CN105440036A - Method for extracting folic acid from soybeans - Google Patents
Method for extracting folic acid from soybeans Download PDFInfo
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- CN105440036A CN105440036A CN201610004854.6A CN201610004854A CN105440036A CN 105440036 A CN105440036 A CN 105440036A CN 201610004854 A CN201610004854 A CN 201610004854A CN 105440036 A CN105440036 A CN 105440036A
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Abstract
The invention belongs to the field of preparation of plant extracts, and in particular relates to a method for extracting folic acid from soybean seeds. Human and animals cannot synthesize folic acid per self, and various crops are main resources for obtaining the vitamin by human and animals. A single-enzyme method and a three-enzyme method are commonly used for extraction of folic acid from the crops. The extraction conditions of the three-enzyme method are different in different food, and optimization of folic acid extraction is required to be performed on each kind of food respectively. The invention provides an optimized method for extracting the folic acid from the soybean seeds, by using the method, hundreds of samples can be extracted at the same time, and the extracting process is rapid and effective.
Description
Technical field
The invention belongs to the preparation of plant milk extract, relate to a kind of extraction of soybean seeds and the measuring method of content particularly.
Background technology
Folic acid is water-soluble B VITAMIN, and folic acid deficiency can cause health problem (Kimetal., 1998 that baby's neural tube defect, cancer and cardiovascular disorder etc. are serious; Geisel, 2003; Choi & Friso, 2005; Marisaetal., 2005).Human and animal self can not synthesize folic acid, and various crop is the main source that human and animal obtains this VITAMIN.But usually very low at the content of crop Folic Acid, especially in cereal, content is lower, and the authentic data how obtaining various folic acid derivatives content in crop is the key issue of this research field.The extraction of crop Folic Acid, conventional has single enzyme process and three enzyme process.Single enzyme process extracts effectively (Iwatanietal., 2003 for the folic acid in the foods such as spinach, cucumber, Caulis et Folium Brassicae capitatae and banana; Zhangetal., 2005).Three enzyme process are to cereals, potato, the extraction efficiency of the folate content of beans etc. is higher than single enzyme process, and three enzyme process can extract folic acid (DeSouza & Eitenmiller, 1990 of being rich in carbohydrate or albumen substrate more completely relative to single enzyme process; Tamuraetal, 1997; Pffeiferetal, 1997; Raderetal, 1998; Aiso & Tamura, 1998).The food folic acid extraction step that Hyun etc. (2005) study, appropriate amount of sample grinds to form homogenate in extracting solution, frozen in-70 DEG C until folic acid extracts after homogenate packing, after adding protease treatment sample, 100 DEG C are boiled sample inactivated proteases, recycling α-amylase and folic acid conjugase hatch sample 2 hours jointly, obtain folic acid sample, with-70 DEG C of frozen samples after centrifugal.But the three Enzymatic Extraction conditions used in different food are different (Aiso & Tamura, 1998; Pandrangietal., 2004), be necessary the optimization every type food being carried out respectively to folic acid extraction.
Crop Folic Acid detection method popular is both at home and abroad microbial method and high performance liquid chromatography at present.High performance liquid chromatography utilizes C
18after chromatographic column carries out sample separation, in conjunction with uv-absorbing, fluoroscopic examination and Electrochemical Detection can carry out quantitative analysis to single folic acid component.High performance liquid chromatography can be separated different folic acid derivatives fast and efficiently, amount of samples is few, but the sensitivity of the method is lower than microbial method, and endogenous folic acid can disturb folic acid, difficulty (Zhangetal., 2005 are quantitatively brought to the folic acid of some form; P ó o-Prietoetal., 2006).Microbial method (MicrobiologicalAssay) is the classical way detecting folic acid, its principle is that all derivatives of indicator to folic acid have identical growth response speed, under certain condition, microbial growth breeding and the proportional relation of substratum Folic Acid, according to microorganism, the degree of absorption of sample Folic Acid is carried out quantitatively (Chang Naning etc., 2010) folic acid.Lactobacillus casei is all responsive to single L-glutamic acid folic acid, two or three L-glutamic acid folic acid and reduced form derivative thereof, is the bacterial classification (Hyunetal., 2005) commonly used the most.Require in folic acid analysis that different sample has suitable extension rate, use 96 hole enzyme plates to carry out 18-24h cultivation to indicator, and after spectrophotometer reading numerical values the content of calculation sample Folic Acid.Microbial method is highly sensitive, and result is accurate, but repeatability is poor, and can only detect total folate content, but advantage does not need valuable instrument or medicine, so cost is lower, is applicable to large batch of sample detection.
