CN104181243B - A kind of method measuring mannose content in mannatide - Google Patents
A kind of method measuring mannose content in mannatide Download PDFInfo
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Abstract
The invention discloses a kind of method measuring mannose content in mannatide, belong to detection technique field.Technical solution of the present invention comprises the thick extraction of fermentation liquor, the acidolysis of sample, derivatization, the content of mannose monomer that is hydrolyzed by gas chromatographic detection, and then mannan peptide content in sign fermentation liquor, get rid of the interference of other impurity, for the real-time monitoring of sweat provides reliable method.Method provided by the invention is reliable repeatable high, more simply, quick and highly sensitive, characterizes mannatide fermentation situation by measuring mannose content.
Description
Technical field
The present invention relates to a kind of method measuring mannose content in mannatide, especially a kind of method of the sign mannan peptide content based on gas chromatography, belongs to detection technique field.
Background technology
Mannatide (Mannatide) former name: polyactin (PolyactinA, PAA), be by alpha-Hemolytic streptococcus (alpha-Hemolyticstreptococci) 33# bacterial strain through fermented and cultured, extract the polysaccharose substance of preparation.It has inhibition tumor cell Growth and metabolism, promotes hematopoietic stem cell growth, strengthens activated immune cell propagation and specific antibody generation, improves peripheral white blood cell, improve the multiple physiologically active such as resisting stress capability and anti-inflammatory.This medicine, as the ancillary drug for the treatment of tumour, can alleviate the toxic and side effect of Radiotherapy chemotherapy, strengthens and immunity moderation function; Good result for the treatment of is had to non-tumor diseases such as anaemia, cytopenia, allergic arthritis, multiple diseases of oral mucosa.
Mannatide not only can treat the various diseases of human body, and in husbandry sector, this medicine of reasonable utilization, also can play good effect, has bright market outlook.But in the middle of fermenting and producing, rely on empirical values such as the morphological observation of thalline in fermentation liquor and fermentation times to the monitoring of fermentation situation more, lack the Efficient Evaluation quality standard to fermentation situation, and with tunning mannatide, content is a kind of feasible scheme as quality standard.Mannatide can be decomposed into basic polypeptide and mannan two parts, and it is 87.7-90.3% that mannatide records containing mannose content after hydrolysis, and the characteristic according to mannan can detect to tunning the content characterizing mannatide.Feng Suomin utilizes Molisch reaction that mannose and alpha-Naphthol are developed the color and forms orange red thing, detect mannose absorbance log at 490nm place and characterize product assay, but other reducing sugars added in nutrient culture media also can with alpha-Naphthol generation chromogenic reaction, thus interference measurement result.Zhang Yuntao establishes the method that ultraviolet spectrometry directly measures mannose content in brewer's yeast.Mannose is at 90%H
2sO
4in solution, dehydration generates 5-methylol-2-Furan Aldehydes, adds NaCl-H
3bO
3, make 5-methylol-2-Furan Aldehydes be converted into 5-chloromethyl-2-Furan Aldehydes, the amount of this inversion quantity and mannose is linear, measure before and after twice in the difference of the light absorption value at 280nm place, draw mannose content.
But fermentation broth contents is more complicated, the assay of the material of uv absorption to mannose may be had to have interference containing other, have very large limitation, can not the content of accurate response mannan.For this reason, the present invention is by first slightly purifying to fermentation liquor, the acidolysis of mannan, derivatization and gas chromatography (GC) separation-hydrogen flame ion assessor (FID) detect mannose monomer content in fermentation liquor, get rid of the interference of other monose factors, characterize the content of product mannatide, for the real-time monitoring of sweat provides evidence-based data.
Summary of the invention
The technical problem to be solved in the present invention be to provide a kind of accurately and reliably, easier and have the method for mannose content in the mensuration mannatide of using value, carry out acidolysis to mannatide to obtain mannose monomer, again mannose monomer is carried out derivatization, utilize gas chromatographic detection mannose content, and then characterize the content of mannatide.
Mannose content (%) in content (%)=mannose content (the %)/mannatide of described mannatide, described number percent is mass percent.
