Abstract

The invention relates to a method for extracting and separating bromelain from pineapple peels with a reverse micelle having a Gemini surfactant and belongs to the technical field of reverse micelle extraction in biological engineering. According to the method, bromelain is extracted from the pineapple peels with reverse micelle having the quaternary ammonium salt Gemini surfactant; during extraction, a small amount of the surfactant is only needed, bromelain in the pineapple peel crude extraction liquid can be transferred to the reverse micelle by controlling the pH of forward-extraction liquid and the pollution of the surfactant to bromelain can be decreased; and by adjusting the ratio of n-hexane to hexanol, the activity of bromelain can also be increased. The method is simple in operation process, the activity loss of bromelain is low, the activity recovery rate of bromelain is higher, the activity recovery rate of bromelain obtained by extraction can reach 163%, and the purification factor can reach 4.

Description

With the method for Gemini surface active reverse micelle extracting and separating bromeline from pineapple peel
Technical field
The invention belongs to inverse micelle abstraction technique field in biotechnology, also relate to the recovery and utilization technology of pineapple peel.
Background technology
Bromeline is typical thiol proteinase, extensively be present in pineapple fruit, bud, leaf, stem, it for the production of cheese, gelatin, protolysate, in the food industry as a kind of foodstuff additive, makes meat tenderization, clarify beer in biochemical industry; In medicine industry, can treat oedema and inflammation, normal tissue is harmless, does not affect skin-grafting, is applicable to the treatment of small areas deep burn.
At present, the method for suitability for industrialized production, extraction bromeline mainly contains three kinds: kaolin absorption method, the tannin precipitator method, ultrafiltration and concentration method; But these three kinds of methods not only production cost high, easily pollute environment, production technique and complicated operation, and gained enzymic activity is also not enough to require higher application to enzymic activity in satisfied medical rank and food.And correlative study focuses mostly in extracting bromeline from pineapple stem and pulp.
Although China's pineapple aboundresources, annual production is more than 1,400,000 tons, and for processing the food such as pineapple tin, fruit juice, what adopt is pineapple pulp fraction completely, the parts such as remaining pineapple peel are just thrown away as waste, so not only cause the waste of resource, and contaminate environment.
Summary of the invention
The object of the invention is to propose one and make full use of pineapple peel waste, carry out the method for efficient bromelain enzyme extraction with Gemini surface active reverse micelle.
The present invention includes following steps:
1) prepare bromelain enzyme extract: after the phosphate buffer solution being 5.5 ~ 6.5 by clean pineapple peel and pH value mixes and shatters, be separated through scum juice, obtain crude enzyme liquid;
2) front extraction: be 0.05 ~ 0.2M by NaCl concentration, pH value be 8 ~ 10.5 Glycine-NaOH damping fluid mix with crude enzyme liquid after again with reverse micelle with equal-volume than after mixing and vibrating, get upper organic phase through centrifugation; Wherein, reverse micelle is made up of Quaternary ammonium gemini surfactants and the mixing of normal hexane, n-hexyl alcohol and water; The concentration of described Quaternary ammonium gemini surfactants in reverse micelle is 0.01g/mL ~ 0.03g/mL;
3) extract afterwards: be 0.25 ~ 1M by KBr concentration, pH value be HAC-NaAC damping fluid and the organic phase equal-volume of 3.8 ~ 4.2 than after mixing and vibrating, a layer aqueous phase is taken off in centrifugation;
4) lyophilize: by lower floor's aqueous phase pressure be 50 ± 2Pa, temperature be-60 DEG C ~-55 DEG C at lyophilize, obtain pure bromeline.
Quaternary ammonium gemini surfactants is with C m-s-C mrepresent, structural formula is as follows:
In above formula, m=12 ~ 16, s=5 ~ 12.
Quaternary ammonium gemini surfactants is different from conventional surfactant, has two hydrophilic head bases and two hydrophobic tail chains, and two hydrophilic head bases are connected with covalent linkage by a linking group.
Reverse micelle is by water droplet stable dispersion orderly molecular aggregates in organic solvent by tensio-active agent.The present invention adopts the reverse micelle with Quaternary ammonium gemini surfactants to extract the bromeline in pineapple peel, a small amount of tensio-active agent is only needed in extraction process, the bromeline in pineapple peel crude extract can be made to be transferred to reverse micelle by the pH value controlling forward extraction liquid, the pollution of tensio-active agent to bromeline can be reduced again; By regulating the ratio of normal hexane and n-hexyl alcohol, the activity of bromeline can also be improved.PH value afterwards by controlling strip liquor can make bromeline from reverse micelle transfer and be enriched in efficiently aqueous phase.