To sum up, be necessary to be optimized the method for soybean folic acid three Enzymatic Extraction and microorganism detection, determine the optimum proportioning of three kinds of enzymes and the step of microbial method detection, the soybean excellent germplasm for screening homofolic acid content provides the foundation of science.
Summary of the invention
Invention is intended to solve the technical problem existed in prior art, the object of the present invention is to provide a kind of extraction and detection method of content of soybean seeds folic acid, be intended to the method setting up three Enzymatic Extraction and microorganism detection soybean seeds Folic Acid, the soybean excellent germplasm for screening homofolic acid content provides the foundation of science.
The material that the present invention uses and instrument as follows:
One, material and reagent
Conventional medication is domestic analytical reagent; Phosphoric acid buffer (production of AMERESCO company); Folic acid (Sigma company); α-amylase (deriving from aspergillus oryzae) and proteolytic enzyme (deriving from Streptomycin sulphate) (Sigma company); Rat blood serum (space in morning (Beijing) trade Co., Ltd of middle section); Milk-acid bacteria liquid nutrient medium and folic acid substratum are purchased from Beijing Ding Guo Bioisystech Co., Ltd; Lactic bacterium strains is purchased from Central Plains, Beijing He Ju Trade Co., Ltd.;
Two, instrument and equipment
AXYGEN series EP pipe (company of HTC of section of hanging oneself); Folic acid detection kit (P1001) is visitd purchased from Germany and thing company limited is occurred; High-flux tissue grinder SPEXGeno2000 (production of SPEX company of the U.S.); 3-30K high speed desktop refrigerated centrifuge (German SIGMA); Microplate reader (U.S. DynexMagellanBiosciences).
The invention provides a kind of extracting method of soybean seeds folic acid, comprise the following steps:
(1) soybean sample preparation: soybean seeds is pulverized and obtained bean powder, take 0.2g bean powder, be placed in AXYGENEP pipe, add 750 μ L folic acid Extraction buffers in the sample to which, hatch 15min for 95 DEG C and be placed in cooled on ice, add 5mm steel ball (2), ground sample in tissue grinder, obtain sample homogenization liquid;
(2) Soybean Leaves acid extraction: add 8 μ L α-amylase and 750 μ L folic acid Extraction buffers to avoid thickness in step (1) sample homogenization liquid, room temperature places 10min, adds 100 μ L proteolytic enzyme, hatches 1h for 37 DEG C; 100 DEG C are boiled 10min, cool 10min rapidly on ice; At 4 DEG C, under 14000rpm condition, centrifugal 10min; Draw supernatant, the volume ratio according to 2% adds rat blood serum 10 μ L in supernatant, after mixing, cultivates 2h in 37 DEG C; 100 DEG C are boiled deactivation 10min, cool 10min rapidly on ice; At 4 DEG C, under 14000rpm condition, centrifugal 15min; Draw supernatant and be sub-packed in direct mensuration or frozen in-80 DEG C in sterile tube, obtain Soybean Leaves acid extraction liquid.
In addition, following additional technical characteristic can also be had according to the extracting method of the above embodiment of the present invention soybean seeds folic acid:
The rotating speed ground in tissue grinder described in described soybean sample preparation process one is 1400rpm, and the time is 5min.Grind described in described soybean sample preparation process one and all need operate under lucifuge condition, Soybean Leaves acid extraction step all need operate under lucifuge condition.
Described in described soybean sample preparation process two, the component of damping fluid is the 50mM phosphoric acid buffer of pH value 6.5, adds the SODIUM ASCORBATE of 1%, and the 2 mercapto ethanol of 0.1%.
