CN105153321B - A kind of fast separating process of the oligomeric sugar monomer of the lotus seeds with prebiotic effect - Google Patents
A kind of fast separating process of the oligomeric sugar monomer of the lotus seeds with prebiotic effect Download PDFInfo
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- CN105153321B CN105153321B CN201510572614.1A CN201510572614A CN105153321B CN 105153321 B CN105153321 B CN 105153321B CN 201510572614 A CN201510572614 A CN 201510572614A CN 105153321 B CN105153321 B CN 105153321B
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Abstract
The invention discloses a kind of fast separating process of the oligomeric sugar monomer of the lotus seeds with prebiotic effect, belong to the extraction and separation technology field of active components of plants, it is with fresh lotus seed as raw material, dried through sorting, pulverized and sieved, the compounding that adds water, washing starch, constant temperature refluxing extraction, suction filtration, alcohol analysis, centrifugal concentrating, freeze-drying, MPLC is separated, MPLC secondary separations obtain the identical or different lotus seeds oligosaccharide monomer powders of the degree of polymerization, through the purity of isolated each monomer of lotus seeds oligosaccharide of the invention up to more than 95%.Compared to high performance liquid chromatography and high-speed countercurrent chromatography, the present invention is separated using MPLC, and its preparation amount is big, and sample applied sample amount can be from milligram level to hectogram, and disengaging time is shorter, while separating column packing can be filled independently, increased separation selectivity；Its stable technical process, easy to operate, separative efficiency are greatly improved, and can be used for the separation preparation of high-purity, the oligomeric sugar monomer of a large amount of lotus seeds.
Description
Technical field
The invention belongs to active components of plants extraction and separation technology field, and in particular to a kind of lotus with prebiotic effect
The fast separating process of the oligomeric sugar monomer of son.
Background technology
With the growth and the continuous improvement of human life quality of countries in the world economic strength, people want to food function
Ask also constantly changing, and the demand of health food is consequently increased.In numerous health foods, oligosaccharide due to
With clear and definite chemical constitution and significant physiological function, the extensive concern of domestic and international researcher is caused.Functional oligose
The low polymerization sugar being formed by connecting through specific glycosidic bond by 2 ~ 10 monose, have been found to for effective Bifidobacterium propagation because
One of son, and with excellent physiologically actives such as proliferation of probiotics, anti-caries tooth and reducing blood lipid in promotion human body intestinal canal, thus extremely
Catch people's attention, volume of production and marketing comes out at the top in functional food.And the degree of polymerization of oligosaccharide also has to the utilization rate of probiotics
Material impact, its absorbability typically increases with the reduction of molecular weight, while resist pathogenic bacteria ability also have a certain impact,
The lower oligosaccharide antibacterial activity of the degree of polymerization is stronger.
The feature of oligosaccharide is closely related with the chemical constitution and configuration of sugar, therefore, qualitative and quantitative point of oligosaccharide
Analyse and important influence is presented with terms of biomedical and functional food to it.Nowadays, silica gel, activated carbon, anion
The a series of separation method such as exchange and gel chromatography technology is used for by independent or united application in field of functional food
Prepare prebiotics.The separation of current different polymerization degree oligosaccharide is done step-by-step in above-mentioned separation method, but due to adjacent
The oligosaccharide of the degree of polymerization is sufficiently close to due to physicochemical property, does not have UV absorption, and heat endurance is low, does not contain charged groups,
This makes while the oligosaccharide of quick separating same polymeric degree is still a challenging job.Therefore, setting up has height
Resolution ratio, automation and attainable preparation method just seem particularly necessary.And there is complex operation with traditional column chromatography, fill out
The problems such as material is expensive is compared, liquid chromatography has that resolution is high, speed is fast, repeatability is high, chromatographic column can Reusability, can be from
The features such as dynamicization is operated, analytical precision is high, can relatively accurately identify product and separation situation.Flash preparative chromatography technology,
Flash chromatography can be divided into according to pressure applied（0.1-5/10 bars）Or middle pressure preparative chromatography（MPLC, 5/10-50
bars）.Flash chromatography system is used primarily for the application that fast purifying synthesizes compound, and this system is gradually applied in recent years
In the separation of complicated natural product extraction mixture.Compared to high performance liquid chromatography and high-speed countercurrent chromatography, its preparation amount
Greatly, sample applied sample amount can be from milligram level to hectogram, and disengaging time is shorter, while separating column packing can be filled independently, increases
Added separation selectivity, can effectively save production cost, natural products isolate and purify research work in play important work
With.Meanwhile, general preparative chromatography is only furnished with ultraviolet detector, and this is not particularly suited for detecting the chemical combination without UV absorption
Thing, such as carbohydrate；And the offline inspection methods such as phend-sulphuric acid colorimetric are used, often using harmful chemical reagent, and specifically
Property poor, analysis time is more long.
Lotus seeds are one of important foreign exchange earning characteristic agricultural byproducts of China, in addition to Fujian, Jiangxi provinces are for major production areas,
Being saved in Zhejiang, Hunan, Jiangsu, Hubei, Hebei, Taiwan etc. also has business to cultivate.Lotus seeds are medicine-food two-purpose food, except containing rich
Outside rich starch, protein, alkaloid and flavonoids, also containing superoxide dismutase, oligosaccharide, polysaccharide the effects such as composition.
And oligosaccharide is used as one of the active component of lotus seeds, research report is less at present, has and largely prepares further to further investigate structure
The demand of effect relation.Therefore, the present invention joins flash preparative chromatography combination evaporative light-scattering and hydrophilic chromatographic with lotus seeds as material
Use technology（MPLC-ELSD-HILIC）Apply to monitor on-line the separation process of the oligomeric sugar monomer of lotus seeds, set up in a kind of lotus seeds
The rapid separation and purification method of oligomeric sugar monomer, it has the advantages that automation high, high-resolution, high yield and security.
The content of the invention
It is an object of the invention to provide a kind of fast separating process of the oligomeric sugar monomer of the lotus seeds with prebiotic effect,
The method is simple, it is easy to operate, and can realize the separation of the oligosaccharide of same polymeric degree, and the oligomeric sugar monomer of gained has prebiotics
Effect.The present invention can fill up the blank of the efficient preparative separation technology of domestic lotus seeds oligosaccharide, be the exploitation of lotus seeds oligosaccharide
Basis is provided, helps to lift lotus seeds industrial value, expand the effective way of lotus seeds deep processing by-product utilized, while or
The efficient quick preparation of other natural oligosaccharide provides new direction.
