CN102851271B - Sweep frequency ultrasonic assisted extraction method for alliinase in garlic - Google Patents
Sweep frequency ultrasonic assisted extraction method for alliinase in garlic Download PDFInfo
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- CN102851271B CN102851271B CN201210322824.1A CN201210322824A CN102851271B CN 102851271 B CN102851271 B CN 102851271B CN 201210322824 A CN201210322824 A CN 201210322824A CN 102851271 B CN102851271 B CN 102851271B
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- 235000004611 garlic Nutrition 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 15
- 238000002137 ultrasound extraction Methods 0.000 title claims abstract description 9
- 108010092760 Alliin lyase Proteins 0.000 title abstract description 6
- 244000245420 ail Species 0.000 title 1
- 240000002234 Allium sativum Species 0.000 claims abstract description 31
- 238000000605 extraction Methods 0.000 claims abstract description 25
- 239000000706 filtrate Substances 0.000 claims abstract description 14
- 238000007789 sealing Methods 0.000 claims abstract description 7
- 239000006228 supernatant Substances 0.000 claims abstract description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000000047 product Substances 0.000 claims description 9
- 238000004140 cleaning Methods 0.000 claims description 6
- 238000005360 mashing Methods 0.000 claims description 6
- 238000005057 refrigeration Methods 0.000 claims description 6
- 230000001105 regulatory effect Effects 0.000 claims description 6
- 239000002893 slag Substances 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims description 2
- 235000011187 glycerol Nutrition 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 abstract description 9
- 102000004190 Enzymes Human genes 0.000 abstract description 9
- 239000008399 tap water Substances 0.000 abstract description 6
- 235000020679 tap water Nutrition 0.000 abstract description 6
- 238000005516 engineering process Methods 0.000 abstract description 4
- 235000013305 food Nutrition 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 abstract description 2
- 238000001816 cooling Methods 0.000 abstract 2
- 238000005119 centrifugation Methods 0.000 abstract 1
- 239000012043 crude product Substances 0.000 abstract 1
- 238000001914 filtration Methods 0.000 abstract 1
- 238000004321 preservation Methods 0.000 abstract 1
- 238000009210 therapy by ultrasound Methods 0.000 abstract 1
- 238000005406 washing Methods 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 7
- 230000008569 process Effects 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 235000015295 alliin Nutrition 0.000 description 4
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 4
- XUHLIQGRKRUKPH-GCXOYZPQSA-N Alliin Natural products N[C@H](C[S@@](=O)CC=C)C(O)=O XUHLIQGRKRUKPH-GCXOYZPQSA-N 0.000 description 3
- XUHLIQGRKRUKPH-UHFFFAOYSA-N S-allyl-L-cysteine sulfoxide Natural products OC(=O)C(N)CS(=O)CC=C XUHLIQGRKRUKPH-UHFFFAOYSA-N 0.000 description 3
- XUHLIQGRKRUKPH-DYEAUMGKSA-N alliin Chemical compound OC(=O)[C@@H](N)C[S@@](=O)CC=C XUHLIQGRKRUKPH-DYEAUMGKSA-N 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- JDLKFOPOAOFWQN-VIFPVBQESA-N Allicin Natural products C=CCS[S@](=O)CC=C JDLKFOPOAOFWQN-VIFPVBQESA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 239000004201 L-cysteine Substances 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- JDLKFOPOAOFWQN-UHFFFAOYSA-N allicin Chemical compound C=CCSS(=O)CC=C JDLKFOPOAOFWQN-UHFFFAOYSA-N 0.000 description 1
- 235000010081 allicin Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 238000006068 polycondensation reaction Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
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- Preparation Of Fruits And Vegetables (AREA)
Abstract
The invention relates to a sweep frequency ultrasonic assisted extraction method for alliinase in garlic, and belongs to the field of food processing. The method is performed according to the following steps: peeling and washing fresh garlic; placing the washed garlic in a low temperature refrigerator to carry out cold preservation for 12 hours; adding a pre-cooling crude enzyme extraction liquid; carrying out crushing homogenate in a pre-cooling tissue triturator; filling the resulting homogenate into a plastic bag, and sealing; placing the bag in an extraction tank of a sweep frequency ultrasonic device to carry out a sweep frequency ultrasonic treatment, wherein the extraction tank is filled with tap water; after completing ultrasonic extraction, filtering through four layers of gauzes, removing garlic residue, and collecting filtrate; and carrying out centrifugation on the filtrate for 20 minutes at a rotation speed of 10000 g at a temperature of 4 DEG C, wherein the resulting supernatant is the alliinase crude product. According to the present invention, the sweep frequency ultrasonic technology is adopted to extract the alliinase in the garlic at a maximum, such that characteristics of convenient operation, short extraction time, high efficiency and reduced cost are provided.