Described acidolysis is by mannatide 1-2MH
2sO
4acidolysis reaction is carried out, 1M and 2MH in 105 DEG C
2sO
4same hydrolysis effect can be reached, add mark in inositol, mixing after reaction terminates, use BaCO
3gradation adds neutralization, and vibration, centrifuging and taking supernatant, carry out freeze drying.Described mannatide, through thick purifying, is removed molecular weight and is less than 10KD and is greater than the impurity of 100KD.
The derivatization of described mannose monomer, is take 10mg oxammonium hydrochloride to be dissolved in 0.5mL pyridine, then joins in hydroxylamine hydrochloride solution by the lyophilized products of mannatide acid hydrolysate, abundant dissolving, 90 DEG C of water-bath 30min, are cooled to room temperature, add 0.5mL acetic anhydride, 90 DEG C of water-bath 30min.
The condition of described gas chromatographic detection mannose content is:
Chromatographic column: OV1701 quartz capillary column, 30m, I.D.0.53mm;
Detecting device: FID (hydrogen flame ion assessor);
Vaporizer temperature: 260 DEG C, detector temperature: 250 DEG C;
Column temperature: temperature programme: initial temperature 120 DEG C, keeps 3min, liter per minute 10 DEG C, to 195 DEG C, retains 0.1min, liter per minute 3 DEG C, to 240 DEG C, retains 10min;
Nebulizer gas pressure (N
2): 0.60kg/cm
2;
Gaseous-pressure (H
2): 0.65kg/cm
2;
Combustion-supporting atmospheric pressure (air): 0.50kg/cm
2;
Split ratio is 30: 1.
Described method is applied to the step measuring mannose content in mannatide in fermentation liquor:
(1) fermentation liquor is slightly purified, utilize the super filter tube of 10KD and 100KD to carry out molecule to fermentation liquor and retain, remove Small molecular and large molecular impurity, realize the thick purification to mannatide;
(2) acidolysis is carried out to mannatide, obtain mannose monomer;
(3) by mannose monomer derivatization, carry out gas chromatographic detection, draw mannose content, characterize the content of mannatide.
Described method is applied to the concrete steps measuring mannose content in mannatide in fermentation liquor:
(1) the thick purification of tunning:
Measure fermentation liquor 20-30mL in 50mL centrifuge tube, the centrifugal 20min of 10000rpm, goes precipitation to get supernatant, goes to 100KD super filter tube, the centrifugal 20min of 5000g, collects efflux, then adds 5mL deionized water to 100KD super filter tube, the centrifugal 20min of 5000g, repeats once, merges efflux.Be transferred to by efflux in 10KD super filter tube, the centrifugal 20min of 7500g, trapped fluid is transferred to new centrifuge tube, repeatedly rinses 10KD super filter tube with deionized water, merges trapped fluid, trapped fluid is carried out freeze drying.
(2) acidolysis of mannatide:
Take 20mg lyophilized products in 15mL centrifuge tube, add 2mL1-2MH
2sO
4in 105 DEG C of reaction 8h (with preservative film sealing, film stabbing a little aperture), after reaction, often pipe adds the interior mark of inositol of 1mL1mg/mL, and mixing, takes 2gBaCO
3add neutralization at twice, vibration, the centrifugal 20min of 12000rpm, gets supernatant in new 1.5mL centrifuge tube, carries out freeze drying.
(3) derivatization of acidolysis sample:
Take 10mg oxammonium hydrochloride to be dissolved in 0.5mL pyridine, then join in hydroxylamine hydrochloride solution by lyophilized products, fully dissolve, 90 DEG C of water-bath 30min, are cooled to room temperature, add 0.5mL acetic anhydride, and 90 DEG C of water-bath 30min cool laggard promoting the circulation of qi analysis of hplc.
(4) GC conditions:
Chromatographic column: OV1701 quartz capillary column, 30m, I.D.0.53mm;
Detecting device: FID (hydrogen flame ion assessor);
Vaporizer temperature: 260 DEG C, detector temperature: 250 DEG C;
Column temperature; Temperature programme: initial temperature 120 DEG C, keeps 3min, liter per minute 10 DEG C, to 195 DEG C, retains 0.1min.Liter per minute 3 DEG C, to 240 DEG C, retains 10min;
Nebulizer gas pressure (N
2): 0.60kg/cm
2;
Gaseous-pressure (H
2): 0.65kg/cm
2;
Combustion-supporting atmospheric pressure (air): 0.50kg/cm
2;
Split ratio: 30: 1.