Operating process of the present invention is simple, and dosage of surfactant is low, and extraction conditions scope is wide; be easy to large-scale production, bromelain loss of enzyme activity is few, and activity recovery is higher; after extraction, the activity recovery of gained bromeline can reach 163%, and purification can reach 4.The present invention greatly can increase the utility value of pineapple peel, also can reduce the pollution of pineapple waste to environment to a certain extent.
In addition, the volume ratio of normal hexane and n-hexyl alcohol reflects the content size of solubility promoter n-hexyl alcohol, and the content of solubility promoter is too small or excessive, all cannot obtain higher percentage extraction and the rate of recovery.Solubility promoter can insert between surfactant molecule, thus weakens the electrostatic repulsion between surfactant molecule.But when solubility promoter excessive concentration, the ratio shared by solubility promoter molecule on reverse micelle surface is excessive, and reverse micelle can be caused to diminish, forms spatial obstacle, so that cannot protein molecule be wrapped up, finally cause percentage extraction to reduce.
Mol ratio (the ω of water and Quaternary ammonium gemini surfactants 0) be an important parameter of reverse micelle, along with ω 0increase, the hydraulic radius of reverse micelle also increases gradually, the water core of reverse micelle is increased, but works as ω 0when increasing to certain value, reverse micelle because water deposition and become opaque, show now excessive water can not resolubilization to reverse micelle.
Therefore, the present invention is in reverse micelle, and the volume ratio of preferred normal hexane and n-hexyl alcohol is 6 ~ 9.5:1, and the mol ratio of water and Quaternary ammonium gemini surfactants is 10 ~ 20:1, to guarantee that front extraction obtains more desirable effect.
The preferred concentration of described Quaternary ammonium gemini surfactants in reverse micelle is 0.025/mL ~ 0.03/mL.Now, after extraction, the activity recovery of gained bromeline can reach 163%, and purification can reach 4.
In addition, in front extraction, the mixed volume ratio of described Glycine-NaOH damping fluid and crude enzyme liquid is 22 ~ 25:1.In crude enzyme liquid to be extracted, the content of enzyme is also determine that reverse micelle extracts an important factor of effect.Find that under this condition, bromeline can show good activity recovery, and purification also reaches maximum value after deliberation.
Accompanying drawing explanation
Fig. 1 is extraction efficiency after bromeline, the variation relation figure of the pH value of activity recovery, purification and front Glycine-NaOH damping fluid when extracting.
Fig. 2 is extraction efficiency after bromeline, the variation relation figure of the pH value of activity recovery, purification and front Glycine-NaOH damping fluid when extracting.
Fig. 3 is tensio-active agent dibrominated-bis-(dimethyl hexadecyl base) pentamethylene diamine (C in extraction efficiency, activity recovery, purification and reverse micelle after bromeline 16c 5c 16) the variation relation figure of concentration.
Fig. 4 is tensio-active agent dibrominated-bis-(dimethyl hexadecyl base) octamethylenediamine (C in extraction efficiency, activity recovery, purification and reverse micelle after bromeline 16c 8c 16) the variation relation figure of concentration.
Embodiment
One, concrete preparation technology:
1, reverse micelle preparation:
3 ~ 90g Quaternary ammonium gemini surfactants is dissolved in 1 L non-polar solvent, then adds 1 ~ 100mL water, fully vibration is to solution clear, is reverse micelle.
Be made into Quaternary ammonium gemini surfactants accounting in each reverse micelle with this and be respectively 0.003g/ml ~ 0.05g/ml, and, above non-polar solvent is made up of normal hexane and n-hexyl alcohol, the volume ratio of normal hexane and n-hexyl alcohol is 5 ~ 12:1, and the mol ratio of water and Quaternary ammonium gemini surfactants is 5 ~ 20:1.
2, the preparation of bromelain enzyme extract:
The preparation of damping fluid: in conventional manner, adopts Sodium phosphate dibasic and SODIUM PHOSPHATE, MONOBASIC to configure the phosphoric acid buffer that pH scope is 5.5 ~ 6.5.
Pineapple peel is cleaned, drains, be cut into segment and be placed on-20 DEG C of freezen protective.Get a certain amount of pineapple peel, add 1 times of phosphoric acid buffer, put into juice extractor, after pulverizing 3 ~ 5 min, carry out scum juice separation with four layers of gauze extruding, elimination fibrous material, filtrate is under 10000 rpm conditions, and centrifugal 25 min, get supernatant liquor, i.e. bromelain enzyme extract, puts into 4 DEG C of refrigerators for subsequent use.