Description
Technical field
The present invention relates to food processing field, refer in particular to the method for utilizing allinase in frequency sweep ultrasonic wave technology assisted extraction fresh garlic.
Background technology
Ultrasonic extraction is to utilize ultrasonic wave to increase molecular motion of material frequency and speed, increases solvent penetration power, improves solute dissolution rate and stripping number of times, shortens the leach extraction method of extraction time.It has utilized the effects such as judder, high acceleration and cavitation phenomenon that ultrasonic wave produces, accelerates target compound and enters extraction solvent, shortens extraction time and also saves solvent load.Frequency sweep ultrasonic ripple is that sound wave changes with the specific cycle around mid-frequency, makes medium that more complicated physicochemical change occur, and effectively promotes the stripping of target substance.
Allinase (Alliinase, EC 4.4.1.4) in garlic bulblet is the dimer that contains two same subunit, exists with the form of (+S)-allyl-L-cysteine crystal, forms dendron shape monoclinic crystal.Allinase is a kind of PLP (P5P) dependency α, β-restriction enzyme, and its activity depends on PLP prothetic group.PLP-dependence protein is brought into play very important effect in the absorption of nitrogen and sulfocompound and metabolism switching process, the various reactions such as elimination, exchange and polycondensation are participated in, particularly containing α, in the amino acid of β and gamma carbon and other reactions participating in containing aminocompound.In garlic, the activity of allinase is about 36 DEG C maximums, and more than 42 DEG C activity reduces gradually.This kind of enzyme is comparatively responsive to pH, and keeping active required optimal pH is 6.5 left and right, and when pH<3.6, activity almost completely loses.After raw garlic is damaged, membranolysis, after decomposing, the alliin that allinase just exists in catalysis garlic generates this compound that has special odor of allicin, this is also the reason that garlic has many bioactive functions, so can the output of allinase most important for the garlic products that produce high-quality, and produce for it report that also there is no suitability for industrialized production at present.
Summary of the invention
The present invention aims to provide a kind of efficient method from extracting alliin from garlic enzyme, to meet medicine and the demand of health products trade to allinase.For reaching this object, the present invention, taking fresh garlic as raw material, adopts the technical scheme of frequency sweep ultrasonic assisted extraction allinase to comprise the steps:
The frequency sweep ultrasonic assisted extraction method of allinase in garlic, carries out according to following step:
Cryogenic refrigerator (0 ~ 5 DEG C) refrigeration 12 h will be positioned over after fresh garlic peeling, cleaning, add vat liquor broken homogenate in the tissue mashing machine of precooling of precooling, after regulating pH, homogenate packs plastic bag sealing into, be positioned in frequency sweep ultrasonic equipment and carry out single-frequency or the processing of double frequency frequency sweep ultrasonic, after supersound extraction finishes, four layers of filtered through gauze, discard garlic slag and collect filtrate, centrifugal 20 min of filtrate 10,000 g at 4 DEG C, supernatant liquor lyophilize is allinase raw product.
The range of frequency of the upper and lower vibration plate of frequency sweep equipment described in such scheme is 22 ± 2 kHz～68 ± 2 kHz, single-frequency is processed and is only used lower vibration plate, double frequency is processed and is used upper and lower vibration plate, every liter for the treatment of solution ultrasonic power is 8 W, frequency sweep cycle 10 ~ 100 ms, ultrasonic work total times 10～50 min, ultrasonic work/intermittent time is than 0.5:1～10:1.
The pH scope of the homogenate described in such scheme is 6～8, with 1 M NaOH or 1 M HCl adjusting.
Such scheme vat liquor used is phosphoric acid buffer, by Na
2hPO
4, KH
2pO
4, glycerine, NaCl and water are formulated.
Advantage of the present invention is: adopt the allinase in frequency sweep ultrasonic technology assisted extraction garlic, efficient pollution-free, has extracted the allinase in garlic to greatest extent.
Embodiment
The measuring method of the allinase vigor in the present invention is as follows: get 1 mL standard reaction system, this standard reaction system is by 60 mmol/L damping fluid (KH
2pO
4and Na
2hPO
4), 25 mmol/L PLP and 20 mmol/L alliins composition, its pH is 6.5.At 25 DEG C, be incubated 5 min, add 0.05 mL allinase liquid, under 25 DEG C of conditions, react 5 min, add immediately 1.5 mL 10% trichoroacetic acid(TCA) termination reactions, then add 2 of 0.5 mL, 4-dinitrobenzene hydrazine solution, under 25 DEG C of conditions, be incubated 5 min, add afterwards 5 mL 0.5 mol/L NaOH solution, be incubated 10 min at 25 DEG C, in wavelength, 520 nm places survey light absorption value, taking air as blank.Enzymic activity definition: under 25 DEG C of conditions, taking alliin as substrate.In catalytic reaction process, every milliliter of extracting solution per minute produces the contained enzyme amount of 1 μ mol pyruvic acid and is defined as 1 U.