Second object of the present invention, there is provided a kind of measuring method of soybean seeds folate content, comprises the following steps:
One, the preparation of milk-acid bacteria liquid nutrient medium: 4.85g milk-acid bacteria liquid nutrient medium is added in 100mL water, 121 DEG C of sterilizing 15min;
Two, the preparation of low folic acid substratum: take 4.7g folic acid culture medium powder, 0.03g ascorbic acid, 0.3mL folic acid standard storage dosing, be dissolved in water and be settled to 100mL, 0.22 μm of filter membrane sterilizing is for subsequent use excessively;
Three, the preparation of analysis buffer: take Na
2hPO
4.2H
2o0.38g, NaH
2pO
40.93g, ascorbic acid 1g are dissolved in water and are settled to 100mL, and 0.22 μm of filter membrane sterilizing is for subsequent use excessively;
Four, the preparation of folic acid substratum: take folic acid culture medium powder 9.4g, be dissolved in water and be settled to 100mL, boil 5min, 0.22 μm of filter membrane sterilizing is for subsequent use excessively;
Five, folic acid preparation of standard sample: take the K that 28mg folic acid is dissolved in 4mL5%
2hPO
4in solution, be then settled to 250mL by analysis buffer, then the analysis buffer drawing this solution step 3 of 0.5mL obtained is settled to 100mL, obtains the standard reserving solution FA that concentration is 0.5 μ g/mL, is dispensed in 1.5mLEP pipe and is stored in-20 DEG C;
Six, the preparation of indicator: draw milk-acid bacteria preservation bacterium liquid 500 μ L and join in 10mL milk-acid bacteria liquid nutrient medium, 24h cultivated by 37 DEG C of shaking tables, again draw cultured bacterium liquid 0.5mL and join in the low folic acid substratum of 100mL, and 18h cultivated by 37 DEG C of shaking tables; Triangular flask is placed in cooled on ice 20min; The glycerine of precooling is mixed with culture (volume ratio is 40:60), stir; Packing bacterium liquid in 1.5mLEP pipe, in-80 DEG C of preservations after liquid nitrogen flash freezer;
Seven, folate content measures: the analysis buffer utilizing step one obtained, by FA solution dilution 250 times obtained for step 5, obtains FA-1 solution, utilizes analysis buffer to dilute according to 2:1 ratio, obtain FA-2 solution; FA-1 and FA-2 solution is all for production standard curve; By of short duration for folic acid sample centrifugal, with analysis buffer dilute sample 30 times; In 96 orifice plates, after 150 μ L analysis buffer are added each sample well, again 150 μ LFA-1, FA-2 and soybean folic acid liquid to be measured are added respectively the 1st row of 96 orifice plates, mixed solution 150 μ l is drawn to the 2nd row from the 1st row, mixed solution 150 μ l is being drawn to the 3rd row from the 2nd row, mixed solution 150 μ l is drawn to the 4th row again from the 3rd row, mixed solution 150 μ l is being drawn to the 5th row from the 4th row, draw mixed solution 150 μ l to the 6th row from the 5th row again, finally the sample mix liquid that the 6th arranges is discarded wherein 150 μ l; The FACM substratum 150 μ L adding indicator is added in 96 orifice plates; Culture plate is placed in 37 DEG C and cultivates 18-20h; Under wavelength is 580nm condition, the OD value of working sample; According to measuring numerical value Criterion curve and calculating the folic acid concentration comprised in sample.
Technique effect of the present invention is:
1, the sample Ginding process that the present invention adopts comes alternative manual grind away or stirrer Ginding process, can extract simultaneously simultaneously, while efficiently completing leaching process, decrease testing error to up to a hundred samples.
2, the present invention choose the α-amylase that can derive from aspergillus oryzae and from the proteolytic enzyme of streptomycete and rat blood serum to extract the folic acid in soybean.
What 3, the present invention determined α-amylase, proteolytic enzyme and rat blood serum is combined in 8 μ L, farthest can extract the folic acid in soybean seeds under the condition of 100 μ L and 10 μ L.