(5) monose interpretation of result:
Monosaccharide component ratio is interior mark with inositol, determines mannose content with internal standard method, mannose content (%) in content (%)=mannose content (the %)/mannatide of mannatide.
Described method is applied to and measures in mannatide purified product in mannatide during mannose content, and do not need to carry out purifying, other steps are identical with the concrete steps of mannose content in mannatide in mensuration fermentation liquor.
Other measure the method for mannose content in mannatide at present, all can there is the disturbing factor of such as assorted monose etc., thus can not get testing result accurately.The present invention by first slightly purifying to fermentation liquor, the acidolysis of mannan, derivatization and gas chromatography (GC) separation-hydrogen flame ion assessor (FID) detects mannose monomer content in fermentation liquor, the content of product mannatide is characterized according to the content of mannose in mannatide, eliminate other impurity, especially the interference of other monose, for the real-time monitoring of sweat provides evidence-based data, and be applicable to the mensuration of monosaccharide component content in saccharide complex in other sweats, there is technical progress significantly.
Accompanying drawing explanation
Fig. 1: six kinds of monose standard specimen gas chromatography peaks
Fig. 2: mannatide is refined sample (140401) and surveyed mannose content by gas chromatography
Fig. 3: mannatide is refined sample (140501) and surveyed mannose content by gas chromatography
The fermentation crude extract of Fig. 4: 48h surveys mannose content by gas chromatography
The fermentation crude extract of Fig. 5: 60h surveys mannose content by gas chromatography
Embodiment
Below in conjunction with refining mannan peptide product, the present invention will be further described.
Embodiment 1 measures the content of mannose in refining mannatide sample
Sample: the refining mannatide sample of two batches provides by Lier Pharmaceutical Co., Ltd., Chengdu City, fermentation liquor is crossed mannatide sample that multistep separation and purification obtains as refining mannatide sample by Lier Pharmaceutical Co., Ltd., Chengdu City.
Reagent: the concentrated sulphuric acid, NaOH, ethanol, BaCO
3, inositol, oxammonium hydrochloride, pyridine, acetic acid is purchased from Chemical Reagent Co., Ltd., Sinopharm Group.
(1) acidolysis of refining mannatide sample
Take 20mg mannatide sample in 15mL centrifuge tube, add 2mL1-2MH
2sO
4in 105 DEG C of reaction 8hr (with preservative film sealing, film stabbing a little aperture), after reaction, often pipe adds the interior mark of inositol of 1mL1mg/mL, and mixing, takes 2gBaCO
3add neutralization at twice, vibration, the centrifugal 20min of 12000rpm, gets supernatant in new 1.5mL centrifuge tube, carries out freeze drying.
(2) derivatization of acidolysis sample
Take 10mg oxammonium hydrochloride to be dissolved in 0.5mL pyridine, then join in hydroxylamine hydrochloride solution by lyophilized products, fully dissolve, 90 DEG C of water-bath 30min, are cooled to room temperature, add 0.5mL acetic anhydride, and 90 DEG C of water-bath 30min cool laggard promoting the circulation of qi analysis of hplc.
(3) efficient liquid phase chromatographic analysis
Chromatographic column: OV1701 quartz capillary column, 30m, I.D.0.53mm; Detecting device: FID (hydrogen flame ion assessor); Vaporizer temperature: 260 DEG C, detector temperature: 250 DEG C; Column temperature, temperature programme: initial temperature 120 DEG C, keeps 3min, liter per minute 10 DEG C, to 195 DEG C, retains 0.1min.Liter per minute 3 DEG C, to 240 DEG C, retains 10min; Nebulizer gas pressure (N
2): 0.60kg/cm
2, gaseous-pressure (H
2): 0.65kg/cm
2, combustion-supporting atmospheric pressure (air): 0.50kg/cm
2, split ratio: 30: 1
(4) monose interpretation of result
Monosaccharide component ratio is interior mark with inositol, within mark method make each contents of monosaccharides.