3, front extraction:
The preparation of forward extraction liquid: in conventional manner, glycine and sodium hydroxide is adopted to configure the Glycine-NaOH damping fluid that pH scope is 7 ~ 12, in Glycine-NaOH damping fluid, add the NaCl of inequality again, make the concentration of NaCl in Glycine-NaOH damping fluid reach the non-equivalence of 0 ~ 0.5 M.
To containing after adding bromelain enzyme extract respectively in each Glycine-NaOH damping fluid of different NaCl concentration, then add isopyknic reverse micelle, through abundant concussion and centrifugal after, get upper strata reverse micelle clear liquid.
4, rear extraction:
The preparation of rear extraction liquid: in conventional manner, adopt acetic acid and sodium-acetate, join the Acetic acid-sodium acetate damping fluid that pH scope is 3.8 ~ 5.6, then add the KBr of inequality, make the concentration of KBr in Acetic acid-sodium acetate damping fluid reach the non-equivalence of 0 ~ 2 M, each solution of gained is rear extraction liquid.
Be 1:1 by volume, rear extraction liquid added the upper strata reverse micelle clear liquid that step 3 is got, fully concussion and centrifugal after, take off a layer bromelain enzyme solution.
5, lyophilize:
By the bromelain enzyme solution of step 4 gained, pressure be 50 ± 2 Pa, temperature be-60 DEG C ~-55 DEG C at lyophilize 36 h, obtain highly active pure bromelain enzyme preparation.
Two, the extraction efficiency (EF) of bromeline of the present invention, activity recovery (AR) and purification (PF) calculation formula are as follows;
Three, comparative analysis result:
1, tensio-active agent dibrominated in reverse micelle-bis-(dimethyl dodecyl base) octamethylenediamine (C 12c 8c 12) concentration the impact of purifying technique is tested:
Each test is carried out, wherein: the volume ratio of normal hexane and n-hexyl alcohol is 9:1 by above processing step; ω 0=20; PH=8.0 of front aqueous phase extracted; The pH=4.2 of rear aqueous phase extracted.Change dibrominated-bis-(dimethyl dodecyl base) octamethylenediamine (C 12c 8c 12) accounting in reverse micelle, get 0.0015g/ml, 0.01g/ml, 0.02g/ml, 0.025g/ml, 0.03g/ml, 0.035g/ml, 0.04g/ml, 0.045g/ml, 0.05g/ml respectively.
Test-results is in table 1:
As seen from Table 1: as dibrominated-bis-(dimethyl dodecyl base) octamethylenediamine (C 12c 8c 12) concentration in reverse micelle is when being 0.03g/mL, bromeline extraction efficiency and activity recovery reach maximum value; As dibrominated-bis-(dimethyl dodecyl base) octamethylenediamine (C 12c 8c 12) concentration in reverse micelle is when being 0.04g/mL, the purification (PF) of bromeline reaches maximum value.
2, during front extraction, the impact of pH value on the extraction efficiency of bromeline and activity recovery of Glycine-NaOH damping fluid is tested:
Each test is carried out, wherein: the volume ratio of normal hexane and n-hexyl alcohol is 9:1 by above processing step; ω 0=20; PH=8.0 of front aqueous phase extracted; The pH=4.2 of rear aqueous phase extracted.When extracting before only changing, the pH value of Glycine-NaOH damping fluid, gets 7.5,8.0,8.5,9,9.5,10,10.5,11,12 respectively.
Test-results is in table 2:
As seen from Table 2: during front extraction, when the pH value of Glycine-NaOH damping fluid is 8.0, bromelain activity recovery and purification reach maximum value.
3, during rear extraction, the impact of pH value on the extraction efficiency of bromeline and activity recovery of HAC-NaAC damping fluid is tested:
Each test is carried out, wherein: the volume ratio of normal hexane and n-hexyl alcohol is 9:1 by above processing step; ω 0=20; PH=8.0 of front aqueous phase extracted.When extracting after only changing, the pH value of HAC-NaAC damping fluid, gets 3.8,4.2,4.6,5.0,5.6 respectively.
Test-results is in table 3:
As seen from Table 3: when the pH value of HAC-NaAC damping fluid is 4.2, bromeline extraction efficiency, activity recovery and purification reach maximum value.
4, the concentration on extraction efficiency of NaCl in damping fluid during front extraction, activity recovery reach, the impact test of purification:
Each test is carried out, wherein: the volume ratio of normal hexane and n-hexyl alcohol is 9:1 by above processing step; ω 0=20; PH=8.0 of front aqueous phase extracted; The pH=4.2 of rear aqueous phase extracted.Before adopting the method for the NaCl adding different amount to change, the concentration of NaCl in Glycine-NaOH damping fluid during extraction, gets 0.05M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M respectively.