To achieve the above object, the present invention is adopted the following technical scheme that：
A kind of fast separating process of the oligomeric sugar monomer of lotus seeds with prebiotic effect, it is with fresh lotus seed as former
Material, dry through sorting, pulverized and sieved, the compounding that adds water, wash starch, constant temperature refluxing extraction, suction filtration, alcohol analysis, centrifugal concentrating, freezing
Dry, MPLC is separated, MPLC secondary separations obtain lotus seeds oligosaccharide monomer powders.
The method specifically includes following steps：
1）Sorting is dried：Full grains, undamaged fresh lotus seed are chosen, are put into air dry oven after screening coring,
It is 4 ~ 7% in moisture is dried at 40 ~ 50 DEG C；
2）Pulverize and sieve：By step 1）Dried lotus seeds are crushed, and cross 40 mesh sieves；
3）Add water compounding：Take step 2）Gained lotus seeds powder, by solid-liquid ratio 1:5 addition deionized waters are mixed and compounded；
4）Washing starch：By step 3）Gained compounding feed liquid crosses 100 mesh sieves, and with the deionized water of 4 ~ 6 times of lotus seeds weight
Filter residue is rinsed, 8 h, centrifugation removal precipitation is staticly settled under the conditions of 4 DEG C are placed in after merging filtrate, then by gained supernatant and rinse
Lotus seeds filter residue afterwards merges：
5）Constant temperature refluxing extraction：By step 4）Gained supernatant and lotus seeds filter residue are in constant temperature refluxing extraction at 75 ~ 85 DEG C
1.5~2 h；
6）Suction filtration：Extraction terminate after by extract solution vacuum filtration, discard filter residue；
7）Alcohol is analysed：By step 6）Gained filtrate added after the 1/5 ~ 1/4 of original volume is concentrated at 60 ~ 65 DEG C concentrate 3 ~
5 times of volumes, ethanol that mass concentration is 95%, mix after 10 ~ 12 h of standing at 4 DEG C；
8）Centrifugal concentrating：By step 7）Precipitation is removed after resulting solution centrifugation, supernatant steams in vacuum rotating at 55 ~ 65 DEG C
Hair is concentrated into the 1/3 ~ 2/5 of original volume；
9）Freeze-drying：By step 8）Gained concentrate is freeze-dried, obtains lotus seeds oligosaccharide raw sugar powder；
10）MPLC is separated：By step 9）Gained lotus seeds oligosaccharide raw sugar powder is dissolved in deionized water, and being configured to concentration is
The solution of 0.1 ~ 0.2 g/mL, then injects solution MPLC and is separated, according to reservation after 0.22 μm of filtering with microporous membrane
Time pointedly collects each target compound, collection liquid respectively at after rotary evaporation in vacuo recycling design at 55 ~ 65 DEG C, through cold
The lyophilized dry freeze-dried powder that must contain specific compound；
11）MPLC secondary separations：Take step 10）Freeze-dried powder of the gained containing specific compound, respectively by step 10）Behaviour
Make and chromatographic condition carries out sub-sampling, collect each target peak of identical retention time, lotus is obtained final product through solvent recovery, freeze-drying
Sub- oligosaccharide monomer powders.
The piece-rate system of MPLC includes middle pressure special glass post（36×460 mm）, the ultraviolet full wavelength scanner of chromatogram pump, DAD
Device（Detection wavelength scope is 190-840 nm）With FLASH ELSD EISDs, automatic fraction collector and manually
Sample load-on module；
Medium pressure special glass post is the Claricep Flash hydrophilic posts of HILIC, and its packing material size is 20-35 μm, hole
Footpath 100A, the m of specific surface 3202/ g, carbon content 5.0%；
The testing conditions of the FLASH ELSD EISDs are：Purging nitrogen pressure is 2.5 ~ 3.5 bar,
85 ~ 95 DEG C of drift tube temperature.
Loading volume during MPLC is separated is 1 mL, and mobile phase is the binary mixture of acetonitrile and water, its flow velocity is 30 ~
45mL/min, column temperature is 25 DEG C；
Mobile phase ratio change specific procedure be：Acetonitrile ratio is 85 ~ 90% 15 ~ 20min of holding in mobile phase, then
Acetonitrile ratio is down to 65 ~ 60% in 30 ~ 35min, 50 ~ 60min is kept, then acetonitrile ratio is risen to 85 ~ 90%, keep 15
~20min。
Gained lotus seeds oligosaccharide monomer powders can be used to prepare the health food with regulation gastrointestinal bacterial flora effect.
The fast separating process of the oligomeric sugar monomer of the lotus seeds with prebiotic effect of the present invention has advantages below：
（1）The present invention is isolated and purified using preparative medium pressure liquid chromatography system to the oligomeric sugar monomer of lotus seeds, and is led to
Crossing DAD and FLASH ELSD carries out real time on-line monitoring, can make impurity component and object definite ingredients in extract solution, reaches
Good separating effect；And preparative chromatograph pointedly can automatically collect lotus seeds oligosaccharide objective monomer peak, from without making
It is collected with Traditional Man is manually operated, is also not required to use chemical method（Such as Phenol sulfuric acid procedure）Detection carbohydrate, can
Environmental pollution is prevented effectively from, and reduces human cost, while being also convenient for controlling product quality, collected each lotus seeds oligosaccharide list
Body purity is up to more than 95%.Additionally, the separating column packing for using can be filled independently, separation selectivity is increased.
（2）The present invention is separated using preparative medium pressure liquid chromatography technology, can make lotus seeds oligosaccharide from extract solution
It is transferred in the mixed solution of acetonitrile and water, the concentration of object not only can be in a short time improved, simultaneously because acetonitrile solution
It is easy to evaporation to reclaim, reduces the energy ezpenditure in traditional water evaporation operation.
（3）The method that conventional sugar is separate is needed by the pre-treatment work such as degreasing, de- albumen, macroporous absorbent resin adsorpting pigment
Skill, and need coupled ion displacement chromatography, gel filtration or the exclusion chromatography to carry out separating for several times or pure to the sample of each collection step
Change, its complex operation step, and finished product preparation amount is very low.Loading is at most only needed 5 times using the inventive method, separated preparation
The demand of structural analysis is substantially met by the oligomeric sugar monomer of lotus seeds for going out, meanwhile, it can be to same point of small degree of polymerization oligosaccharide
Isomers is effectively separated, and this is opened for further studying lotus seeds oligosaccharide structure-activity relationship, making full use of China's lotus seeds resource
The hair oligomeric sugar product of lotus seeds is significant.