Below in conjunction with embodiment, the present invention is described in further detail, but working of an invention mode is not limited to this.
embodiment 1
Fresh garlic is removed the peel, after cleaning, be positioned over cryogenic refrigerator (0 ~ 5 DEG C) refrigeration 12 h, take 100 g, add thick enzyme vat liquor 200 mL of 4 DEG C of precoolings, broken homogenate in the tissue mashing machine of precooling, after regulating homogenate pH to 6.0, pack plastic bag sealing into, single-frequency supersound process is carried out in the extraction pond that is full of tap water that is positioned over frequency sweep ultrasonic equipment, lower plate frequency is 40 ± 2 kHz, supersound extraction 50 min, frequency sweep cycle 10 ms, ultrasonic work/intermittent time is compared 4:1, after ultrasonic end, use four layers of filtered through gauze, discard garlic slag and collect filtrate, filtrate is at 4 DEG C 10, centrifugal 20 min of 000 g, collect supernatant liquor and measure its allinase vigor.
Controlled trial: above-mentioned processing condition do not add supersound process, is placed directly in lixiviate in tap water, carries out controlled trial, measures allinase vigor in product.
Obtain after measured, the vigor of the allinase in the every milliliter of extracting solution obtaining without frequency sweep ultrasonic extraction process is 45.6 U/mL, extracting through frequency sweep ultrasonic the vigor obtaining is 90.3 U/mL, visible, compared with direct extraction, frequency sweep ultrasonic processing makes the allinase vigor of product improve 1.98 times.
embodiment 2
Fresh garlic is removed the peel, after cleaning, be positioned over cryogenic refrigerator (0 ~ 5 DEG C) refrigeration 12 h, take 100 g, add thick enzyme vat liquor 200 mL of 4 DEG C of precoolings, broken homogenate in the tissue mashing machine of precooling, after regulating homogenate pH to 6.0, pack plastic bag sealing into, single-frequency supersound process is carried out in the extraction pond that is full of tap water that is positioned over frequency sweep ultrasonic equipment, lower plate frequency is 68 ± 2 kHz, supersound extraction 30 min, frequency sweep cycle 30 ms, ultrasonic work/intermittent time is compared 0.5:1, after ultrasonic end, use four layers of filtered through gauze, discard garlic slag and collect filtrate, filtrate is at 4 DEG C 10, centrifugal 20 min of 000 g, collect supernatant liquor and measure its allinase vigor.Controlled trial is with embodiment 1.
Obtain after measured, extract the vigor obtaining be 88.3 U/mL through frequency sweep ultrasonic, visible, compared with direct extraction, frequency sweep ultrasonic processing makes the allinase vigor of product improve 1.93 times.
embodiment 3
Fresh garlic is removed the peel, after cleaning, be positioned over cryogenic refrigerator (0 ~ 5 DEG C) refrigeration 12 h, take 100 g, add thick enzyme vat liquor 200 mL of 4 DEG C of precoolings, broken homogenate in the tissue mashing machine of precooling, after regulating homogenate pH to 8.0, pack plastic bag sealing into, double-frequency ultrasound processing is carried out in the extraction pond that is full of tap water that is positioned over frequency sweep ultrasonic equipment, upper plate frequency is 22 ± 2 kHz, lower plate frequency is 33 ± 2 kHz, supersound extraction 10 min, frequency sweep cycle 100 ms, ultrasonic work/intermittent time is compared 10:1, after ultrasonic end, use four layers of filtered through gauze, discard garlic slag and collect filtrate, filtrate is at 4 DEG C 10, centrifugal 20 min of 000 g, collect supernatant liquor and measure its allinase vigor.Controlled trial is with embodiment 1.