The results are shown in Table 4:
As seen from Table 4: when the concentration of NaCl in damping fluid is 0.1M, activity recovery and the purification of bromeline reach maximum value.
5, the concentration on extraction efficiency of KBr in HAC-NaAC damping fluid during rear extraction, activity recovery reach, the impact test of purification:
Each test is carried out, wherein: the volume ratio of normal hexane and n-hexyl alcohol is 9:1 by above processing step; ω 0=20; PH=8.0 of front aqueous phase extracted; The pH=4.2 of rear aqueous phase extracted.Extract the concentration of KBr in HAC-NaAC damping fluid after adopting the method adding KBr to change, get 0.25M, 0.5M, 1.0M respectively.
The results are shown in Table 5:
As seen from Table 5: when the concentration of KBr in HAC-NaAC damping fluid is 0.5M, bromelain activity recovery reaches maximum value; When the concentration of KBr in HAC-NaAC damping fluid is 0.25M, bromeline extraction efficiency reaches maximum value; When the concentration of KBr in HAC-NaAC damping fluid is 1M, the purification of bromeline reaches maximum value.
6, during front extraction the mixed volume of Glycine-NaOH damping fluid and crude enzyme liquid than the impact with extraction efficiency and activity recovery, purification:
Each test is carried out, wherein: the volume ratio of normal hexane and n-hexyl alcohol is 9:1 by above processing step; ω 0=20; PH=8.0 of front aqueous phase extracted; The pH=4.2 of rear aqueous phase extracted.Only change the volume ratio (i.e. extension rate) of crude enzyme liquid and Glycine-NaOH damping fluid, get respectively: 5,10,15,20,25,30,35.
The results are shown in Table the volume ratio of 6:(extension rate and crude enzyme liquid and Glycine-NaOH damping fluid)
As seen from Table 6: when bromeline extension rate is 25, bromelain activity recovery and purification reach maximum value.
7, during front extraction, the impact of pH value on the extraction efficiency of bromeline and activity recovery of Glycine-NaOH damping fluid is tested:
Each test is carried out, wherein: C by above processing step 12c 5c 12form reverse micelle system, the volume ratio of normal hexane and n-hexyl alcohol is 9:1; ω 0=20; [C 12c 5c 12]=0.01g/ml; The pH=4.2 of rear aqueous phase extracted.When extracting before only changing, the pH value of Glycine-NaOH damping fluid, gets 8.0,8.5,9,9.5,10,10.5,11,12 respectively.
As seen from Figure 1: during front extraction, when the pH value of Glycine-NaOH damping fluid is 10.5, bromelain activity recovery and purification reach maximum value.
8, during front extraction, the impact of pH value on the extraction efficiency of bromeline and activity recovery of Glycine-NaOH damping fluid is tested:
Each test is carried out, wherein: C by above processing step 12c 12c 12form reverse micelle system, the volume ratio of normal hexane and n-hexyl alcohol is 9:1; ω 0=10; [C 12c 12c 12]=0.03g/ml; The pH=4.2 of rear aqueous phase extracted.When extracting before only changing, the pH value of Glycine-NaOH damping fluid, gets 8.0,8.5,9,9.5,10,10.5,11,12 respectively.
As seen from Figure 2: during front extraction, when the pH value of Glycine-NaOH damping fluid is 10.0, bromelain activity recovery and purification reach maximum value.
9, tensio-active agent dibrominated in reverse micelle-bis-(dimethyl hexadecyl base) pentamethylene diamine (C 16c 5c 16) concentration the impact of purifying technique is tested:
Each test is carried out, wherein: C by above processing step 16c 5c 16form reverse micelle system, the volume ratio of normal hexane and n-hexyl alcohol is 9:1; ω 0=15; PH=10.5 of front aqueous phase extracted; The pH=4.2 of rear aqueous phase extracted.Change dibrominated-bis-(dimethyl hexadecyl base) pentamethylene diamine (C 16c 5c 16) accounting in reverse micelle, get 0.005g/ml, 0.01g/ml, 0.02g/ml, 0.03g/ml, 0.04g/ml respectively.
As seen from Figure 3: as tensio-active agent dibrominated-bis-(dimethyl hexadecyl base) pentamethylene diamine (C 16c 5c 16) concentration in reverse micelle is when being 0.01g/mL, bromeline extraction efficiency and activity recovery reach maximum value; As dibrominated-bis-(dimethyl hexadecyl base) pentamethylene diamine (C 16c 5c 16) concentration in reverse micelle is when being 0.005g/mL, the purification (PF) of bromeline reaches maximum value.