（4）Amino bonded is generally mutually used for the analysis of liquid chromatogram small molecular carbohydrate in the prior art.But
Amino bonded silica gel needs equilibration time more long, while silica gel solid phase property is more active in nh 2 column, is easily made when using
Into Irreversible Adsorption phenomenon, spectrogram peak type is caused to deteriorate.The present invention selects the hydrophilic chromatographic that amide group is main group, with amino
Relatively low compared to its activity, alkalescence is weaker, and reservation of the material on amide groups Bonded Phase is influenceed smaller by pH, is not likely to produce and is matched somebody with somebody
Body is lost in, with higher recovery, while using the bonded silica gel of propyl amides as filler, positive silicon is used when this filler is manufactured
Acid esters processes Silica Surface, reduces the quantity of the isolated silicone hydroxyl in surface, reduces the activity of Silica Surface silicone hydroxyl, can be effective
Polar compound is avoided to adsorb too strong problem.This column material and C in anti-phase system18Material has opposite reservation special
Property, and it is weaker compared with the retention of amino in positive phase system, be difficult to elute under the conditions of positive such that it is able to eluting or being retained in
Or retain very weak material under antiphasic condition.And it has good stability in aqueous mobile phase, compensate for tradition
Amino sugar post shortcoming in this regard.The chromatographic column filler cost for being used is relatively low, and operating pressure is smaller.
（5）The country has used silicagel column that the research for preparing is separated for larger dose oligosaccharide and has reported, its separating technology
The problems such as remaining not high in product separating degree, detection means is cumbersome, while can only be separated to the oligosaccharide of different polymerization degree.
The present invention can effectively make up above-mentioned deficiency, the isomer for separating lotus seeds oligosaccharide, and its separating effect has compared with the former
Significantly lifted.
（6）Due to other highly sophisticated devices such as high performance liquid chromatography color and luster, the purity etc. of analyzing sample are required it is higher,
The extract solution obtained by simple process is unable to direct injection analysis, and if using this hair before high performance liquid chromatography separation is carried out
Bright preprocess method carries out pre-treatment to sample introduction solution（Can be applied to other small molecule carbohydrate）, can avoid to high-accuracy
Instrument is polluted（Such as shorten device longevity）, and then reduce the production cost of product.
（7）For the physicochemical property of various composition present in lotus seeds, the present invention is analysed by washing starch, alcohol, MPLC divides
From the order collocation of, MPLC secondary separations, and use suitable technological parameter, can effectively extract the oligomeric sugar monomer of lotus seeds into
Point, and the impurity such as lotus seed starch, polyphenol, protein, pigment are removed, so as to reach the effect for separating and purifying simultaneously, shorten
Production cycle, save production cost.
（8）Chromatographic condition mainly includes chromatographic column（Including filler, column length, column temperature etc.）, mobile phase（Including composition, flow velocity
Deng）, detector and Detection wavelength etc..In MPLC separation processes, selection of chromatographic condition and combinations thereof is particularly significant, and it can be straight
Connect appearance time, peak shape of influence material etc..The present invention is by substantial amounts of experimental study and comparative analysis, it is determined that suitable color
Spectral condition, so that the appearance time of material, peak shape, separating effect etc. reach optimization, realizes the oligomeric sugar monomer of lotus seeds
Efficiently separate.
Brief description of the drawings
Fig. 1 is the chromatogram of first time MPLC separation in the embodiment of the present invention 1.
Fig. 2 is the HPLC collection of illustrative plates of the present invention gained oligomeric sugar monomer of lotus seeds, wherein 1 is LOS4,2 is LOS3-2, and 3 are
LOS3-1,4 is LOS2, and 5 is LOS1.
Fig. 3 is the monosaccharide composition analysis chromatogram of the present invention gained oligomeric sugar monomer of lotus seeds.
Fig. 4 is influence of the different carbon source to lactobacillus acidophilus growth course.
Fig. 5 is influence of the different carbon source to bifidobacterium adolescentis growth course.
Fig. 6 is influence of the different carbon source to bifidobacterium longum growth course.
Fig. 7 adheres to the influence of Caco-2 cells for the carbon source of various concentrations to enteropathogenic E. Coli, wherein different capitalization
To thalline adhesion inhibition rate significant difference between the various dose of letter expression same carbon source（p<0.05）；Different lowercase letters
To thalline adhesion inhibition rate significant difference between different carbon source same dose（p<0.05）.
Specific embodiment
In order that content of the present invention easily facilitates understanding, with reference to specific embodiment to of the present invention
Technical scheme is described further, but the present invention is not limited only to this.
The piece-rate system of MPLC used includes middle pressure special glass post in embodiment（36×460 mm）, chromatogram pump, DAD are purple
Outer full wavelength scanner device（Detection wavelength scope is 190-840 nm）With FLASH ELSD EISDs, automatic cut
Collector and manual sample load-on module；Middle pressure special glass post used is the Claricep Flash hydrophilic posts of HILIC, and it is filled out
Material particle diameter is 20-35 μm, aperture 100A, the m of specific surface 3202/ g, carbon content 5.0%.
Embodiment 1
A kind of fast separating process of the oligomeric sugar monomer of lotus seeds with prebiotic effect, it specifically includes following steps：
1）Sorting is dried：Full grains, undamaged fresh lotus seed are chosen, are put into air dry oven after screening coring,
It is 4% in moisture is dried at 40 DEG C；
2）Pulverize and sieve：By step 1）Dried lotus seeds are crushed, and cross 40 mesh sieves；
3）Add water compounding：Take step 2）Gained lotus seeds powder, by solid-liquid ratio 1:5 addition deionized waters are mixed and compounded；
4）Washing starch：By step 3）Gained compounding feed liquid crosses 100 mesh sieves, and with the deionized water punching of 4 times of lotus seeds weight
Filter wash slag, staticly settles 8 h under the conditions of 4 DEG C are placed in after merging filtrate, centrifugation removal is precipitated, then by after gained supernatant and flushing
Lotus seeds filter residue merge：
5）Constant temperature refluxing extraction：By step 4）Gained supernatant and lotus seeds filter residue are in the h of constant temperature refluxing extraction 1.5 at 75 DEG C；
6）Suction filtration：Extraction terminate after by extract solution vacuum filtration, discard filter residue；
7）Alcohol is analysed：By step 6）Gained filtrate added after the 1/5 of original volume is concentrated at 60 DEG C 3 times of volumes of concentrate,
Mass concentration is 95% ethanol, is mixed after standing 10 h at 4 DEG C；
8）Centrifugal concentrating：By step 7）Precipitation is removed after resulting solution centrifugation, supernatant is in rotary evaporation in vacuo at 55 DEG C
It is concentrated into the 1/3 of original volume；
9）Freeze-drying：By step 8）Gained concentrate is freeze-dried, obtains lotus seeds oligosaccharide raw sugar powder；
10）MPLC is separated：By step 9）Gained lotus seeds oligosaccharide raw sugar powder is dissolved in deionized water, and being configured to concentration is
The solution of 0.1 g/mL, then injects MPLC, using Claricep Flash by solution after 0.22 μm of filtering with microporous membrane
The hydrophilic posts of HILIC are isolated and purified, and ultraviolet and evaporative light-scattering is carried out online（Testing conditions are：Purging nitrogen pressure is 2.5
Bar, 85 DEG C of drift tube temperature）Detection, each target compound is pointedly collected according to retention time, and collection liquid is respectively at 55 DEG C
After lower rotary evaporation in vacuo recycling design, the freeze-dried freeze-dried powder that must contain specific compound；
11）MPLC secondary separations：Take step 10）Freeze-dried powder of the gained containing specific compound, respectively by step 10）Behaviour
Make and chromatographic condition carries out sub-sampling, collect each target peak of identical retention time, lotus is obtained final product through solvent recovery, freeze-drying
Sub- oligosaccharide monomer powders.