4, the present invention determines the method for microbiology polymorphism folate content: folic acid standard model analysis buffer is diluted 250 times, obtains FA-1 solution, utilize analysis buffer to dilute according to 2:1 ratio, obtain FA-2 solution; FA-1 and FA-2 solution is all for production standard curve; By of short duration for folic acid sample centrifugal, with analysis buffer dilute sample 30 times; In 96 orifice plates, after 150 μ L analysis buffer are added each sample well, again 150 μ LFA-1, FA-2 and soybean folic acid liquid to be measured are added respectively the 1st row of 96 orifice plates, mixed solution 150 μ l is drawn to the 2nd row from the 1st row, mixed solution 150 μ l is being drawn to the 3rd row from the 2nd row, mixed solution 150 μ l is drawn to the 4th row again from the 3rd row, mixed solution 150 μ l is being drawn to the 5th row from the 4th row, draw mixed solution 150 μ l to the 6th row from the 5th row again, finally the sample mix liquid that the 6th arranges is discarded wherein 150 μ l; The FACM substratum 150 μ L adding indicator is added in 96 orifice plates; Culture plate is placed in 37 DEG C and cultivates 18-20h; Under wavelength is 580nm condition, the OD value of working sample; According to measuring numerical value Criterion curve and calculating the folic acid concentration comprised in sample.
Embodiment
Embodiments of the invention are described below in detail, used only for explaining the present invention, and can not limitation of the present invention be interpreted as.
Prepared by embodiment 1 soybean sample
Soybean seeds is pulverized and is obtained bean powder, takes bean powder 0.2g, is placed in 2mLAXYGENEP pipe, repeats for three times.750 μ L folic acid Extraction buffer (50mM phosphoric acid buffers are added in every increment product, PH=6.5,1% SODIUM ASCORBATE, 0.1%2-mercaptoethanol), hatch 15min at 95 DEG C and be placed in cooled on ice, add 5mm steel ball afterwards, ground sample (rotating speed is 1400rpm, and the time is 5min) in tissue grinder.Sample process of lapping all needs lucifuge to operate.
The acid extraction of embodiment 2 Soybean Leaves
In sample homogenization liquid, add 8 μ L α-amylase and 750 μ L folic acid Extraction buffers to avoid thickness, room temperature places 10min, adds 100 μ L proteolytic enzyme, hatches 1hr for 37 DEG C; 100 DEG C are boiled said mixture 10min to stop enzyme effect, cool 10min rapidly on ice; At 4 DEG C, under 14000rpm condition, centrifugal 10min; Draw supernatant, the volume according to 2% adds rat blood serum 10 μ L in supernatant, after mixing, cultivates 2h in 37 DEG C; 100 DEG C are boiled deactivation 10min, cool 10min rapidly on ice; At 4 DEG C, under 14000rpm condition, centrifugal 15min; Draw supernatant and be sub-packed in direct mensuration or frozen in-80 DEG C in sterile tube.Sample extraction process all needs lucifuge to operate.
The microorganism detection method of embodiment 3 soybean folic acid
(1) preparation of reagents
Milk-acid bacteria liquid nutrient medium: 4.85g lactic acid bacteria culturing medium matter adds in 100mL water, 121 DEG C of sterilizing 15min.
Low folic acid substratum: take 4.7g folic acid culture medium powder, 0.03g ascorbic acid, 0.3mL folic acid standard storage dosing, be dissolved in water and be settled to 100mL, 0.22 μm of filter membrane sterilizing is for subsequent use excessively.
Analysis buffer: take Na
2hPO
4.2H
2o0.38g, NaH
2pO
40.93g, ascorbic acid 1g are dissolved in water and are settled to 100mL, and 0.22 μm of filter membrane sterilizing is for subsequent use excessively.
Folic acid substratum: take folic acid culture medium powder 9.4g, be dissolved in water and be settled to 100mL, boil 5min, 0.22 μm of filter membrane sterilizing is for subsequent use excessively.
(2) folic acid preparation of standard sample
Take the K that 28mg folic acid is dissolved in 4mL5%
2hPO
4in solution, be then settled to 250mL by analysis buffer, be settled to 100mL in absorption 0.5mL this solution analysis buffer, obtain the standard reserving solution FA that concentration is 0.5 μ g/mL, to be dispensed in 1.5mLEP pipe and to be stored in-20 DEG C.