Certain contents of monosaccharides (%)=(Ai/AIi) × (AIs/As) × (Ws/WIs) × (WIi/W) × 100
In formula: Ai represents certain monose sample peak area,
AIi represents the inositol interior mark peak area added in sample,
AIs represents the inositol interior mark peak area added in standard specimen,
As represents certain monose standard specimen peak area,
Ws represents the quality mg of certain monose standard specimen,
WIs represents target quality mg in the inositol that adds in standard specimen,
WIi represents target quality mg in the inositol that adds in sample,
W represents the quality mg taking sugared sample.
Wherein, six kinds of monose standard specimen gas chromatography information are as following table:
Table 1: the gas chromatography information of six kinds of hexose standard specimens
Sample (140401) carries out gas chromatographic analysis, obtains information as follows:
Table 2: mannatide refines sample (140401) gas chromatography information
| Peak | Retention time | Peak area | Peak height | Peak area ratio % |
| 1 | 22.222 | 6458514.8 | 658630.7 | 81.8039 |
| 2 | 23.070 | 745706.4 | 73315.8 | 9.4452 |
| 3 | 23.728 | 36150.7 | 3242.9 | 0.4579 |
| 4 | 25.695 | 654749.9 | 60903.9 | 8.2931 |
According to formula, calculating wherein mannose content is: 47.7%.
Sample (140501) carries out gas chromatographic analysis, obtains information as follows:
Table 3: mannatide refines sample (100603) gas chromatography information
| Peak | Retention time | Peak area | Peak height | Peak area ratio % |
| 1 | 22.229 | 7464023.4 | 732459.2 | 87.4106 |
| 2 | 23.058 | 392058.7 | 37477.7 | 4.5914 |
| 3 | 23.720 | 44850.3 | 4195.6 | 0.5252 |
| 4 | 25.688 | 638107.7 | 60522.2 | 7.4728 |
According to formula, calculating wherein mannose content is: 56.6%.
Can see in gas chromatogram, mannose chromatographic peak obviously separates with other monose chromatographic peaks, can get rid of the impact of other monose in mensuration process accordingly.Such as glucose, it is as the primary carbon source in nutrient culture media, in the additive method that mannose content measures in mannatide (such as Molisch reaction), very large interference can be caused to result, so this method can accurate and narrow spectrum analysis mannose content, characterize mannan peptide content, the real-time control for sweat provides the Data support of reliable digit.Application context of methods measures mannose respectively, and to refine in sample (140401) and (140501) mannose content in mannatide be 47.7% and 56.6%, and it is substantially identical often to criticize mannose content in refining sample, also illustrate that the method is comparatively reliable, repeatability is high.
Embodiment 2 measures the content of mannose in mannatide in fermentation liquor crude extract
Sample: be that strain fermentation produces mannatide with hemolytic streptococcus, by 0.5% glucose, 0.5%NaCl, 0.6% peptone, 0.2% tryptone, 0.5% beef extract and 0.3% yeast extract, pH is adjusted to be that 7.2-7.6 is as nutrient culture media, the fermentation liquor of different fermentations time point is slightly carried fermentation liquor through simple purge process, utilizes method of the present invention to analyze.
Reagent: the concentrated sulphuric acid, NaOH, ethanol, BaCO
3, inositol, oxammonium hydrochloride, pyridine, acetic acid is purchased from Chemical Reagent Co., Ltd., Sinopharm Group; 100KD, 10KD super filter tube, Millipore company.
(1) the thick purification of tunning:
Measure fermentation liquor 30mL in 50mL centrifuge tube, the centrifugal 20min of 10000rpm, goes precipitation to get supernatant, goes to 100KD super filter tube, the centrifugal 20min of 5000g, collects efflux, then adds 5mL deionized water to 100KD super filter tube, the centrifugal 20min of 5000g, repeats once, merges efflux.Efflux is transferred in 10KD super filter tube, the centrifugal 20min of 7500g, and trapped fluid is transferred to new centrifuge tube, repeatedly rinses 10KD super filter tube with deionized water, merges trapped fluid, trapped fluid is carried out freeze drying.