Loading volume during MPLC is separated is 1 mL, and mobile phase is the binary mixture of acetonitrile and water, and its flow velocity is
30mL/min, column temperature is 25 DEG C；Mobile phase ratio change specific procedure be：Acetonitrile ratio is 85% holding in mobile phase
15min, is then down to 65% by acetonitrile ratio in 30 min, keeps 50 min, then acetonitrile ratio is risen into 85%, keeps
15min。
Embodiment 2
A kind of fast separating process of the oligomeric sugar monomer of lotus seeds with prebiotic effect, it specifically includes following steps：
1）Sorting is dried：Full grains, undamaged fresh lotus seed are chosen, are put into air dry oven after screening coring,
It is 5.5% in moisture is dried at 45 DEG C；
2）Pulverize and sieve：By step 1）Dried lotus seeds are crushed, and cross 40 mesh sieves；
3）Add water compounding：Take step 2）Gained lotus seeds powder, by solid-liquid ratio 1:5 addition deionized waters are mixed and compounded；
4）Washing starch：By step 3）Gained compounding feed liquid crosses 100 mesh sieves, and with the deionized water punching of 5 times of lotus seeds weight
Filter wash slag, staticly settles 8 h under the conditions of 4 DEG C are placed in after merging filtrate, centrifugation removal is precipitated, then by after gained supernatant and flushing
Lotus seeds filter residue merge：
5）Constant temperature refluxing extraction：By step 4）Gained supernatant and lotus seeds filter residue are in constant temperature refluxing extraction 1.75 at 80 DEG C
h；
6）Suction filtration：Extraction terminate after by extract solution vacuum filtration, discard filter residue；
7）Alcohol is analysed：By step 6）Gained filtrate added after the 2/9 of original volume is concentrated at 63 DEG C 4 times of volumes of concentrate,
Mass concentration is 95% ethanol, is mixed after standing 11 h at 4 DEG C；
8）Centrifugal concentrating：By step 7）Precipitation is removed after resulting solution centrifugation, supernatant is in rotary evaporation in vacuo at 60 DEG C
It is concentrated into the 3/8 of original volume；
9）Freeze-drying：By step 8）Gained concentrate is freeze-dried, obtains lotus seeds oligosaccharide raw sugar powder；
10）MPLC is separated：By step 9）Gained lotus seeds oligosaccharide raw sugar powder is dissolved in deionized water, and being configured to concentration is
The solution of 0.15 g/mL, then injects MPLC, using Claricep Flash by solution after 0.22 μm of filtering with microporous membrane
The hydrophilic posts of HILIC are isolated and purified, and ultraviolet and evaporative light-scattering is carried out online（Testing conditions are：Purging nitrogen pressure is 3.0
Bar, 90 DEG C of drift tube temperature）Detection, each target compound is pointedly collected according to retention time, and collection liquid is respectively at 60 DEG C
After lower rotary evaporation in vacuo recycling design, the freeze-dried freeze-dried powder that must contain specific compound；
11）MPLC secondary separations：Take step 10）Freeze-dried powder of the gained containing specific compound, respectively by step 10）Behaviour
Make and chromatographic condition carries out sub-sampling, collect each target peak of identical retention time, lotus is obtained final product through solvent recovery, freeze-drying
Sub- oligosaccharide monomer powders.
Loading volume during MPLC is separated is 1 mL, and mobile phase is the binary mixture of acetonitrile and water, and its flow velocity is
40mL/min, column temperature is 25 DEG C；Mobile phase ratio change specific procedure be：Acetonitrile ratio is 87% holding in mobile phase
18min, is then down to 63% by acetonitrile ratio in 32 min, keeps 55 min, then acetonitrile ratio is risen into 87%, keeps
18min。
Embodiment 3
A kind of fast separating process of the oligomeric sugar monomer of lotus seeds with prebiotic effect, it specifically includes following steps：
1）Sorting is dried：Full grains, undamaged fresh lotus seed are chosen, are put into air dry oven after screening coring,
It is 7% in moisture is dried at 50 DEG C；
2）Pulverize and sieve：By step 1）Dried lotus seeds are crushed, and cross 40 mesh sieves；
3）Add water compounding：Take step 2）Gained lotus seeds powder, by solid-liquid ratio 1:5 addition deionized waters are mixed and compounded；
4）Washing starch：By step 3）Gained compounding feed liquid crosses 100 mesh sieves, and with the deionized water punching of 6 times of lotus seeds weight
Filter wash slag, staticly settles 8 h under the conditions of 4 DEG C are placed in after merging filtrate, centrifugation removal is precipitated, then by after gained supernatant and flushing
Lotus seeds filter residue merge：
5）Constant temperature refluxing extraction：By step 4）Gained supernatant and lotus seeds filter residue are in the h of constant temperature refluxing extraction 2 at 85 DEG C；
6）Suction filtration：Extraction terminate after by extract solution vacuum filtration, discard filter residue；
7）Alcohol is analysed：By step 6）Gained filtrate added after the 1/4 of original volume is concentrated at 65 DEG C 5 times of volumes of concentrate,
Mass concentration is 95% ethanol, is mixed after standing 12 h at 4 DEG C；
8）Centrifugal concentrating：By step 7）Precipitation is removed after resulting solution centrifugation, supernatant is in rotary evaporation in vacuo at 65 DEG C
It is concentrated into the 2/5 of original volume；
9）Freeze-drying：By step 8）Gained concentrate is freeze-dried, obtains lotus seeds oligosaccharide raw sugar powder；
10）MPLC is separated：By step 9）Gained lotus seeds oligosaccharide raw sugar powder is dissolved in deionized water, and being configured to concentration is
The solution of 0.2 g/mL, then injects MPLC, using Claricep Flash by solution after 0.22 μm of filtering with microporous membrane
The hydrophilic posts of HILIC are isolated and purified, and ultraviolet and evaporative light-scattering is carried out online（Testing conditions are：Purging nitrogen pressure is 3.5
Bar, 95 DEG C of drift tube temperature）Detection, each target compound is pointedly collected according to retention time, and collection liquid is respectively at 65 DEG C
After lower rotary evaporation in vacuo recycling design, the freeze-dried freeze-dried powder that must contain specific compound；
11）MPLC secondary separations：Take step 10）Freeze-dried powder of the gained containing specific compound, respectively by step 10）Behaviour
Make and chromatographic condition carries out sub-sampling, collect each target peak of identical retention time, lotus is obtained final product through solvent recovery, freeze-drying
Sub- oligosaccharide monomer powders.