(3) preparation of indicator
Drawing milk-acid bacteria preservation bacterium liquid 500 μ L joins in 10mL milk-acid bacteria liquid nutrient medium, and 24h cultivated by 37 DEG C of shaking tables, again draw cultured bacterium liquid 0.5mL and join in the low folic acid substratum of 100mL, and 18h cultivated by 37 DEG C of shaking tables; Triangular flask is placed in cooled on ice 20min.The glycerine of precooling is mixed with culture (volume ratio is 40:60), stir; Packing bacterium liquid in 1.5mLEP pipe, in-80 DEG C of preservations after liquid nitrogen flash freezer.
(4) folate content measures
Utilize analysis buffer FA solution dilution 250 times, obtain FA-1 solution, utilize analysis buffer to dilute according to 2:1 ratio, obtain FA-2 solution; FA-1 and FA-2 solution is all for production standard curve; By of short duration for folic acid sample centrifugal, with analysis buffer dilute sample 30 times; In 96 orifice plates, after 150 μ L analysis buffer are added each sample well, again 150 μ LFA-1, FA-2 and soybean folic acid liquid to be measured are added respectively the 1st row of 96 orifice plates, mixed solution 150 μ l is drawn to the 2nd row from the 1st row, mixed solution 150 μ l is being drawn to the 3rd row from the 2nd row, mixed solution 150 μ l is drawn to the 4th row again from the 3rd row, mixed solution 150 μ l is being drawn to the 5th row from the 4th row, draw mixed solution 150 μ l to the 6th row from the 5th row again, finally the sample mix liquid that the 6th arranges is discarded wherein 150 μ l; The FACM substratum 150 μ L adding indicator is added in 96 orifice plates; Culture plate is placed in 37 DEG C and cultivates 18-20h; Under wavelength is 580nm condition, the OD value of working sample; According to measuring numerical value Criterion curve and calculating the folic acid concentration comprised in sample.
Embodiment 4: the detection of invention effect
The usage quantity of α-amylase, proteolytic enzyme and rat blood serum three kinds of enzymes in the present invention is groped and optimized, the usage quantity of α-amylase is respectively 2,4,8,10 and 20 μ L, the usage quantity of proteolytic enzyme (working concentration is 10mg/mL) is respectively 40,80,100,150 and 200 μ L, and the usage quantity of rat blood serum is respectively 0,10,20,30,40 μ L.Different enzyme usage quantitys is combined according to Three factors five level, totally 125 groups.Folate content mensuration is carried out to the soybean varieties Williams82 of 125 combinations, repeats, the results are shown in Table 1 for 3 times.Folate content average as calculated under different concns condition finds, α-amylase, proteolytic enzyme and rat blood serum be combined in 8 μ L, farthest can extract the folic acid in soybean seeds under the condition of 100 μ L and 10 μ L.
The folate content (unit: μ g/100g) of table 1 soybean varieties Williams82 in different enzyme combination
Take different soybean varieties 0.2g respectively, be placed in 2mLEP pipe, repeat for three times.In every increment product, add 750 μ L folic acid Extraction buffers, hatch 15min at 95 DEG C and be placed in cooled on ice, add 5mm steel ball afterwards, ground sample (rotating speed is 1400rpm, and the time is 5min) in tissue grinder.
In sample homogenization liquid, add 8 μ L α-amylase and 750 μ L folic acid Extraction buffers to avoid thickness, room temperature places 10min, adds 100 μ L α-proteolytic enzyme, hatches 1hr for 37 DEG C; 100 DEG C are boiled said mixture 10min to stop enzyme effect, cool 10min rapidly on ice; At 4 DEG C, under 14000rpm condition, centrifugal 10min; In supernatant, add rat blood serum 10 μ L, after mixing, cultivate 2hr in 37 DEG C; 100 DEG C are boiled deactivation 10min, cool 10min rapidly on ice; At 4 DEG C, under 14000rpm condition, centrifugal 15min; Absorption supernatant is sub-packed in sterile tube and directly measures.