(2) slightly get sample the acidolysis of product
Take 20mg mannatide sample in 15mL centrifuge tube, add 2mL1-2MH
2sO
4in 105 DEG C of reaction 8hr (with preservative film sealing, film stabbing a little aperture), after reaction, often pipe adds the interior mark of inositol of 1mL1mg/mL, and mixing, takes 2gBaCO
3add neutralization at twice, vibration, the centrifugal 20min of 12000rpm, gets supernatant in new 1.5mL centrifuge tube, carries out freeze drying.
(3) derivatization of acidolysis sample
Take 10mg oxammonium hydrochloride to be dissolved in 0.5mL pyridine, then join in hydroxylamine hydrochloride solution by lyophilized products, fully dissolve, 90 DEG C of water-bath 30min, are cooled to room temperature, add 0.5mL acetic anhydride, and 90 DEG C of water-bath 30min cool laggard promoting the circulation of qi analysis of hplc.
(4) efficient liquid phase chromatographic analysis
Chromatographic column: OV1701 quartz capillary column, 30m, I.D.0.53mm; Detecting device: FID (hydrogen flame ion assessor); Vaporizer temperature: 260 DEG C, detector temperature: 250 DEG C; Column temperature, temperature programme: initial temperature 120 DEG C, keeps 3min, liter per minute 10 DEG C, to 195 DEG C, retains 0.1min.Liter per minute 3 DEG C, to 240 DEG C, retains 10min; Nebulizer gas pressure (N
2): 0.60kg/cm
2, gaseous-pressure (H
2): 0.65kg/cm
2, combustion-supporting atmospheric pressure (air): 0.50kg/cm
2, split ratio: 30: 1
(5) monose interpretation of result
Monosaccharide component ratio is interior mark with inositol, within mark method make each contents of monosaccharides.
Certain contents of monosaccharides (%)=(Ai/AIi) × (AIs/As) × (Ws/WIs) × (WIi/W) × 100
The crude extract of fermentation 48h carries out gas chromatographic analysis, obtains information as follows:
Table 4: the crude extract gas chromatography information of fermentation 48h
| Peak | Retention time | Peak area | Peak height | Peak area ratio % |
| 1 | 22.254 | 432444.8 | 43951.5 | 10.1684 |
| 2 | 23.152 | 653414.4 | 63212.6 | 15.3642 |
| 3 | 23.800 | 37517.2 | 3528.2 | 0.8822 |
| 4 | 25.818 | 3129450.6 | 293155.7 | 73.5852 |
According to formula, calculating wherein mannose content is: 0.71%.
The crude extract of fermentation 60h carries out gas chromatographic analysis, obtains information as follows:
Table 5: the crude extract gas chromatography information of fermentation 60h
| Peak | Retention time | Peak area | Peak height | Peak area ratio % |
| 1 | 22.255 | 616683.8 | 62928.6 | 21.4044 |
| 2 | 23.147 | 506335.2 | 49280.5 | 17.5743 |
| 3 | 23.797 | 49188.5 | 4769.7 | 1.7073 |
| 4 | 25.796 | 1708899.4 | 160371.9 | 59.3140 |
According to formula, calculating wherein mannose content is: 1.86%.
From the data that gas chromatography draws, known, in the fermentation crude extract of fermentation 60h, mannose content is than about many twices in fermentation 48h crude extract, and can find out the prolongation along with fermentation time, the mannatide in fermentation liquor accumulates to some extent.And slightly to carry in fermentation liquor mannose content than low in refining fermenting liquid, owing to eliminating most impurity in fermentation liquor in refining sample, and slightly carry fermentation liquor and just simply carried out molecule and retain, do not refine, object is the mannose content in more simple and quick acquisition fermentation liquor, for the real-time control of sweat provides reliable method.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (4)
1. one kind measures the method for mannose content in mannatide, be slightly purify to the mannatide in fermentation liquor, then acidolysis obtains mannose monomer, then mannose monomer is carried out derivatization, by gas chromatographic detection mannose content, and then characterize the content of mannatide; Described mannatide is purified through thick, removes molecular weight and is less than 10KD and is greater than the impurity of 100KD;
The GC conditions of described gas chromatographic detection mannose content is:
Chromatographic column: OV1701 quartz capillary column, 30m, I.D.0.53mm;
Detecting device: hydrogen flame ion assessor;
Vaporizer temperature: 260 DEG C, detector temperature: 250 DEG C;
Column temperature: temperature programme: initial temperature 120 DEG C, keeps 3min, liter per minute 10 DEG C, to 195 DEG C, retains 0.1min, liter per minute 3 DEG C, to 240 DEG C, retains 10min;
Nebulizer gas pressure: 0.60kg/cm
2, carrier gas is N
2;
Gaseous-pressure: 0.65kg/cm
2, combustion gas is H
2;
Combustion-supporting atmospheric pressure: 0.50kg/cm
2, combustion-supporting gas is air;
Split ratio is 30: 1.