Loading volume during MPLC is separated is 1 mL, and mobile phase is the binary mixture of acetonitrile and water, and its flow velocity is
45mL/min, column temperature is 25 DEG C；Mobile phase ratio change specific procedure be：Acetonitrile ratio is 90% holding in mobile phase
20min, is then down to 60% by acetonitrile ratio in 35 min, keeps 60 min, then acetonitrile ratio is risen into 90%, keeps
20min。
Embodiment 4
Fig. 1 is the chromatogram that first time MPLC is separate in embodiment 1, by it is separated go out peak 1, peak 2, peak 3 and peak 4 make
Secondary MPLC separation is carried out after being collected for target product, then by the peak 1 of identical retention time, peak 2, peak 3 and peak 4 difference again
Recycling design, freeze-drying are carried out after collection and obtain five kinds of oligosaccharide monomer powders, be denoted as LOS1, LOS2, LOS3-1,
LOS3-2、LOS4。
It is mixed due to existing during the ratio of mobile phase reclaimed water increases 0 → 100% simultaneously for HILIC chromatograms
Conjunction mechanism, the reservation of solute will be presented " U " type curve.Organic solvent mixes during as mobile phase with water, exists between solvent molecule
Interior complexing action power is acted on including hydrogen bond action, dipole.When the ratio of mobile phase reclaimed water is when 0 increases to 50% (v/v),
Chromatographic column embodies the characteristic feature of HILIC reservations, and water is strong eluant, eluent；After the ratio of water continues to increase to 50%, solute is protected
The trend that increase is presented on the contrary is stayed, water is changed into weak eluant, eluent, and solute reservation is changed into reverse-phase chromatography retention mechanism, is unfavorable for saccharification
The reservation of compound.Accordingly, it is determined that the change ratio of mobile phase reclaimed water is 15-35% changing as the solvent ratios in separation process
Scope.
Embodiment 5
Separated LOS1, LOS2, LOS3-1, LOS3-2, LOS4 oligosaccharide monomer powders are dissolved in deionized water, are entered
Row efficient liquid phase chromatographic analysis, and the lotus seeds oligosaccharide each component collected by tracking and measuring.Its chromatographic condition is as follows：Loading is dense
Degree：2 mg/mL；Chromatogram column type number：Hi-plex Na(Octo)；Detector：Differential refraction detector；Mobile phase：Distilled water；Stream
Speed：0.6 mL/min.Gained HPLC liquid chromatograms are shown in Fig. 2.
As seen from Figure 2, it is separated go out five compound separating degrees it is good, chromatographic peak is more single, sharp smooth
And it is symmetrical, its retention time is respectively 7.032,7.6213,7.689,8.799 and 12.149 min, purity is respectively 96.76%,
98.07%th, 98.18%, 99.74% and 92.67%.
Embodiment 6
Weigh separated each 20 mg of LOS2, LOS3-1, LOS3-2, LOS4 oligosaccharide monomer powders, add 2 mol/
Tube sealing after L trifluoroacetic acids, 100 DEG C of 4 h of hydrolysis take out, BaCO3Supernatant is neutralized to neutrality, after 0.22 μm of filtering with microporous membrane
Monosaccharide composition analysis are carried out using high performance liquid chromatography, quantified by external standard method is carried out using monose hybrid standard product.Its chromatostrip
Part is as follows：Chromatographic column：Agilent Zorbax Carbohydrate （250×4.6 mm）；Mobile phase（Acetonitrile-water）80:20；
Flow velocity：1 mL/min；Temperature：25 ℃；Sample size：25 μL；Analysis time：30 min.Its monosaccharide composition analysis chromatogram is shown in
Fig. 3.
Compareed with monose hybrid standard product from each oligosaccharide hydrolysis monosaccharide products in Fig. 3, LOS2 mainly contains grape
Sugar and 2 kinds of saccharide residues of galactolipin, both are about 1 at mol ratio:1.LOS3-1 mainly contains fructofuranose, mannose and glucose 3
Saccharide residue is planted, three's mol ratio is about 1:1:1.LOS3-2 mainly contains 2 kinds of saccharide residues of mannose and glucose, both mol ratios
About 2:1.LOS4 mainly contains fructofuranose, 3 kinds of saccharide residues of mannose and glucose, and three's mol ratio is about 1:2:1.Wherein
LOS3-1 and LOS3-2 is isomer.
Embodiment 7
The carbohydrate of some particular sources, such as arabinooligosaccharides（AOS）, galactooligosaccharide（GOS）, FOS
（FOS）, xylo-oligosaccharide（XOS）All it is proved with inulin with good prebiotic effect, to being colonized the bifid in human body intestinal canal
Bacillus and lactobacillus have different fermentabilities, and this mechanism for consuming carbohydrate with them is closely related.
Strain mother liquor preparation method：Under aseptic technique, the lyophilized bacterium powder in ampoul tube is moved into and fills the aseptic lifes of 9mL
Manage in the test tube of salt solution, fully vibration is well mixed thalline.The corresponding Spawn incubations of 15mL are inoculated in 16% bacterium amount that connects
Base, 37 DEG C of Anaerobic culturel culture 48h are passed on 2 times, and strain mother liquor is obtained after renewed vaccination fermentation 18h.
Lactobacillus acidophilus culture medium prescription：The g of casein peptone 10, the g of powdered beef 8, dusty yeast 4 g, D (+)-glucose 20
G, the g of magnesium sulfate 0.2, the g of sodium acetate 5, the g of Triammonium citrate 2, Tween-80 1 g, K2HPO42 g, the g of manganese sulfate 0.05, go
The mL of ionized water 1000, pH=6.2 ± 0.2.