Obtain after measured, extract the vigor obtaining be 92.8 U/mL through frequency sweep ultrasonic, visible, compared with direct extraction, frequency sweep ultrasonic processing makes the allinase vigor of product improve 2.03 times.
embodiment 4
Fresh garlic is removed the peel, after cleaning, be positioned over cryogenic refrigerator (0 ~ 5 DEG C) refrigeration 12 h, take 100 g, add thick enzyme vat liquor 200 mL of 4 DEG C of precoolings, broken homogenate in the tissue mashing machine of precooling, after regulating homogenate pH to 7.0, pack plastic bag sealing into, double-frequency ultrasound processing is carried out in the extraction pond that is full of tap water that is positioned over frequency sweep ultrasonic equipment, upper plate frequency is 40 ± 2 kHz, lower plate frequency is 68 ± 2 kHz, supersound extraction 30 min, frequency sweep cycle 50 ms, ultrasonic work/intermittent time is compared 0.5:1, after ultrasonic end, use four layers of filtered through gauze, discard garlic slag and collect filtrate, filtrate is at 4 DEG C 10, centrifugal 20 min of 000 g, collect supernatant liquor and measure its allinase vigor.Controlled trial is with embodiment 1.
Obtain after measured, the vigor obtaining through supersound extraction is 93.7 U/mL, and visible, compared with direct extraction, frequency sweep ultrasonic processing makes the allinase vigor of product improve 2.05 times.
Claims (1)
1. the frequency sweep ultrasonic assisted extraction method of allinase in garlic, is characterized in that carrying out according to following step:
0 ~ 5 DEG C of refrigeration 12 h of cryogenic refrigerator will be positioned over after fresh garlic peeling, cleaning, add vat liquor broken homogenate in the tissue mashing machine of precooling of precooling, after regulating pH, homogenate packs plastic bag sealing into, be positioned in frequency sweep ultrasonic equipment and carry out single-frequency or the processing of double frequency frequency sweep ultrasonic, after supersound extraction finishes, filter, discard garlic slag and collect filtrate, centrifugal 20 min of filtrate 10,000 g at 4 DEG C, supernatant liquor lyophilize is allinase raw product;
The range of frequency of the upper and lower vibration plate of frequency sweep equipment is 22 ± 2 kHz～68 ± 2 kHz, single-frequency is processed and is only used lower vibration plate, double frequency is processed and is used upper and lower vibration plate, every liter for the treatment of solution ultrasonic power is 8 W, frequency sweep cycle 10 ~ 100 ms, ultrasonic work total times 10～50 min, ultrasonic work/intermittent time is than 0.5:1～10:1;
The pH scope of described homogenate is 6～8, with 1 M NaOH or 1 M HCl adjusting;
Vat liquor used is phosphoric acid buffer, by Na
2hPO
4, KH
2pO
4, glycerine, NaCl and water are formulated.
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| CN103875598B (en) * | 2014-04-08 | 2016-01-13 | 贵州大学 | A kind of preparation method reducing kind of the method for duck feather picking and solution used thereof |
| CN105995705B (en) * | 2016-05-24 | 2019-08-02 | 江苏大学 | A kind of environmentally protective preparation method of deodorized garlic powder |
| CN107114483A (en) * | 2017-04-18 | 2017-09-01 | 江苏大学 | A kind of ultrasonic wave added of butter rapidly and efficiently preparation method |
| CN108913678A (en) * | 2018-07-23 | 2018-11-30 | 江苏挺卫实业有限公司 | A kind of separating and extracting process of the garlic allinase for cereal beverage |
| CN112779244A (en) * | 2021-02-15 | 2021-05-11 | 巨野恒丰果蔬有限公司 | Sweep frequency ultrasonic assisted extraction method based on alliinase in garlic |
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|---|---|---|---|---|
| CN101696435A (en) * | 2009-10-30 | 2010-04-21 | 北京工商大学 | Method for continuously preparing garlic flavor substance by ultrasound enhanced endogenesis allinase aqueous enzymatic reaction |
| CN101781644A (en) * | 2009-10-30 | 2010-07-21 | 北京工商大学 | Immobilized allinase and method for preparing allicin by ultrasonic field enzymatic reaction |
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| CN101711969B (en) * | 2009-10-30 | 2012-08-29 | 江苏大学 | Device and method for biological processing with pulsed multi-frequency ultrasonic waves in sweep-frequency mode and use thereof |
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|---|---|---|---|---|
| CN101696435A (en) * | 2009-10-30 | 2010-04-21 | 北京工商大学 | Method for continuously preparing garlic flavor substance by ultrasound enhanced endogenesis allinase aqueous enzymatic reaction |
| CN101781644A (en) * | 2009-10-30 | 2010-07-21 | 北京工商大学 | Immobilized allinase and method for preparing allicin by ultrasonic field enzymatic reaction |
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Effective date of registration: 20240423 Address after: Room 1501-6, No. 137 Qingyang Road, Liangxi District, Wuxi City, Jiangsu Province, 214021 Patentee after: Wuxi Laikang Biotechnology Co.,Ltd. Country or region after: China Address before: Zhenjiang City, Jiangsu Province, 212013 Jingkou District Road No. 301 Patentee before: JIANGSU University Country or region before: China |