2. method according to claim 1, is characterized in that, described acidolysis is by mannatide 1-2MH
2sO
4carry out acidolysis reaction in 105 DEG C, after reaction terminates, use BaCO
3gradation adds neutralization, vibration, centrifuging and taking supernatant, adds mark in inositol, mixing, carries out freeze drying.
3. method according to claim 1, it is characterized in that, the derivatization of described mannose monomer, be take 10mg oxammonium hydrochloride to be dissolved in 0.5mL pyridine, then the lyophilized products of mannatide acid hydrolysate is joined in hydroxylamine hydrochloride solution, fully dissolve, 90 DEG C of water-bath 30min, be cooled to room temperature, add 0.5mL acetic anhydride, 90 DEG C of water-bath 30min.
4. method according to claim 1, is characterized in that, in described fermentation liquor, in mannatide, the determination step of mannose content is:
(1) the thick purification of tunning: measure fermentation liquor 20-30mL in 50mL centrifuge tube, the centrifugal 20min of 10000rpm, precipitation is gone to get supernatant, go to 100KD super filter tube, the centrifugal 20min of 5000g, collect efflux, add 5mL deionized water again to 100KD super filter tube, the centrifugal 20min of 5000g, repeats once, merges efflux; Be transferred to by efflux in 10KD super filter tube, the centrifugal 20min of 7500g, trapped fluid is transferred to new centrifuge tube, repeatedly rinses 10KD super filter tube with deionized water, merges trapped fluid, trapped fluid is carried out freeze drying;
(2) acidolysis of mannatide: take 20mg lyophilized products in 15mL centrifuge tube, add 2mL1MH
2sO
4in 105 DEG C of reaction 8h, after reaction, often pipe adds the interior mark of inositol of 1mL1mg/mL, and mixing, takes 2gBaCO
3add neutralization at twice, vibration, the centrifugal 20min of 12000rpm, gets supernatant in new 1.5mL centrifuge tube, carries out freeze drying;
(3) derivatization of acidolysis sample: take 10mg oxammonium hydrochloride and be dissolved in 0.5mL pyridine, then lyophilized products is joined in hydroxylamine hydrochloride solution, fully dissolve, 90 DEG C of water-bath 30min, are cooled to room temperature, add 0.5mL acetic anhydride, 90 DEG C of water-bath 30min, cool laggard promoting the circulation of qi analysis of hplc;
(4) GC conditions:
Chromatographic column: OV1701 quartz capillary column, 30m, I.D.0.53mm;
Detecting device: hydrogen flame ion assessor;
Vaporizer temperature: 260 DEG C, detector temperature: 250 DEG C;
Column temperature: temperature programme: initial temperature 120 DEG C, keeps 3min, liter per minute 10 DEG C, to 195 DEG C, retains 0.1min, liter per minute 3 DEG C, to 240 DEG C, retains 10min;
Nebulizer gas pressure: 0.60kg/cm
2, carrier gas is N
2;
Gaseous-pressure: 0.65kg/cm
2, combustion gas is H
2;
Combustion-supporting atmospheric pressure: 0.50kg/cm
2, combustion-supporting gas is air;
Split ratio is 30: 1;
(5) monose interpretation of result:
Monosaccharide component ratio is interior mark with inositol, determines mannose content with internal standard method, mannose content (%) in content (%)=mannose content (the %)/mannatide of mannatide.
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| 气相色谱检测酵母多糖中的葡萄糖及甘露糖组分;朱海华等;《食品科技》;20111231;第36卷(第1期);275-276，281 * |
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