Bifidobacterium adolescentis culture medium prescription：The g of beef extract 10, the g of peptone 5, the g of dusty yeast 3 g, D (+)-glucose 5,
The g of starch 1, the g of sodium chloride 5, the g of sodium acetate 3, the g of L-cysteine hydrochloride 0.5, mL, 115 oC are damp and hot for deionized water 1000
Sterilize 20 min, the g of PB 0.02, pH=6.8 ± 0.2 (25 DEG C).
Bifidobacterium longum culture medium prescription is：The g of broth extract 3, the g of proteose peptone 10, the g of tryptone 5, plant ketone 3
G, the g of dusty yeast 5, the g of liver extract 150 mL, D (+)-glucose 10, the g of soluble starch 0.5, the mL of solution A 10, solution B
5 mL, Tween-80 1 g, agar 15g, the mL of L-cysteine hydrochloride 10, the mL of horse blood 50, the mL of deionized water 825.（Wherein
Liver extract preparation method is：Plus 10 g hepar siccatums in 170 mL deionized waters, 1 h is incubated at 50 ~ 60 DEG C, be heated to boiling,
Maintain 5 min, pH=6.9 ± 0.1, filtering.Solution A formulation：K2HPO4 10 g, KH2PO410 g, the mL of distilled water 100.Solution B
Formula：MgSO4·7H2O 4 g, NaCl 0.2g, FeSO4·7H2O 0.2g, MnSO4·xH2O 0.2g, the mL of distilled water 100.）
Take the oligomeric sugar monomer of lotus seeds prepared by the equivalent present invention respectively simultaneously（LOS3-1、LOS3-2、LOS4）With oligomeric fruit
Sugar（FOS）For replacing D (+)-glucose carbon source in different strain culture medium（Glc）As test medium, strain is carried out
Iii vitro proliferation assay, without D (+)-glucose carbon source in the culture medium of blank control group.
Fluid nutrient medium is taken after 2,4,6,8,10,12,14,16,20,24 h are cultivated respectively, with 115 DEG C of moist heat sterilizations
20min, takes strain mother liquor and is inoculated with each culture medium with 16% bacterium amount that connects.37 DEG C of Anaerobic culturel 48h.Through the Liquid Culture after culture
Base is centrifuged 15min with 3000r/min, removes supernatant.Tool plug screw thread test tube is moved to after deionized water washing precipitation thalline 2 times to go
Ionized water is settled to 15mL, and spiral oscillator fully vibrates test tube 20s, is made thalline suspension, and extinction is determined at wavelength 600nm
Degree, makes bacteria suspension absorbance fall in the standard curve range of linearity during measure, the thalline of bacteria suspension is calculated by standard curve
Concentration, each treatment parallel determination 3 times.
SCFA carries out quantitative analysis using gas chromatography, and chromatographic condition is：HP-INNOWAX chromatographic columns（30 m
×0.320 mm×0.25 μm）；100 DEG C of initial temperature, keeps 0.5 min, then be heated to the programming rate of 4 DEG C/min
200 DEG C, the min of total process operation 20.3.The μ L of sample size 0.2, carrier gas is nitrogen, the mL/min of flow velocity 20；Combustion gas hydrogen flow rate
30 mL/min, the combustion-supporting mL/min of gas air velocity 300, the mL/min of tail nitrogen flushing throughput 19；240 DEG C of fid detector temperature,
240 DEG C of injector temperature；By the way of not shunting.Take 5 mL cultures during determination sample to be based in centrifuge tube, the min of ice bath 10
Afterwards, 4 mL sterile deionized waters, more than the min of magnetic force Stirring 2 are added.5000 × g of culture medium（At 4 DEG C）20 min are centrifuged
Afterwards, supernatant repeated centrifugation is taken once.Collect secondary centrifuging supernatant and inject 1.5 mL gas phase sample introductions after crossing 0.45 μm of filter membrane
In bottle, each sample is independently repeated three times, and the content of excrement Short-Chain Fatty Acids is determined with similarity condition.
When determining lactic acid, by 25 mL, the H of 4.5 mmol2SO4It is added in the culture medium solution of 5 mL, is sufficiently mixed and carries
1 h is taken, and 5 min are centrifuged with 12000 × g.Take 50 μ L of supernatant liquid use HPX-87H Aminex ion exchange columns (300 ×
7.8 mm, Bio-Rad company, the U.S.) isocratic separation is carried out, separation condition is flow velocity：0.7 mL/min, column temperature：65 DEG C, stream
Dynamic phase：H2SO4（3 mmol/L）, detector：RID, using quantified by external standard method.
1. Fig. 4 is influence of the different carbon source to lactobacillus acidophilus growth course.From fig. 4, it can be seen that lactobacillus acidophilus gives birth to
Logarithmic phase long is up to 12 h, in logarithmic phase is grown, lactobacillus acidophilus growth rate in the culture medium with glucose as carbon source
Highest, logarithmic phase is only 6 h, is secondly the culture medium with LOS3-1 as carbon source.Carbon source is to the final cultivation effect of lactobacillus acidophilus
Influence be successively LOS3-1>Glc>LOS4>FOS>LOS3-2.
The different carbon source of table 1 produces the influence of SCFA and lactic acid to lactobacillus acidophilus
Table 1 is influence of the different carbon source to lactobacillus acidophilus product SCFA and lactic acid after culture 10h and 24h.From table 1
As can be seen that lactobacillus acidophilus produces acetic acid and lactic acid in growth course using different carbon source metabolism is main, except Glc is carbon source
Culture medium outside, with the extension of fermentation time, lactobacillus acidophilus metabolism produce acetic acid and lactic acid dramatically increase（p<
0.05）.Wherein, lactobacillus acidophilus quickly can produce lactic acid using LOS3-1 and Glc fermentations, but at later stages, only LOS3-
Lactobacillus acidophilus in 1 culture medium can continued growth breeding produce lactic acid, and in the culture medium lactic acid content with Glc as carbon source simultaneously
Do not increase significantly（p>0.05）, culture medium its thalline for illustrating at later stages with Glc as carbon source increases to be stagnated.With
During LOS3-1 is for the culture medium of carbon source, after lactobacillus acidophilus cultivates 10 h, 24 h, its acetic acid and lactic acid content are all remarkably higher than
Other culture mediums, its content is corresponding consistent with thalline quantity in lactobacillus acidophilus growth curve, shows LOS3-1 to promoting acidophilus
The effect of lactobacillus propagation is notable（p<0.05）.The lactic acid production difference of lactobacillus acidophilus is not notable in Glc and LOS4 culture mediums
（p>0.0）.