10, tensio-active agent dibrominated in reverse micelle-bis-(dimethyl hexadecyl base) octamethylenediamine (C 16c 8c 16) concentration the impact of purifying technique is tested:
Each test is carried out, wherein: C by above processing step 16c 8c 16form reverse micelle system, the volume ratio of normal hexane and n-hexyl alcohol is 9:1; ω 0=15; PH=10.5 of front aqueous phase extracted; The pH=4.2 of rear aqueous phase extracted.Change dibrominated-bis-(dimethyl hexadecyl base) pentamethylene diamine (C 16c 5c 16) accounting in reverse micelle, get 0.005g/ml, 0.01g/ml, 0.02g/ml, 0.03g/ml, 0.04g/ml respectively.
As seen from Figure 4: as tensio-active agent dibrominated-bis-(dimethyl hexadecyl base) octamethylenediamine (C 16c 8c 16) concentration in reverse micelle is when being 0.01g/mL, bromeline extraction efficiency and activity recovery reach maximum value; As dibrominated-bis-(dimethyl hexadecyl base) octamethylenediamine (C 16c 8c 16) concentration in reverse micelle is when being 0.005g/mL, the purification (PF) of bromeline reaches maximum value.
By testing the top condition and best extraction efficiency, activity recovery and the purification that obtain bromeline in Gemini surface active inverse micelle abstraction pineapple peel above, as following table:

Claims (6)

1., with the method for Gemini surface active reverse micelle extracting and separating bromeline from pineapple peel, after the phosphate buffer solution that to comprise clean pineapple peel and pH value be 5.5 ~ 6.5 mixes and shatters, be separated through scum juice, obtain crude enzyme liquid; Crude enzyme liquid is mixed with Glycine-NaOH damping fluid and reverse micelle and carries out forward extraction and get, the upper organic phase front extraction obtained again mixes with HAC-NaAC damping fluid carries out rear extraction, the lower floor's aqueous phase lyophilize finally rear extraction obtained, obtains pure bromeline; It is characterized in that:
Described reverse micelle is mixed formed by Quaternary ammonium gemini surfactants and normal hexane, n-hexyl alcohol, water; The concentration of described Quaternary ammonium gemini surfactants in reverse micelle is 0.01g/mL ~ 0.03g/mL;
After the Glycine-NaOH damping fluid that before described, extraction is is 0.05 ~ 0.2M by NaCl concentration, pH value is 8 ~ 10.5 mixes with crude enzyme liquid again with reverse micelle with equal-volume than after mixing and vibrating, get upper organic phase through centrifugation;
Described rear extraction is is 0.25 ~ 1M by KBr concentration, pH value be HAC-NaAC damping fluid and the organic phase equal-volume of 3.8 ~ 4.2 than after mixing and vibrating, a layer aqueous phase is taken off in centrifugation;
Described lyophilize be by lower floor's aqueous phase pressure be 50 ± 2Pa, temperature be-60 DEG C ~-55 DEG C at lyophilize.
2. method according to claim 1, it is characterized in that: in reverse micelle, the volume ratio of normal hexane and n-hexyl alcohol is 6 ~ 9.5:1, and the mol ratio of water and Quaternary ammonium gemini surfactants is 10 ~ 20:1.
3. method according to claim 1 or 2, is characterized in that: the concentration of described Quaternary ammonium gemini surfactants in reverse micelle is 0.025g/mL ~ 0.03g/mL.
4. method according to claim 1, it is characterized in that described before in extraction, the mixed volume ratio of described Glycine-NaOH damping fluid and crude enzyme liquid is 22 ~ 25:1.
5. method according to claim 1, before it is characterized in that during extraction, in described Glycine-NaOH damping fluid, the concentration of NaCl is 0.1M.
6. method according to claim 1, when it is characterized in that rear extraction, in described HAC-NaAC damping fluid, the concentration of KBr is 0.25 ~ 0.5M.
CN201510028564.0A 2015-01-21 2015-01-21 Method for extracting and separating bromelain from pineapple peels with reverse micelle having Gemini surfactant Pending CN104531642A (en)

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CN112280765B (en) * 2020-09-27 2021-10-08 扬州大学 Application of sulfobetaine surfactant in improving bromelinase activity
CN115078739A (en) * 2022-08-22 2022-09-20 上海执诚生物科技有限公司 Kit for detecting apolipoprotein A1 and application thereof

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Application publication date: 20150422