By analysis buffer by standard reserving solution FA solution dilution 250 times, obtain FA-1 solution, utilize analysis buffer to dilute according to 2:1 ratio, obtain FA-2 solution.FA-1 and FA-2 solution is all for production standard curve; By of short duration for folic acid sample centrifugal, with analysis buffer dilute sample 30 times.In 96 orifice plates, after 150 μ L analysis buffer are added each sample well, again 150 μ LFA-1, FA-2 and extraction paddy rice folic acid sample are added respectively the 1st row or the 7th row of 96 orifice plates, mixed solution 150 μ l is drawn to the 2nd hole or the 8th hole from the 1st row or the 7th row, the like, until the 6th row or the 12nd row sample mix liquid amass be 300 μ L after, discard wherein 150 μ l; The FACM substratum 150 μ L adding indicator is added (ratio of indicator/FACM substratum is 1:100) in 96 orifice plates; Culture plate is placed in 37 DEG C and cultivates 18-20hr; Under wavelength is 580nm condition, the OD value of working sample; According to measuring numerical value Criterion curve and calculating the folic acid concentration comprised in sample.The results are shown in Table 2.
The folate content (unit: μ g/100g) of table 2121 part northern area soybean seeds
Claims (3)
1. from soybean, extract the method for folic acid, comprise the following steps:
(1) soybean sample preparation: soybean seeds is pulverized and obtained bean powder, take 0.2g bean powder, be placed in AXYGENEP pipe, add 750 μ L folic acid Extraction buffers in the sample to which, hatch 15min for 95 DEG C and be placed in cooled on ice, add 5mm steel ball (2), ground sample in tissue grinder, obtain sample homogenization liquid;
(2) Soybean Leaves acid extraction: add 8 μ L α-amylase and 750 μ L folic acid Extraction buffers to avoid thickness in step (1) sample homogenization liquid, room temperature places 10min, adds 100 μ L proteolytic enzyme, hatches 1h for 37 DEG C; 100 DEG C are boiled 10min, cool 10min rapidly on ice; At 4 DEG C, under 14000rpm condition, centrifugal 10min; Draw supernatant, the volume ratio according to 2% adds rat blood serum 10 μ L in supernatant, after mixing, cultivates 2h in 37 DEG C; 100 DEG C are boiled deactivation 10min, cool 10min rapidly on ice; At 4 DEG C, under 14000rpm condition, centrifugal 15min; Draw supernatant and be sub-packed in direct mensuration or frozen in-80 DEG C in sterile tube, obtain Soybean Leaves acid extraction liquid.
2. the method extracting folic acid from soybean according to claim 1, is characterized in that, the preparation of described step (1) soybean sample and step (2) Soybean Leaves acid extraction, all carry out under lucifuge condition.
3. according to claim from the method extracting folic acid from soybean described in 1, it is characterized in that, the component of damping fluid described in step (2) is in the 50mM phosphoric acid buffer of pH value 6.5, adds the SODIUM ASCORBATE of 1%, and the 2 mercapto ethanol of 0.1%.
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| CN106083857A (en) * | 2016-06-06 | 2016-11-09 | 大连民族大学 | A kind of method extracting folic acid from Actinidia arguta Sieb.et Zucc |
| CN106226137A (en) * | 2016-08-10 | 2016-12-14 | 浙江大学 | A kind of method of quick detection Brassica genus hexaploid new germ plasm ploidy |
| CN106518878A (en) * | 2016-10-14 | 2017-03-22 | 天津理工大学 | Method for extracting folic acid from spinach leaf powder |
| CN109633029A (en) * | 2019-01-11 | 2019-04-16 | 山西农业大学 | A kind of measuring method of millet seed folic acid extracting method and folate content |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106083857A (en) * | 2016-06-06 | 2016-11-09 | 大连民族大学 | A kind of method extracting folic acid from Actinidia arguta Sieb.et Zucc |
| CN106226137A (en) * | 2016-08-10 | 2016-12-14 | 浙江大学 | A kind of method of quick detection Brassica genus hexaploid new germ plasm ploidy |
| CN106226137B (en) * | 2016-08-10 | 2019-04-23 | 浙江大学 | A kind of method of quick detection Brassica genus hexaploid new germ plasm ploidy |
| CN106518878A (en) * | 2016-10-14 | 2017-03-22 | 天津理工大学 | Method for extracting folic acid from spinach leaf powder |
| CN109633029A (en) * | 2019-01-11 | 2019-04-16 | 山西农业大学 | A kind of measuring method of millet seed folic acid extracting method and folate content |
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| CN105440036B (en) | 2017-03-08 |
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