2. Fig. 5 is influence of the different carbon source to bifidobacterium adolescentis growth course.From fig. 5, it can be seen that youth bifid bar
Bacterium thalline quantity in LOS4 is for the culture medium of carbon source is all remarkably higher than the culture medium for adding other carbon sources, and Glc is to youth bifid
The proliferation function of bacillus takes second place, and only LOS3-2 is less than positive controls.It follows that lotus seeds oligosaccharide is to bifidobacterium adolescentis
With preferable proliferation function, mainly caused by monomer LOS4.
The different carbon source of table 2 produces the influence of SCFA and lactic acid to bifidobacterium adolescentis
Acetic acid and lactic acid are considered as typical bifidobacterium fermentation end-product.Bifidobacterium adolescentis profit in growth course
Acetic acid, lactic acid, propionic acid, butyric acid are produced with different carbon source is metabolizable.Table 2 is that different carbon source produces short-chain fat to bifidobacterium adolescentis
Acid and the influence of lactic acid.As shown in table 2, extension over time, bifidobacterium adolescentis produces acetic acid using different carbon source metabolism
Dramatically increased with lactic acid content（p<0.05）；Except Glc, LOS4 and FOS for carbon source culture medium, in remaining culture medium propionic acid and
Butyric acid content is not dramatically increased（p>0.05）.In the culture medium with LOS4 as carbon source, acetic acid, lactic acid and butyric acid content are notable
Higher than other culture mediums, but propionic acid content is substantially less than the culture medium that Glc is carbon source（p<0.05）.Third is removed in each carbon source culture medium
Outside acid content, the variation tendency of remaining content of fatty acid is corresponding with bifidobacterium adolescentis growth curve, shows LOS4 to green grass or young crops
Spring Bifidobacterium has notable proliferation function（p<0.05）.
3. Fig. 6 is influence of the different carbon source to bifidobacterium longum growth course.From fig. 6, it can be seen that bifidobacterium longum exists
The cultivation effect in culture medium with LOS4 as carbon source is significantly higher than the culture medium for adding other carbon sources.Can by final thalline quantity
Know, 3 kinds of carbon sources of FOS, Glc, LOS4 are having no significant difference to the influence of bifidobacterium longum cultivation effect（p>0.05）.
The different carbon source of table 3 produces the influence of SCFA and lactic acid to bifidobacterium longum
Bifidobacterium longum produces acetic acid, lactic acid, propionic acid, butyric acid in growth course using different carbon source is metabolizable.Table 3 is
Different carbon source produces the influence of SCFA and lactic acid to bifidobacterium longum.As shown in table 3, bifidobacterium longum profit when cultivating 10 h
Fermented with LOS4 and produce acetic acid, lactic acid content to be significantly higher than other carbon sources（p<0.05）, bifidobacterium longum is utilized after 24 h
The butyric acid content that LOS4 and LOS3-1 fermentations are produced is significantly higher than other carbon sources（p<0.05）；Culture medium with LOS4 as carbon source
In, though propionic acid content is in dramatically increase trend in fermentation process, its content is substantially less than the culture that LOS3-1, FOS are carbon source
Base（p<0.05）, show that the produced propionic acid in growth course of bifidobacterium longum may be utilized generation butyric acid.
Embodiment 8
Human body infectious disease generally begins at adhesion of the pathogen to host cell, and a series of pathology are just caused thereafter
Physiology course.Adhesion of the pathogen to host depends on the specificity knot between its surface receptor and host surface saccharide compound
Close, the difference of host surface oligosaccharide receptor sequence determines that the host range and tissue of Microorganism colonization tend to.Such as large intestine
Bacillus ORN178 can be by the mannose on the FimH specific recognition enteron aisle hosts surface on its pili and then in combination and invade
Dye host cell；Actinomyces WVU45 then causes the mouth diseases such as carious tooth with reference to the galactolipin on host buccal surface；Influenza virus is then
By the acceptor on its surface HA combination respiratory tracts surface.This microorganism interacts to expand with the specificity of oligomeric saccharide acceptor
The application of exogenous oligosaccharide provides new possibility.Because lotus seeds oligosaccharide has mannose residue, the present invention is anti-stick to its
Attached effect is tested.
Drawn under germ-free condition in LB culture mediums to the enteropathogenic E. Coli freeze-dried vaccine preservation pipe of 1 mL, blown and beaten repeatedly mixed
It is even.400 μ L strain mother liquors are drawn to add into the sterile centrifugation tube of the LB culture mediums containing 3 mL, after the alcolhol burner sterilization mouth of pipe,
Tube sealing, is put into 37 DEG C of incubated 12 h in incubator.Then 1mL bacterium solutions are taken and is seeded to 9 mL green meat soup fluid nutrient mediums
In, the h of static state constant-temperature culture 12 is continued after mixing.Collecting add glycerine to freeze after bacterium solution is dispensed again, standby.Mark is pathogenic
During Escherichia coli, the centrifuge tube of 400 μ L strains mother liquors of absorption to the LB culture mediums equipped with 3 mL, 37 DEG C of incubated 12 h,
2500 × g is centrifuged 15 min, and abandoning supernatant under germ-free condition is using aseptic PBS solution washing thalline twice, resuspended by thalline
In the aseptic PBS solutions of 10 mL（Thalline final concentration is about 6 × 108cfu/mL）.FITC solution is subsequently added, makes FITC in solution
In final concentration of 10 mg/mL, lucifuge be incubated 12 h.Thalline after mark is centrifuged 10 min, collects thalline by 6000 × g
Precipitate and be resuspended in containing the 0.1% aseptic PBS solution of Tween-20, centrifugation and suspension thalline, repeat this process to wash again
Wash thalline 3 times.Use flow cytometer（The argon laser of 488 nm is used as excitation wavelength）Detection research fluorescence labeling is deposited to thalline
The influence of motility rate and fluorescence labeling rate.The bacteria suspension that will the have been marked h of avoid light place 1 ~ 5 at room temperature, uses sepectrophotofluorometer
The situation of thalline fluorescent quenching is studied, while carrying out influence of the plate count research FITC marks to Survival probability of bacteria to bacterium.
The Caco-2 cells of recovery are washed twice in aseptic PBS solution, add 96 orifice plates（Holding cell concentration about 1 ×
104Cells/well）.Then by 0.5 mL bacterial suspensions（Keep bacterial concentration about 5 × 106Cfu/ holes）It is added to cell plates each
Kong Zhong, is eventually adding the oligosaccharide monomer sample prepared by the present invention（LOS3-1、LOS3-2、LOS4）, make sugar-like product in every hole
Concentration is about 1.625mg/mL, 3.25 mg/mL, 7.5 mg/mL, 15 mg/mL, 30 mg/mL, and plate then is positioned over into CO2Training
37 DEG C of 3 h of culture in case are supported, after bacterial adhesion quantitatively processes 2 h using pancreas enzyme -EDTA, is containing the aseptic of 0.1% Tween-20
Washed 3 times in PBS, remove nonadherent bacterium.Fluorescence microscope bacterial adhesion cell situation, while using ELIASA
（Excitation wavelength is 488 nm, and Detection wavelength is 528 nm）Fluorescence intensity is determined, experiment is in triplicate.Enteropathogenic E. Coli pair
The adhesion rate of Caco-2 cells is calculated according to the following formula：
。
Fig. 7 adheres to the influence of Caco-2 cells for the carbon source of various concentrations to enteropathogenic E. Coli.From fig.7, it can be seen that sweet
Dew sugar（Man）, LOS3-1, LOS3-2, LOS4 and mannan-oligosaccharides（MOS）With the increase of additive capacity, to suppressing pathogenic
The effect of E. coli adhesion Caco-2 cells also constantly strengthens, and wherein mannose, LOS4 and mannan-oligosaccharides reach in dosage
Inhibition increase is not notable after 15 mg/mL（p>0.05）.Overall inhibition LOS4>Mannan-oligosaccharides>Mannose>LOS3-
2>LOS3-1, when additive capacity is 15 mg/mL, LOS4 and mannan-oligosaccharides inhibition difference is not notable（p>0.05）.
From above-mentioned experiment, the separating obtained oligomeric sugar monomer of different lotus seeds of the present invention can be in human gastrointestinal tract
Probiotics（Such as lactobacillus and Bifidobacterium）Proliferation function is played, while suppressing harmful bacteria（Such as enteropathogenic E. Coli）Adhere to
Enterocyte surface, is expected to turn into a kind of potential prebiotics product.
The present invention is separated through the rapid extraction that appropriate transformation can be used for various natural function oligosaccharide compositions, the lotus of gained
The oligomeric sugar monomer of son can be of the invention for expanding lotus seeds further technological processing way as functional food, with huge market potential.
Embodiment described above only expresses several embodiments of the invention, and its description is more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made.Therefore, the guarantor of patent of the present invention
Shield scope should be determined by the appended claims.Greatly simplified with technological process, substantially reduce, produce without enzyme-added hydrolysis, cost
Produce rate advantage high.
Claims (1)
1. the fast separating process of the oligomeric sugar monomer of a kind of lotus seeds with prebiotic effect, it is characterised in that：Methods described has
Body is comprised the following steps：
1）Sorting is dried：Full grains, undamaged fresh lotus seed are chosen, screening coring contains after being dried to moisture at 40 ~ 50 DEG C
Measure is 4 ~ 7%；
2）Pulverize and sieve：By step 1）Dried lotus seeds are crushed, and cross 40 mesh sieves；
3）Add water compounding：Take step 2）Gained lotus seeds powder, by solid-liquid ratio 1:5 addition deionized waters are mixed and compounded；
4）Washing starch：By step 3）Gained compounding feed liquid crosses 100 mesh sieves, and with the deionized water rinsing of 4 ~ 6 times of lotus seeds weight
Filter residue, staticly settles 8 h under the conditions of 4 DEG C are placed in after merging filtrate, centrifugation removal is precipitated, then by after gained supernatant and flushing
Lotus seeds filter residue merges：
5）Constant temperature refluxing extraction：By step 4）Gained supernatant and lotus seeds filter residue are in constant temperature refluxing extraction 1.5 ~ 2 at 75 ~ 85 DEG C
h；
6）Suction filtration：Extraction terminate after by extract solution vacuum filtration, discard filter residue；
7）Alcohol is analysed：By step 6）Gained filtrate adds 3 ~ 5 times of concentrate after the 1/5 ~ 1/4 of original volume is concentrated at 60 ~ 65 DEG C
Volume, mass concentration are 95% ethanol, are mixed after 10 ~ 12 h of standing at 4 DEG C；
8）Centrifugal concentrating：By step 7）Precipitation is removed after resulting solution centrifugation, supernatant at 55 ~ 65 DEG C in being concentrated into original volume
1/3~2/5；
9）Freeze-drying：By step 8）Gained concentrate is freeze-dried, obtains lotus seeds oligosaccharide raw sugar powder；
10）MPLC is separated：By step 9）Gained lotus seeds oligosaccharide raw sugar powder is dissolved in deionized water, be configured to concentration for 0.1 ~
The solution of 0.2 g/mL, then injects solution MPLC and is separated, according to retention time after 0.22 μm of filtering with microporous membrane
Each target compound is pointedly collected, collection liquid is chilled dry respectively at after rotary evaporation in vacuo recycling design at 55 ~ 65 DEG C
The dry freeze-dried powder that must contain specific compound；
11）MPLC secondary separations：Take step 10）Freeze-dried powder of the gained containing specific compound, respectively by step 10）Operation and
Chromatographic condition carries out sub-sampling, collects each target peak of identical retention time, and through solvent recovery, that freeze-drying obtains final product lotus seeds is low
Glycan monomer powders；
The piece-rate system of MPLC includes middle pressure special glass post, and the ultraviolet full wavelength scanner device of chromatogram pump, DAD and FLASH ELSD steam
Light Scattering Detector, automatic fraction collector and manual sample load-on module；
Medium pressure special glass post is the Claricep Flash hydrophilic posts of HILIC, and its packing material size is 20-35 μm, aperture
100A, the m of specific surface 3202/ g, carbon content 5.0%；
The testing conditions of the FLASH ELSD EISDs are：Purging nitrogen pressure is 2.5 ~ 3.5bar, drift
85 ~ 95 DEG C of pipe temperature；
Loading volume during MPLC is separated is 1 mL, and mobile phase is the binary mixture of acetonitrile and water, its flow velocity is 30 ~
45mL/min, column temperature is 25 DEG C；
Mobile phase ratio change specific procedure be：Acetonitrile ratio is 85 ~ 90% 15 ~ 20min of holding in mobile phase, then by second
Nitrile ratio is down to 65 ~ 60% in 30 ~ 35min, keeps 50 ~ 60min, then acetonitrile ratio is risen into 85 ~ 90%, keep 15 ~
20min。
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| 莲子低聚糖快速制备工艺研究;卢旭等;《中国食品学报》;20150531;第15卷(第5期);第96-104页 * |
| 莲子低聚糖提取工艺优化及其组分分析;卢旭等;《热带作物学报》;20150528;第36卷(第4期);813-820 * |
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